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Tc and Th1 interaction and viral immune response

Tc and Th1 interaction and viral immune response


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Tc is T cell which can give T killer cells and T helper cells. T helper cells (Th1) see the pathogen presented by antigen presenting cells (dendritic cells and macrophages). They then secrete antigens to kill the pathogen. If there is memory from previous interaction, then it will be easier to protect. However, since some viruses replicate fast (RNA particularly), this memory benefit is often lost, like in the case of influenza virus A and B types. However, I am uncertain how to explain this immunology with viruses.

How can you explain this general viral interaction better in the immune response?

I have these pictures in mind:

and


PlaysDice's answer:

Antigen presenting cells migrate from skin/mucosa to secondary lymphoid organs like lymph nodes/spleen/GALT and drive T cell activation and maturation. T helper cells with CD8 and MHCII complex recognise the virus and kill it.

I think this is what is happening, for instance in HIV infection:

where

  • dendritic cell (ADCC) presentation of HIV infected CD4+ to MHCII and T helper cell
  • T helper cell activates cytokine release
  • Natural killer cells come and lyse the HIV infected CD4+ T lymphocyte

Frontiers in Microbiology

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    In vivo antiviral host response to SARS-CoV-2 by viral load, sex, and age

    Despite limited genomic diversity, SARS-CoV-2 has shown a wide range of clinical manifestations in different patient populations. The mechanisms behind these host differences are still unclear. Here, we examined host response gene expression across infection status, viral load, age, and sex among shotgun RNA-sequencing profiles of nasopharyngeal swabs from 430 individuals with PCR-confirmed SARS-CoV-2 and 54 negative controls. SARS-CoV-2 induced a strong antiviral response with upregulation of antiviral factors such as OAS1-3 and IFIT1-3, and Th1 chemokines CXCL9/10/11, as well as a reduction in transcription of ribosomal proteins. SARS-CoV-2 culture in human airway epithelial cultures replicated the in vivo antiviral host response. Patient-matched longitudinal specimens (mean elapsed time = 6.3 days) demonstrated reduction in interferon-induced transcription, recovery of transcription of ribosomal proteins, and initiation of wound healing and humoral immune responses. Expression of interferon-responsive genes, including ACE2, increased as a function of viral load, while transcripts for B cell-specific proteins and neutrophil chemokines were elevated in patients with lower viral load. Older individuals had reduced expression of Th1 chemokines CXCL9/10/11 and their cognate receptor, CXCR3, as well as CD8A and granzyme B, suggesting deficiencies in trafficking and/or function of cytotoxic T cells and natural killer (NK) cells. Relative to females, males had reduced B and NK cell-specific transcripts and an increase in inhibitors of NF-κB signaling, possibly inappropriately throttling antiviral responses. Collectively, our data demonstrate that host responses to SARS-CoV-2 are dependent on viral load and infection time course, with observed differences due to age and sex that may contribute to disease severity.


    Developmental Biology of the Innate Immune Response: Implications for Neonatal and Infant Vaccine Development

    Molecular characterization of mechanisms by which human pattern recognition receptors (PRRs) detect danger signals has greatly expanded our understanding of the innate immune system. PRRs include Toll-like receptors, nucleotide oligomerization domain-like receptors, retinoic acid inducible gene-like receptors, and C-type lectin receptors. Characterization of the developmental expression of these systems in the fetus, newborn, and infant is incomplete but has yielded important insights into neonatal susceptibility to infection. Activation of PRRs on antigen-presenting cells enhances costimulatory function, and thus PRR agonists are potential vaccine adjuvants, some of which are already in clinical use. Thus, study of PRRs has also revealed how previously mysterious immunomodulators are able to mediate their actions, including the vaccine adjuvant aluminum hydroxide that activates a cytosolic protein complex known as the Nacht domain leucine-rich repeat and pyrin domain-containing protein 3 inflammasome leading to interleukin-1β production. Progress in characterizing PRRs is thus informing and expanding the design of improved adjuvants. This review summarizes recent developments in the field of innate immunity emphasizing developmental expression in the fetus, newborn, and infant and its implications for the design of more effective neonatal and infant vaccines.

    Need for Effective Neonatal and Infant Vaccines

    On a global basis, infections result in ∼ 2 million deaths per year in those younger than 6 mo (World Health Organization) (1). Common pathogens in neonates and/or infants include Gram-positive bacteria (e.g., group B Streptococcus, Streptococcus pneumoniae), Gram-negative bacteria (such as Escherichia coli and Bacillus pertussis), herpes simplex virus, respiratory syncitial virus, and rotavirus. This susceptibility highlights the unmet need for effective vaccines for newborns and infants and the functional immunodeficiencies that must be overcome in designing vaccines that adequately protect the very young. As birth is the most reliable point of healthcare contact worldwide, vaccines that are active at birth are a major global health imperative (2). Design and development of such vaccines will require understanding of the developmental expression of innate immune pathways whose activation enhances the adaptive immune response.

    Distinct Aspects of Neonatal Innate Immunity That Pose Challenges to Effective Vaccination Early in Life

    The fetal immune system is heavily Th2-biased, presumably to avoid proinflammatory Th1-type alloimmune responses to maternal tissues that may trigger preterm birth or spontaneous abortion.

    Birth triggers a dramatic shift in environment that places further demands on the neonatal immune system, mediating the transition from a normally sterile intrauterine compartment to a foreign antigen (Ag)-rich external environment. This transition includes the first colonization of the skin and gastrointestinal tract. In contrast to low levels of Th1-type cytokines [e.g., tumor necrosis factor (TNF), interleukin (IL)-12p70, interferon (IFN)-γ], human neonatal plasma contains high levels of the Th2-type cytokine IL-6 in vivo at birth and throughout the first week of life (3). IL-6 induces an acute phase response at birth that may serve to shield against bacterial infections and clear microbial products and/or pattern recognition receptor (PRR) agonists (4).

    The distinct polarization of fetal and early neonatal immune responses presents obstacles to effective neonatal immunization, including impaired antigen-presenting cell (APC) responses (e.g., impaired IFNγ production) to many (but not all) stimuli, a Th2 bias to immune responses, impaired antibody (Ab) affinity maturation (5), and the potential inhibitory effect of maternal Abs (6).

    Quantitative and qualitative differences exist between neonatal and adult APCs. Qualitative differences are evident in human monocytes in utero as assessed by flow cytometry indicating reduced expression levels of major histocompatability complex (MHC) class II molecules (7). Several mechanisms have been implicated in skewing neonatal APCs toward Th2-type responses, including a) the production by placental tissues of transforming growth factor-β, progesterone and prostaglandin E2 that enhance Th2 cytokine production (8) and b) the presence in neonatal blood plasma of relatively high concentrations ( ∼ 300 nM) of adenosine, an immunosuppressive endogenous purine metabolite (4,9). The patterns of neonatal cytokine production appear to be relevant in vivo, in that after birth, during the first week of life, human neonatal peripheral serum levels of TNF remain low (relative to human adult serum) whereas levels of IL-6, a Th2-polarizing cytokine with anti-inflammatory properties, increase.

    Multiple studies document that human neonatal monocytes and APCs function suboptimally when tested in vitro with respect to costimulatory responses to most stimuli (10). Studies of murine neonatal dendritic cells (DCs) have demonstrated that Ag-presentation increases during ontogeny, correlating with increased costimulatory molecule expression and increased responses to protein-conjugated, T cell-dependent polysaccharide Ags (11). However, C57BL/6 neonatal mice (1–7 d) demonstrated stronger LPS-induced inflammatory cytokine production by splenocytes in the presence of T cells in vitro and after intraperitoneal injection in vivo, ascribed to neonatal quantitative deficiency of CD4 + and CD8 + T cells (12). Thus, inflammatory responses in neonates seem to be dependent on the species [noting that innate immune genes are hypervariable between species, including between humans and mice (13,14)], experimental model (in vitro versus in vivo), extracellular culture medium [autologous 100% (vol/vol) adenosine-rich neonatal plasma/serum (15) versus low concentrations of heat-inactivated fetal calf serum that are used in many studies], and particular stimulus studied.

    In contrast to neonatal APCs, neonatal CD4 + /CD25 high T regulatory cells (Treg) are fully functional and found at high abundance in human fetal lymphoid tissues (16) and newborn cord blood (17,18). Neonatal Treg cells suppress both T-responder cell proliferation and Th1-cytokine (e.g., IFN-γ) production, induced by self-Ags, to limit adaptive immune responses. Treg mediate their effects by both cell contact-dependent and contact-independent mechanisms, including secretion of IL-10, CD39, and CD73 (ectonucleotidase)-mediated generation of extracellular adenosine, and adenosine A2A receptor-mediated enhancement of cyclic AMP concentrations in target T-responder cells (19,20). Neonatal inhibition of autoimmunity via Treg suppression has clear advantages as neonates first encounter the foreign-Ag rich world however, these effects may be detrimental to neonatal immunity to infection (21) and to neonatal vaccine responses (22,23).

    Innate Immune Activation Enhances Adaptive Immune Responses

    Activation of PRRs on APCs such as macrophages and DCs enhances Ag-presenting activity and adaptive immune responses via direct and indirect mechanisms (24). PRR signaling is influenced by cross signals mediated via diverse PRR classes, tyrosine kinases, tyrosine phosphatases, ubiquitinating systems, and glucocorticoids, and therefore varies between different cell types (25). On activation, DCs efficiently process and present Ags in the context of MHC, increase production of Th1-polarizing cytokines such as IL-12p70, and up-regulate costimulatory molecules (e.g., CD40, CD80, CD86) and chemokine receptors mediating cell migration from the tissues into the draining lymph nodes. Once inside the lymph nodes, DCs interact with naïve T and B cells inducing their differentiation into effector cells, thereby triggering an acquired immune response.

    In addition to their direct effect on APCs, PRR agonists also enhance the transition from innate to adaptive immune responses via indirect mechanisms. For example, Toll-like receptor (TLR)-mediated DC activation induces IL-6 that renders T-responder cells refractory to inhibition by suppressive Treg (26). Tissue-derived signals may also influence APCs and control the type of effector class generated (27). TLR-mediated activation of DCs enhances lymph node function via triggering DC migration to lymph nodes, expression of vascular endothelial growth factor, increasing high endothelial venule proliferation, remodeling primary feed arterioles, and increasing nodal blood flow and recruitment of rare Ag-specific lymphocytes (28,29).

    The impressive progress in defining the potential roles of PRRs has provided an opportunity to better understand the development of innate immune responses after birth, with potential implications for the optimization of vaccine development. Later, we review the current state of knowledge regarding the developmental expression of key PRRs, highlighting both recent progress and gaps in our knowledge.

    Developmental Expression of PRRs

    Responses to microbial infection are initiated through an innate immune system that features diverse PRRs poised for activation in extracellular and intracellular locations (Fig. 1).

    Mechanisms of innate immune activation induced by vaccine adjuvants. TLR agonists found in multiple neonatal vaccines (Table 1) activate either cell associated or intracellularly located TLRs or NODs. These PRRs then interact with specific adaptor molecules culminating in NF-κB or IRF activation. Viral-derived products (dsRNA or ssRNA) can also activate endosomal TLRs, along with RLRs (such as RIG-I), which induce type I IFN production. The vaccine adjuvant Alum activates the cytosolic NALP3/inflammasome leading to proIL-1β cleavage into bioactive IL-1β.

    Toll-like receptors.

    TLRs are type I transmembrane proteins with an extracellular amino terminus and an intracellular carboxy terminus. They are composed of various domains including extracellular leucine rich repeat (LRR) with one or two cysteine-rich regions and an intracellular toll/IL-1 receptor (TIR) domain, named after its homology with the IL-1 receptor. Humans express 10 TLRs: a) surface expressed TLRs include TLR2 (bacterial lipopeptides), TLR4 (lipopolysaccharide), and TLR5 (flagellin) b) endosomal TLRs include TLR7 and 8 [single stranded RNA (30,31)] and TLR9 (unmethylated CpG DNA). Engagement of TLRs activates intracellular signaling cascades via myeloid differentiation factor 88 (MyD88)-dependent and MyD88-independent pathways (32), including IL-1 receptor-associated kinase-4 (IRAK-4) recruitment, culminating in NF-κB activation and expression of proinflammatory cytokines, such as TNF, IL-6, and pro-IL1β [note that processing to mature IL-1β requires Nacht domain leucine-rich repeat and pyrin domain (PYD)-containing protein 3 (NALP3) inflammasome (INFL) action]. TLR stimulation can also lead to the activation of several other intracellular signaling pathways such as those involving Jun N-terminal kinase, mitogen activated protein kinase (33,34), interferon regulatory factor (IRFs), and the FAS-associated death domain-induced apoptosis pathway (32).

    The importance of the TLR pathway for host defense in newborns and infants is apparent in the clinical consequences of TLR pathway defects. Defects of signaling molecules downstream of TLRs, including IRAK4 and MyD88 deficiency result in selective susceptibility to pyogenic infections (often streptococcal and staphylococcal) during childhood with improvement later in life (35–37).

    Cord blood monocytes of full-term human newborns express normal amounts of TLRs, yet on TLR-mediated stimulation in whole cord blood (i.e., 100% vol/vol autologous, adenosine-rich plasma), agonists of TLRs 1–7 demonstrate a 1–3 log impairment in TNF-α production relative to adult peripheral blood monocytes (15). One of the mechanisms accounting for this impairment is that during the hypoxia and stress accompanying the birth process, plasma concentrations of adenosine, an endogenous immunosuppressive purine metabolite, increase. Adenosine can act via A3 adenosine receptors on neonatal mononuclear cells to induce production of cAMP, a key second messenger that inhibits TLR-mediated production of Th1-polarizing cytokines (4,9). Other studies have demonstrated that LPS-induced responses of newborn mononuclear cells are diminished at birth because of reduced expression of the TLR adaptor molecule MyD88 (38) and by failure of nucleosome remodeling at the IL-12 promoter (39). Impaired TLR agonist-induced production of type I IFNs from human neonatal plasmacytoid DCs (pDCs) and neonatal monocyte-derived DCs (moDC) has also been described (40,41).

    In contrast to agonists of TLRs 1–7, TLR8 agonists, including TLR7/8 agonists, are able to induce robust immune responses by human neonatal APC, comparable with those of healthy adult controls (42). Exposure of human neonatal cord blood-derived monocytes and APCs, including myeloid dendritic cells and moDCs, to TLR8 agonists induces robust (i.e., adult level) phosphorylation of p38 MAP kinase, NF-κB activation, proinflammatory cytokine production (TNF, IL-12), and upregulation of costimulatory molecules (e.g., CD40).

    Retinoic acid inducible gene-like receptors.

    During the infection cycle of some viruses, double stranded RNA is produced that can be detected in the cell cytosol by retinoic acid inducible gene-like receptors (RLRs), such as retinoic acid inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5). These cytoplasmic proteins are composed of amino terminal caspase-recruitment domains (CARDs) and a carboxy-terminal helicase domain (43,44). RLR activation induces CARD domain interaction with the CARD domain-containing adaptor protein, IFN-β promoter-stimulator (IPS)-1 (also known as mitochondrial antiviral signaling protein, virus-induced signaling adaptor, and CARD adaptor inducing IFN-β) leading to IRF3 and NF-κB activation. Little is known regarding the developmental expression of RLRs at birth and in the neonate, an important area of future study.

    Nucleotide oligomerization domain-like receptors.

    Nucleotide oligomerization domain-like receptors (NLRs) detect bacterial components and include members of the nucleotide-binding oligomerization domain (NOD) subfamily and NLRs associated with the INFL. The NOD proteins in humans are a family of >20 cytosolic proteins. Structurally they are composed of: a) a variable N-terminal effector-binding domain, usually consisting of a PYD or CARD, capable of regulating homotypic and heterotypic binding b) a centrally located NOD domain and c) a C-terminal ligand-recognition domain, which can be composed of LRRs. As with the RLRs, little is known regarding the developmental expression of NODs at birth and in the neonate.

    The INFL is a cytosolic multicomponent protein complex, including caspase-1, which on activation cleaves pro-IL-1β to the potent proinflammatory cytokine IL-1β. IL-1β has a central role in the onset of parturition, particularly in the context of intrauterine infection/inflammation. A recent study, found that: a) caspase-1 concentration in the amniotic fluid increases as a function of gestational age b) women with spontaneous term labor had a higher median caspase-1 amniotic fluid concentration than women at term without labor, suggesting that the INFL may be activated in spontaneous parturition at term and c) higher caspase-1 levels were associated with infection/inflammation (45). Human neonatal monocytes, in particular those of preterm newborns, demonstrate impaired IL1-β production to LPS (45a) and to lipoteichoic acids (45b). A search of Pubmed did not yield any publications relating to INFL function at birth.

    C-type lectin receptors.

    C-type lectin receptors (CLRs) can be produced as secreted soluble proteins, including mannose binding lectin (MBL), lung surfactant protein A, or as transmembrane proteins such as selectins, mannose receptor, and the DC-specific ICAM-3 grabbing nonintregrin (DC-SIGN). MBL is an acute phase plasma protein whose expression in the liver is upregulated during inflammation. MBL recognizes a variety of carbohydrate patterns found on infectious microorganisms including bacteria (e.g., Staphylococcus aureus), fungi (e.g., Saccharomyces cerevisiae), and viruses (e.g., HIV) and on altered selfglycoproteins (e.g., aberrant glycosylation of cancer cells). MBL binding to carbohydrate targets triggers activation of MBL-associated serine protease, cleaving complement proteins, and promoting opsonisation/membrane attack complex formation (46). MBL can also act directly as an opsonin by binding to receptors, which promote phagocytosis (46) and can modulate cytokine production in vitro and in vivo (47) thereby playing important roles in neonatal infection. Plasma MBL concentrations in both premature and full-term neonates are lower than those of adults (48), but steadily increase during the first weeks of life, possibly as a consequence of the skew of neonatal cytokine responses toward IL-6, which induces the acute phase response (3,4). MBL deficiency is associated with an increased risk of bacterial sepsis (49).

    From the standpoint of innate immune modulation of vaccine responses, MBL deficiency enhances mouse Ag-specific IgG production after immunization with tetanus toxoid-conjugated group B Streptococcus polysaccharide vaccine or tetanus toxoid alone (50). These data suggest that under certain circumstances MBL can inhibit Ab production. Further characterization of the MBL pathway may inform design of vaccines that minimize such inhibitory MBL effects.

    Developmental expression of antimicrobial proteins and peptides.

    APPs expressed by leukocytes and epithelial cells are ancient components of innate immune defense that are best known for their ability to kill microorganisms and neutralize their surface components. However, some APPs also demonstrate activity in modulating adaptive immune responses. For example, bactericidal/permeability-increasing protein (BPI), a neutrophil-derived primary granule protein with endotoxin binding activity, was recently shown to enhance APC function (50a). Neonates are deficient in BPI expression, increasing the possibility that may also impair delivery of naturally shed Gram-negative bacterial LPS outer membrane blebs to APCs. Defensin peptides can enhance production of antiviral IFNs, which are important to Th1 polarization and cross-presentation (51). Cathelicidin peptides, found in neutrophil secondary granules, enhance adaptive immune responses in vivo. Indeed there is an age-dependent maturation in the expression in human neonatal blood plasma of a broad range of APPs, and thus to the extent that these molecules contribute to modulating adaptive immune responses, deficiencies in their expression could contribute to impaired adaptive responses.

    The recent progress in defining PRRs and APPs involved in triggering innate immune responses and thereby modulating adaptive immunity provides new opportunities to understand that function of adjuvants in currently administered vaccines, as outlined below.

    Engagement of Innate Immunity by Currently Approved Neonatal and Infant Vaccines

    It is increasingly appreciated that an important determinant of vaccine efficacy is the ability of a given vaccine to activate the innate immune system to enhance APC function and Th1-polarizing adaptive responses (52). Currently, the two vaccines that are regularly given at birth in humans are the Bacillus Calmette-Guerin (BCG) vaccine and the hepatitis B (HepB) vaccine (Table 1). BCG is capable of inducing a strong Th1-type immune responses in human neonatal cells in vitro (53) and in vivo (54), which is in part due its ability to activate multiple TLRs expressed by APCs (55). The BCG vaccine demonstrates that under certain conditions, the human neonatal immune system is capable of mounting a protective Th1-type response (56), but the underlying mechanisms of its efficacy at birth is not fully understood. Recent work has indicated that it is very likely that activation of multiple PRRs by BCG plays important roles in its efficacy. The BCG vaccine is a live bacterium and able to activate TLR2 (57,58) and TLR4 (57,58) by virtue of its cell wall that consists of peptidoglycan, arabinogalactan, and mycolic acids (59), and TLR9 through its CpG-rich DNA (60). More recently, the possibility that BCG also engages TLR8 has also been raised based on associations of TLR8 polymorphisms with susceptibility to pulmonary tuberculosis, increased susceptibility in males (TLR8 is encoded on the X chromosome), and the ability of BCG to induce macrophage TLR8 expression (61).

    Hepatitis B virus (HBV) is a global public health threat that has chronically infected >350 million people worldwide (62). The HepB vaccine is composed of a virus-like particle containing the viral envelope protein hepatitis B surface Ag (HBsAg), prepared using recombinant DNA technology. HepB vaccine is composed of an aluminum-containing ajuvant, but is still incompletely immunogenic as ∼ 10% of vaccinated populations fail to mount immune responses to HBsAg after immunization (Table 1).

    Haemophilus influenzae type b (Hib) conjugate vaccines are initially administered at 6–8 wk of age. Hib activates transfected HEK293 cells in a TLR2-dependent and TLR4-dependent manner, likely reflecting expression of bacterial lipopeptides (TLR2) and lipopolysaccharide (TLR4) (63,64) (Table 1). Indeed, the outer membrane protein complex (OMPC) found within the Hib-OMPC glycoconjugate vaccine is TLR2 and MyD88-dependant, and in the absence of TLR2, the immunogenicity of the Hib-OMPC vaccine is significantly reduced (64).

    In addition to TLR-induced vaccine responses (65), activation of RLRs [RIG-I and MDA-5 (66)] has been reported for the wild-type measles virus, suggesting that the attenuated strains used for vaccination may also engage this innate immune pathway (Table 1).

    To date, the US Food and Drug Administration (FDA) have approved a limited number of human vaccine adjuvants, chief among which is aluminum hydroxide, aluminum phosphate (typically referred to as “Alum”). Alum is a commonly used adjuvant present in human and animal vaccines worldwide (HepB, DTaP, Hib, PCV7, and HepA Table 1) and is known for its ability to induce protective Th2-type responses. It was recently demonstrated that the key to Alum's adjuvant activity is its ability to activate the NALP3/INFL (67,68) (Fig. 1). These conclusions were based on the observations that IL-1β and IL-18 production by macrophages in response to Alum in vitro requires intact INFL signaling. Moreover, mice deficient in NALP3, apoptosis-associated speck-like protein containing a CARD or caspase-1 failed to mount a significant Ab response to an Ag administered with Alum, whereas the response to complete Freund's adjuvant remained intact. These data highlight the crucial role of the NALP3/INFL in Alum's adjuvant activity.

    MF59 is an oil-in-water squalene emulsion and the precise mechanism of its adjuvant effects is still largely unknown (69). Recent studies have revealed that MF59 is a very efficient adjuvant due to its ability to activate and sustain tissue-resident DCs (69a). Studies using fluorescently labeled MF59 injected intramuscularly then observed 2 d later indicated partial localization in T cell areas of the draining lymph node (70). MF59 has also been documented to induce a significant influx of macrophages to the site of infection in mice, which is chemokine receptor 2 dependant (71). Of interest, the oil emulsion incomplete Freund's adjuvant (not licensed in humans due to its toxicity) induces optimal IgG1 and IgG2c in a NOD2-dependant manner (72).

    Translational Research Toward Improved Adjuvants for Newborns and Infants

    Given that newborns and young infants are susceptible to multiple bacterial and viral pathogens, neonatal immunization is of particular importance because: a) birth represents a likely point of contact of the newborn with healthcare providers, b) early life immunization is associated with a substantially higher rate of vaccination coverage than immunization given at later time points (2,73), c) vaccines that require multiple dosing regimens throughout infancy increase cost and reduce compliance, and (d) strategies involving immunization of the mother pose substantial logistical and medico-legal challenges. There is, therefore, an unmet medical need to develop vaccines that would be more effective very early in life or that would require fewer doses to generate durable and protective immune responses.

    A novel approach to neonatal vaccination has recently been described, which uses an attenuated strain of the intracellular pathogenic bacterium Listeria monocytogenes to deliver Ag to the cytoplasm of APC (74). Vaccinated neonatal mice induced strong CD8 + and CD4 + T cell responses and were protected after wild-type challenge. Of note, L. monocytogenes is able to activate several PRRs including multiple NODs (IPAF, NALP3) (75) and can induce the expression of RIG-I (76), possibly accounting for its immunostimulatory properties. Therefore, this approach could potentially overcome preexisting hurdles of maternal immune response interfering with neonatal vaccine responses (77). This live attenuated vaccine seemed safe in this murine model in that there was no associated mortality and no bacteria were recovered from the spleens or liver of immunized newborn mice 7 d postvaccination.

    Among the TLR agonists, TLR7/8 agonists hold particular promise as potential adjuvants for use in neonatal and infant vaccines as they induce robust production of the Th1-polarizing cytokines TNFα and IL-12 from neonatal (and adult) APCs that substantially exceeds responses induced by agonists of TLR-2, TLR-4, or TLR-7 (alone) (42,78). Therefore, TLR7/8 agonists have potential as novel neonatal vaccine adjuvants, due to their ability to activate both TLR-dependant and independent pathways (NALP3/INFL) mediating Th1-type responses from APC (78). In addition, human Treg express TLR8 and mediate reversal of Treg function on exposure to TLR8 agonists (79) (Fig. 2). Indeed TLR7/8 agonists, such as the synthetic low-molecular weight (<400 Da) antiviral imidazoquinoline compounds imiquimod and resiquimod (R848), have already been used as immunomodulators for many years against specific viral infections. The US FDA approved imiquimod for the treatment of external genital and perianal warts caused by certain strains of human papilloma virus (80), and it may also have activity against molluscum contagiosum (mulluscipox virus) (81–83). One of the main cellular targets of imiquimod is pDCs (IFN-α producing cells), which express high amounts of TLR7, whose engagement induced IFN-α in a MyD88-dependent manner (80,84).

    TLR8 agonists activate human APCs and reverse human Treg function. TLR8 agonists, such as R848, ssRNA and stabilized immune modulatory RNA (SIMRA) strongly activate human APCs via TLR8-dependent and TLR8-independent mechanisms including activation of the NALP3 inflammasome inducing IL-1β production. Exposure of human neonatal APCs to TLR8 agonists induces robust phosphorylation of p38 MAP kinase and profound/prolonged disappearance of IkB-κ, resulting in robust induction of protective Th1-type immune responses, including production of IL-12 and up-regulation of the costimulatory molecule CD40. TLR8 agonists also reverse suppression mediated by human Treg cells, via both direct action on Treg as well as by induction of APC production of IL-6, a cytokine that renders Tresponder cells refractory to Treg-mediated inhibition.

    R848 is an effective adjuvant for HBsAg vaccination in mice, increasing humoral and cellular immune responses. R848 used in combination with the TLR9 agonists CpG ODN further strengthened the immune response and long-lasting HBsAg-specific T cells displaying effector memory phenotype were detected in mice (85). R848 or topical application of imiquimod administered with Leishmania Ag induced protective Th1 immune responses in mice, compared with Leishmania Ag alone. In addition, s.c. vaccination also induced protective immunity whereas intramuscular vaccination did not (86). Indeed, conjugation of the TLR7/8 agonist to the HIV Gag protein improved the magnitude of Th1 and CD8 responses in adult Rhesus macaques (87). A combined TLR7/8 agonist compared with a pure TLR8 agonist can also induce greater Th1 responses and IFNα production from pDCs, which express TLR7 but not TLR8. IFNα is a key cytokine within a vaccine adjuvant setting, inducing Th1 differentiation (80,88), induction of cytotoxic T lymphocytes, enhancement of cross presentation and of primary Ab responses and DC activation (89). Novel TLR7 and TLR8 agonists referred to as stabilized immune modulatory RNA (SIMRA) compounds have recently developed and have distinct pharmacodynamic characteristics (90). SIMRA compounds demonstrate greater stability in human sera compared with linear RNA, which is rapidly degraded by ubiquitous RNases. In addition, SIMRA compounds and are able to activate TLR7 or TLR8 in HEK293 cells without the need for lipid carriers.

    TLR9 agonists are also undergoing biopharmaceutical development for multiple indications, including as vaccine adjuvants for HBV by linking an immunostimulatory DNA sequence to the recombinant HBsAg. This vaccine formulation, which has currently completed phase III trials, may help drive Th1-type responses and reduce Th2 responses (91).

    A murine study demonstrated that although the IPS-1 signaling pathway seems to be important for initial type I IFN responses, the TLR7/MyD88 pathway is needed for induction of protective immune responses to influenza A infection. Inactivated influenza virus vaccine failed to confer protection against lethal challenge with live influenza virus in TLR7-deficient and MyD88-deficient mice. Thus, protective adaptive immune responses to live attenuated influenza A virus strains are likely dependent on the TLR7-MyD88 pathway (92).


    The molecular biology and immune control of chronic Toxoplasma gondii infection

    Department of Microbiology, Immunology and Cancer Biology and the Carter Immunology Center, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

    Address correspondence to: Sarah E. Ewald, Carter Immunology Center MR-6 3706, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA. Phone: 434.924.1925 Email: [email protected]

    Find articles by Zhao, X. in: JCI | PubMed | Google Scholar | />

    Department of Microbiology, Immunology and Cancer Biology and the Carter Immunology Center, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

    Address correspondence to: Sarah E. Ewald, Carter Immunology Center MR-6 3706, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA. Phone: 434.924.1925 Email: [email protected]

    Toxoplasma gondii is an incredibly successful parasite owing in part to its ability to persist within cells for the life of the host. Remarkably, at least 350 host species of T. gondii have been described to date, and it is estimated that 30% of the global human population is chronically infected. The importance of T. gondii in human health was made clear with the first reports of congenital toxoplasmosis in the 1940s. However, the AIDS crisis in the 1980s revealed the prevalence of chronic infection, as patients presented with reactivated chronic toxoplasmosis, underscoring the importance of an intact immune system for parasite control. In the last 40 years, there has been tremendous progress toward understanding the biology of T. gondii infection using rodent models, human cell experimental systems, and clinical data. However, there are still major holes in our understanding of T. gondii biology, including the genes controlling parasite development, the mechanisms of cell-intrinsic immunity to T. gondii in the brain and muscle, and the long-term effects of infection on host homeostasis. The need to better understand the biology of chronic infection is underscored by the recent rise in ocular disease associated with emerging haplotypes of T. gondii and our lack of effective treatments to sterilize chronic infection. This Review discusses the cell types and molecular mediators, both host and parasite, that facilitate persistent T. gondii infection. We highlight the consequences of chronic infection for tissue-specific pathology and identify open questions in this area of host-Toxoplasma interactions.

    Toxoplasma gondii is a single-cell obligate intracellular protozoan parasite acquired by the eating of contaminated foods. Feline species are T. gondii’s definitive hosts, meaning that cats facilitate sexual recombination of the parasite and shed millions of highly infectious, environmentally stable oocysts ( 1 ). T. gondii is unique in its incredibly broad intermediate host range, which includes humans, livestock (sheep and pigs are particularly important for human transmission), birds, and rodents, among others ( 2 ). These intermediate hosts support the asexual tachyzoite and bradyzoite tissue cyst forms of the parasite. Mollusks, which concentrate oocysts by filtering contaminated water, are an additional vector for transmission to humans ( 3 ). After consumption of bradyzoite tissue cysts or oocysts, T. gondii invades the small intestine of its host ( 4 , 5 ). Recent work from Laura Knoll’s laboratory suggests that the parasite may sense linoleic acid in the feline gut as a critical signal for sexual stage differentiation in these species ( 6 ). Passing through the cat confers a tremendous benefit to the parasite in terms of genetic diversity and range expansion, and facilitating transmission to cats appears to be a major pressure driving parasite evolution. Given the importance of the predator-prey cycle between rodents and cats, rodents may be a particularly important host for T. gondii. As will be discussed, this conclusion is supported by the observations that T. gondii expresses a sophisticated cadre of effectors that intersect mouse immune signaling ( 7 – 9 ) and that infected rodents lose their natural aversion to feline urine ( 10 , 11 ) and can become severely wasted ( 12 – 14 ), all of which may facilitate transmission via predation of a rodent host.

    Rates of human T. gondii infection range from 10% in the United States to over 50% in France, Colombia, and Brazil ( 15 – 17 ). Acute infection can cause flu-like symptoms however, immune-competent individuals clear the majority of parasites during acute infection. Surviving parasites persist as slow-growing bradyzoite tissue cysts, most abundant in tissues with limited immune surveillance, including brain, eye, cardiac, and skeletal muscle ( 18 ). Contracting T. gondii during pregnancy can be lethal to the fetus, which also has a minimal immune system ( 19 ). Tissues that were not classically considered “immune privileged” also harbor parasites, based on the observation that transplant recipients of kidney, liver, heart, or lung have contracted toxoplasmosis from an infected donor ( 20 – 24 ). However, chronic infection in these tissues is almost unstudied, as parasite frequency is incredibly low. The immune response to T. gondii is sustained throughout chronic infection, and this is evident in elevated T. gondii–specific IgG and IFN-γ in the sera, both of which are essential for parasite restriction ( 25 ). If the immune system is suppressed during chemotherapy, organ transplant, or AIDS, for example, T. gondii can revert to tachyzoite replication ( 26 , 27 ). This process, known as recrudescence, can be lethal if parasitemia is not controlled with drugs. The most frequently prescribed regimens are pyrimethamine combined with sulfadiazine or clindamycin trimethoprim in combination with sulfamethoxazole can be used as an alternative ( 28 ). However, these antiparasitic treatments are poorly tolerated, and hypersensitivity to sulfa drugs is particularly common. Currently, no treatments have been developed that clear tissue cysts, maybe because of the slow growth of bradyzoites, their sequestration within neurons, and/or the difficulty of developing drugs that cross the blood-brain barrier. This is an area of outstanding need, as new haplotypes of T. gondii are emerging that associate with severe ocular disease in immune-competent patients ( 29 , 30 ).

    In North America and Europe, environmental isolates of T. gondii predominantly belong to three major strains or types: type I, type II, and type III. These types are notable in that virulence, measured by lethal dose (LD), differs by several logs in inbred strains of mice (e.g., C57BL/6, CBA/J, BALB/c). Type I is the most virulent (LD100 of 1–10 tachyzoites), compared with type II (LD50 of 100–1000) and type III (LD50 of

    100,000 to 1 million), which are substantially less aggressive in vivo ( 31 , 32 ). Human infection is dominated by type II in North America and Europe however, a fourth type, haplogroup 12, was recently isolated from North American patients and wild animals ( 33 ). In Asia and Africa, region-specific clonal lineages have also been isolated ( 34 ). Strains that do not fit the pattern of clonal lineage expansion have been identified in South America belonging to haplogroups 4 through 15 ( 35 – 37 ). Whole genome sequencing indicates that genomic admixture and recombination between a limited number of ancestral strains account for T. gondii’s genetic diversity. The relationship between strains was determined by comparison of the inheritance pattern of large gene haploblocks. These haploblocks encode virulence-associated, secreted parasite effector proteins, suggesting that the unique assortment of effector alleles may also control pathogenesis and/or transmission rates ( 38 ). Importantly, there is evidence of coevolution between virulent haplotypes and mouse IFN-inducible immunity-related GTPase (IRG) genes, which are critical mediators of cell-intrinsic parasite killing in mouse models ( 39 , 40 ).

    Activating a robust immune response is critical to both host and parasite survival. Bradyzoite cysts are resistant to peptic proteases, but tachyzoites are not: if the host dies before chronic infection is established, parasite transmission does not occur. In keeping with this paradigm, the parasite has evolved effectors that selectively activate host immune cell signaling in addition to strategies to avoid sterilizing immunity. In acute and chronic infection, T. gondii grows and persists within a parasitophorous vacuole membrane (PVM). The PVM is generated from the host plasma membrane as the parasite ratchets its way into the cell using injected parasite effector proteins. This process avoids the lytic environment of the endo/lysosomal compartments and accounts for the parasite’s remarkable capacity to infect almost any nucleated cell type in vitro ( 41 ). In vivo, however, the cell types harboring the parasite are more limited. After a parasite cyst is ingested, T. gondii invades the distal jejunum of the small intestine in mice ( 42 , 43 ). The precise host cell types mediating invasion (e.g., M cells, epithelial cells) are not clear however, T. gondii sporozoites and tachyzoites have been observed in intestinal epithelial cells ( 44 ). Tachyzoites are also observed within infiltrating immune cells ( 43 – 45 ). The CCL2/CCR2 chemokine axis is a conserved mechanism of monocyte recruitment in mice and humans ( 46 ). The parasite effectors Tg14-3-3 and TgWIP have been shown to promote hypermotility in infected human and murine dendritic cells ( 47 , 48 ). Dendritic cell hypermotility has been observed in vivo, suggesting that it may be a stealth mechanism that facilitates parasite dissemination while avoiding detection by circulating immune effectors ( 49 , 50 ).

    The long evolutionary relationship between T. gondii and mammalian hosts is evident in analysis of the pathways infected cells use to detect and destroy the parasite ( 39 ). Three main arms of innate immune sensing have been described in T. gondii infection: the Toll-like receptors (TLRs), the IFN-inducible GTPases, and the inflammasomes. TLR and IL-1 receptor (IL-1R) signaling through MyD88 is a central mediator of IL-12 secretion and the protective Th1 response to T. gondii ( 51 ). In mice that are intraperitoneally infected with T. gondii, the parasite protein profilin directly binds and activates TLR11, contributing to IL-12 production and parasite restriction ( 52 ). However, profilin is an actin-modifying protein sequestered within parasites and TLR11 signals from endosomal compartments, suggesting that this pathway may be mostly activated by phagocytosed, dead, or dysfunctional parasites (Figure 1). Consistent with this model, after oral infection, TLR11-deficient mice had minimal defects in their Th1 response compared with mice deficient in MyD88 or TLR2, TLR4, and TLR9 however, treating TLR11-deficient mice with antibiotics phenocopied MyD88-deficient mice ( 53 , 54 ). These data indicate that gut commensal microbiota can prime a protective immune response to T. gondii independent of parasite recognition by TLR11. It is notable that human TLR11 is a pseudogene, indicating alternative innate sensing mechanisms to detect and destroy T. gondii in human cells.

    Innate immune signaling and the influence of parasite effectors. T. gondii grows within a parasitophorous vacuole membrane (PVM) that protects the parasite from cytosolic immune sensors and avoids fusion with the endolysosomal compartments containing Toll-like receptors (TLRs). In the mouse, TLR11 recognizes Tg profilin, an actin-modifying protein that is exposed once dead or damaged parasites are phagocytosed. TLR11 is a pseudogene in humans. TgGRA15 can promote host NF-κB phosphorylation and nuclear translocation. In mice, NF-κB stimulation is necessary for transcriptional regulation of the inflammasome components NLRP1, NLRP3, and IL-1 however, human monocytes can engage an NLRP3 inflammasome independent of NF-κB prestimulation. A detailed mechanism of inflammasome activation, parasite killing, and host cell death remains elusive, particularly in regard to signal integration with IFN-γ. IFN-γ signaling induces STAT1 translocation to the nucleus and upregulation of IFN-responsive genes, including immunity-related GTPases (IRGs, mouse) and guanylate-binding proteins (GBPs, human and mouse), which functions to attack parasite vacuole, leading to parasite killing and host cell death. In human cells, GBP1 is necessary for this process, which leads to AIM2 activation of an alternative apoptosis pathway. The type I parasite rhoptry proteins, TgROP5, 17, and 18, can dismantle the function of the mouse IRGs IRGa6 and IRGb6 at the PVM, inactivating GBP attack and parasite killing. The parasite dense granule effector TgIST is a nuclear repressor of STAT1 transcription. TgROP16 is a kinase that phosphorylates and activates host STAT3 and STAT6. TgEGGR affects host gene expression through E2F3- and E2F4-mediated epigenetic modifications. In infected monocytes and dendritic cells (DCs), TgWIP and 14-3-3 proteins promote cell mobility, a putative mechanism of intracellular parasite dissemination in vivo.

    The inflammasome links detection of microbial components or cell damage associated with infection to the release of IL-1 family cytokines and, often, inflammatory cell death. While the inflammasome response to protozoa is understudied in comparison with bacterial and viral pathogens, what is known about T. gondii recognition suggests major differences ( 55 , 56 ). The inflammasome sensors NLRP1 (in mice) and NLRP3 (in mice and humans) have been shown to process and release IL-1β in response to T. gondii infection (Figure 1 and refs. 57 – 59 ). Mice deficient in NLRP3, caspase-1, and/or caspase-11 have a higher parasite burden in vivo ( 57 , 58 , 60 ). However, unlike better-studied inflammasome triggers (e.g., the NLRP1 protease anthrax lethal toxin or bacterial pathogens that activate NLRP3), pyroptotic host cell death is not observed in the mouse or human cells ( 57 – 59 ). Unlike murine macrophages, the NLRP3 inflammasome in human monocytes is activated independent of the TLR pathway via Syk and CARD9 signaling and IL-1β release is independent of the pore-forming gasdermin D ( 59 , 61 – 63 ). Open questions in the molecular mechanism of the inflammasome response to T. gondii include what parasite signals activate the inflammasome, why proptosis is not engaged, and whether this is the result of active parasite manipulation.

    Recent data also suggest crosstalk between the inflammasome and the IFN-inducible GTPases, a pathway that surveys the cell for foreign or damaged membranes and targets them for clearance downstream of IFN-γ. In human cells the dynamin-superfamily guanylate-binding protein 1 (GBP1) localizes to the PVM this triggers release of parasite DNA into the host cell cytosol, where it is detected by the inflammasome sensor AIM2 (Figure 1 and refs. 63 , 64 ). For reasons that are still unclear, a pyroptotic inflammasome response is not engaged instead, an alternative apoptotic pathway of host cell death is activated ( 63 , 64 ). Unlike the human system, mice rely on an expanded family of p47 IRGs to detect the parasite vacuole downstream of IFN-γ. Mouse IRGM1 and IRGM3 regulate the interaction between IRGa6, IRGb6, and phospholipids at the PVM ( 65 ). A broader range of mouse GBPs have been implicated in T. gondii clearance however, the mechanism of parasite killing and host cell death in mouse cells is not known ( 66 – 68 ). The importance of the IRG system in parasite clearance is underscored by the observation that type I parasites express a triad of secreted effectors, rhoptry protein 5 (ROP5), ROP17, and ROP18, which bind to and inactivate the GTPase function of IRGa6 and IRGb6 ( 69 , 70 ). This inactivation is a major mechanism of type-specific virulence, as type II and type III parasites express alleles of Rop5 or Rop18, respectively, that cannot effectively subvert IRG attack. Although there has been tremendous progress toward identifying the classes of cell-autonomous immune signaling in response to T. gondii, the field lacks an integrated model of cell-autonomous sensing across these pathways for both mouse and human systems, particularly in cell types other than fibroblast, monocyte, and macrophage.

    T.gondii recognition by innate immune sensors triggers a Th1-polarized, CD8 + T cell–dependent immune response that is necessary for host survival. There are many excellent reviews on the immunobiology of infection ( 7 , 8 , 71 , 72 ), so we will touch briefly on aspects of the acute immune response that are necessary for the progression to chronic infection. Mice deficient in IL-12, TNF-α, and IFN-γ or their signaling pathways die of parasite overgrowth in acute infection ( 73 – 76 ). IFN-γ and IL-12 deficiency is rare in humans and has not been correlated with increased susceptibility to toxoplasmosis however, monocyte-derived macrophages from IFNGR1-deficient patients fail to restrict T. gondii after IFN-γ stimulation compared with healthy-donor macrophages ( 77 , 78 ). Mice deficient in the IL-6 pathway fail to mount a protective B cell response and die in early chronic infection ( 79 ). IL-10 and regulatory T cells play an equally important role in host survival by limiting the magnitude of the inflammatory response and bystander damage ( 80 – 85 ).

    A growing number of T. gondii effectors have been identified that are secreted into the host cell to control immune signaling. These effectors are released from secretory organelles known as the rhoptries (ROP) and the dense granules (GRA), and many of the effectors are polymorphic across strains and play a role in virulence (Figure 1). GRA15 activates NF-κB, and GRA24 activates the p38 MAPK pathway to promote expression of IL-12 and IL-18, the upstream regulators of IFN-γ and T cell activation ( 86 , 87 ). ROP16 is a serine-threonine kinase that directly phosphorylates STAT3 and STAT6 and dampens IL-12 production, which may be consistent with the concept that fine-tuning immune response is necessary for host survival and parasite transmission ( 88 , 89 ). However, the effector TgIST was recently identified as an inhibitor of IFN receptor signaling. TgIST binds STAT1 and forms an inhibitory complex with the nucleosome remodeling deacetylase (Mi-2/NuRD) complex. This suppresses transcription of IRF1-dependent cytokines, MHC class II expression and antigen presentation, and inducible nitric oxide synthase (iNOS) expression, which kills parasites by producing reactive nitrogen species ( 90 , 91 ). Similarly, TgTEEGR interacts with E2F3 and E2F4 transcription factors, and forms a nuclear complex with a catalytic subunit of polycomb repressor complex to block NF-κB–mediated expression of proinflammatory cytokines like IL-1β and IL-6 ( 92 ). These effectors are among 200–300 predicted secreted effector proteins in the parasite genome, the majority of which have not been characterized, particularly in the context of chronic infection.

    In chronic infection, the central nervous system contains the highest frequency of parasites per gram of tissue. The potential implications of neural infection for host behavior and homeostasis have led to great interest in understanding the biology of T. gondii infection in the brain. Our understanding of chronic central nervous system infection is almost exclusively based on murine models of infection. There are many open questions, beginning with how the parasite traverses the blood-brain barrier (BBB). Using intravital microscopy, T. gondii has been imaged replicating within brain endothelial cells and then directly entering the brain ( 93 ). Mice infected intravenously with the T. gondii RH strain had a higher brain parasite load than mice infected with the CPS strain, which cannot replicate in vivo, suggesting that T. gondii growth within vascular endothelial cells may be an important stopover before direct entry into the brain (Figure 2A and ref. 93 ). Perfusion of Evans blue dye shows increased BBB permeability during chronic T. gondii infection, accompanied by reduced blood flow and capillary rarefication which may permit immune cell entry into the brain ( 94 ). Using intravital microscopy, CCR2 + monocytes are found to accumulate, exhibiting rolling and cradling behavior at the BBB ( 95 ). This observation, coupled with the high frequency of infection of dendritic cells and their hypermotility phenotype, has led to the Trojan horse hypothesis: that parasites traverse the BBB within immune cells (Figure 2A and refs. 49 , 96 ). Although direct evidence for this model is lacking, antibody depletion of CD11b + leukocytes correlated with reduced brain parasite load and adoptive transfer of T. gondii–infected CD11c + or CD11b + cells into naive mice led to neural infection ( 97 ).

    T. gondii entry and control of persistent infection in the brain. (A) In acute infection, T. gondii is frequently observed in immune cells, including monocytes and dendritic cells, with hypermigratory behavior. During infection, blood-brain barrier (BBB) permeability increases and monocytes accumulate in the endothelial lumen, interacting with endothelial cells. These observations have led to the hypothesis that migratory immune cells deliver T. gondii to the BBB and, perhaps, smuggle them into the brain. Replicating parasites are also observed in brain endothelial cells, whose subsequent lysis may be a mechanism of T. gondii entry into the brain. (B) During acute infection parasites are observed infecting neurons, astrocytes, microglia, and infiltrating immune cells. Astrocytes and microglia as well as peripheral monocytes can clear parasites with cell-autonomous immune pathways. (C) As chronic infection progresses, infected astrocytes and microglia or the parasites within them are cleared and cysts are primarily observed within neurons. Most parasite cysts are not associated with immune infiltrate however, individual parasites or parasite debris can be observed colocalizing with immune infiltrate.

    Analysis of mouse brain sections and an extremely limited number of healthy human brain samples indicates that most intracellular cysts are not associated with immune infiltration ( 98 ). However, within the same brain section, inflammatory foci can be observed containing parasites or parasite debris, activated microglia, macrophages, and T cells ( 99 ). Depleting IFN-γ or CD4 + and CD8 + T cells leads to parasite recrudescence ( 100 ). Taken together these data suggest that intracellular cysts are relatively immunologically silent however, cysts that lyse (spontaneously or through recrudescence) are recognized and quickly contained by infiltrating immune cells (Figure 2C).

    Experiments using parasites engineered to secrete Cre recombinase in Cre reporter mice have demonstrated that neurons are the major cell type interacting with the parasite in the brain, although T cells, monocytes or macrophages, microglia, and astrocytes are reporter-positive early in brain infection (Figure 2B and refs. 101 – 103 ). These data also suggest that rather than having a tropism for neurons, T. gondii is cleared from non-neuronal cell types in the brain. Consistent with this model, disabling the IFN-γ signaling in mouse astrocytes by knocking out the transcription factor STAT1 led to greater incidence of cysts within astrocytes ( 104 ) and IFN-γ depletion increased the percentage of infected astrocytes ( 102 ). Subsequently, the ability of mouse astrocytes to restrict T. gondii growth in response to IFN-γ was shown to depend on IRGM3 (IGTP), not iNOS IRGM3 and IRGa6 disrupted the PVM and, in one study, led to parasite egress ( 105 – 107 ). In human astrocytes, IL-1β in combination with IFN-γ induced iNOS-dependent killing of T. gondii ( 108 ), whereas TNF- and IFN-γ limited T. gondii growth by tryptophan starvation via upregulated indoleamine 2,3-dioxygenase (IDO) ( 109 ). The parasite effector TgGRA15 has been shown to limit IDO-mediated parasite restriction in cultured glioblastoma and neuroblastoma cell lines ( 110 ). IFN-γ in combination with TNF-α or LPS has also been shown to activate parasite killing functions of human and murine microglia through iNOS-dependent and -independent mechanisms ( 111 – 113 ). Microglia can also produce IFN-γ and TNF-α, which are critical for central nervous system restriction of the infection ( 114 ) IFN-γ, in particular, has been shown to induce adhesion molecule expression on vascular endothelial cells and promote the expression of CXCL9, CXCL10, and CCL5, which recruit peripheral immune cells to the brain ( 115 , 116 ). Most of these data are from in vitro experiments, and better tools to study microglia and astrocyte function in vivo will be important to clarify which pathways control central nervous system infection.

    While brain-resident immune cells contribute to T. gondii restriction, the role of cell-autonomous immunity in neurons is less clear. A recent study using OVA-expressing parasites and conditional MHC class I–deficient mice demonstrated that neurons can present T. gondii–derived antigens to initiate a CD8 + T cell response ( 117 ) however, whether endogenous parasite epitopes are efficiently presented on neurons is yet to be examined. Brain-infiltrating CD8 + T cells have been shown to control cyst burden indirectly through IFN-γ secretion, and, to a lesser extent, via perforin-dependent killing of infected cells ( 118 – 120 ). It is notable that perforin has been shown to trigger parasite egress in vitro, suggesting that perforin may limit parasite growth but other cells are responsible for parasite killing, potentially through cell-autonomous immunity ( 121 ). A minimal reliance on perforin-mediated T. gondii clearance also fits a model wherein cellular cytotoxicity should be limited in the brain to promote survival of neurons, which have an extremely limited regenerative capacity.

    Currently there are no therapeutic tools that effectively target bradyzoite cysts and sterilize chronic infection. Our understanding of bradyzoite biology is weaker than our understanding of tachyzoite biology. This is linked to long-standing technical challenges associated with genetic manipulation of bradyzoite-specific genes that are required to perform “necessary and sufficient” experiments. However, the recent bloom in CRISPR/Cas9 tools has led to gains in this arena ( 122 , 123 ).

    The transition between tachyzoite and bradyzoite has commonly been referred to as “switching” however, recent studies suggest that stage conversion is a continuum under epigenetic and transcriptional regulation rather than a finite life stage. Bradyzoite polarization can be induced by cell stressors including alkaline media, heat shock, and oxidative stress (refs. 124 – 126 and Figure 3). IFN-γ treatment has been shown to induce bradyzoite gene expression in infected macrophages but not fibroblasts, suggesting that cell type–specific differentiation signals may also exist ( 127 ). Compared with fibroblasts, infected neuronal or skeletal muscle cells support a stronger expression of bradyzoite markers and a higher frequency of cyst development ( 128 ). It is worth noting that neurons and muscle cell types are historically difficult to culture, suggesting that cell stress signals may be relevant to bradyzoite development in these models. However, terminally differentiated myotubes are reported to support a higher frequency of bradyzoites compared with dividing myoblast progenitor cells, suggesting that cell cycle may provide developmental cues for the parasite development as well ( 129 ).

    Environmental and host cell–specific pressures driving the T. gondiitachyzoite to bradyzoite transition. Left: T. gondii tachyzoites can invade almost any nucleated host cell type and grow within the PVM formed from host plasma membrane. In vitro, a range of tissue culture stress conditions can upregulate bradyzoite-specific genes. As parasites polarize to a bradyzoite transcriptional profile, they synthesize a heavily glycosylated cyst wall beneath the PVM. The frequency and rate of bradyzoite differentiation are also influenced by the host cell type, cell cycle status, the host cell lifespan, and inflammatory signals in vitro. In vivo, cysts are most frequently observed in neurons, cardiac muscle, skeletal muscle, and retinal pigment epithelial cells. If the host is immune-suppressed, parasites shift toward a replicative tachyzoite form in a process referred to as recrudescence, which is associated with tissue damage, particularly in the eye.

    Although the precise signals are unclear, histone methylation and acetylation are important epigenetic regulators of bradyzoite differentiation. Treating tachyzoites with arginine methyltransferase inhibitor, AMI-I, induces a reduction of histone H3R17 methylation and bradyzoite differentiation in vitro ( 130 ). The T. gondii histone acetyltransferase TgGCN5a is enriched at promoter regions of bradyzoite-specific genes, and TgGCN5a-deficient parasites fail to upregulate the bradyzoite markers Bag1 and Ldh2 under stress ( 131 ). Treating infected cells with the histone deacetylase inhibitor FR235222 induces bradyzoite differentiation through inhibiting TgHDAC3 ( 132 ). Phosphorylation of the T. gondii eukaryotic initiation factor 2 α subunit (TgeIF2α) is enhanced under stress conditions and is necessary for bradyzoite differentiation ( 133 ). Guanabenz, an eIF2α dephosphorylation inhibitor, has been shown to impair tachyzoite proliferation and promote bradyzoite differentiation in vitro ( 134 ).

    The ApiAP2 family of transcription factors are emerging as central regulators of bradyzoite differentiation. This family consists of 67 genes, many of which are associated with bradyzoite stage–specific expression. Specifically, AP2XI-4– and AP2IV-3–knockout parasites have reduced expression of bradyzoite-specific genes after in vitro switch and AP2XI-4–null T. gondii forms fewer cysts in mice ( 135 , 136 ). AP2IV-4 knockouts express some bradyzoite-specific genes under tachyzoite culture, but had fewer brain cysts in mice ( 137 ). Using a CRISPR/Cas9 guide RNA library targeting mostly AP2 domain–containing proteins and predicted nucleic acid–binding proteins, bradyzoite formation deficient 1 (BFD1), a Myb-like transcription factor, was recently identified as a key regulator of bradyzoite differentiation in vitro and in vivo in mice. Interestingly, Bfd1 mRNA is expressed in tachyzoites however, protein expression is only induced by stress conditions ( 138 ). It remains to be seen whether immunosuppression induces any parasite recrudescence in mice infected with BFD1-deficient parasites and how BFD1- and AP2-family proteins coordinate bradyzoite differentiation.

    T.gondii bradyzoite cysts are often defined by formation of a cyst wall consisting of heavily glycosylated proteins underneath the PVM ( 139 ). The cyst wall is essential for transmission, protecting the parasite from gastric proteases and the low pH of the stomach. Parasites deficient in the cyst wall–localized bradyzoite pseudokinase 1 (BPK1) were more sensitive to pepsin digestion and less orally infectious than WT parasites ( 140 ). Parasites that were rendered genetically deficient in cyst glycoproteins, including loss of the nucleotide-sugar transporter TgNST1 or the heavily glycosylated cyst wall protein TgCST1, have defects in cyst number, cyst stability, and infectivity during oral infection ( 141 – 143 ). The cyst wall may also protect bradyzoites from enzymatic attack during chronic infection. The Wilson laboratory demonstrated that chitinase-expressing, alternatively activated (M2) macrophages were able to recognize and degrade chitin-like polysaccharides in the cyst wall ( 144 ). Consistent with this observation, a GWAS identified single-nucleotide polymorphisms in the intergenic region of the human CHIA locus, which expresses chitinase, that were significantly associated with T. gondii infection ( 145 ).

    T.gondii infection is the most frequent cause of posterior uveitis, also referred to as chorioretinitis or inflammation of the retina and choroid (pigmented vascular coat of the eye) ( 30 ). This is one area of T. gondii infection that has been more extensively studied in patients than in animal models, which have been limited until recently. Type II strains, most frequently associated with infection in Europe and North America, are associated with chorioretinitis ( 146 , 147 ). Historically, ocular toxoplasmosis was associated with congenital infection however, rates of disease associated with postnatal infection are rising and associated with new T. gondii strains ( 148 ). Over 70% of patients presenting with acute ocular toxoplasmosis already have ocular scars, suggesting that disease progression is driven by the inflammatory response to recrudescent T. gondii leading to the accumulation of tissue damage over time ( 149 ). Immune-competent individuals are able to control ocular infection, but early antiparasitic treatment is critical to limit the extent of retinal damage ( 150 ). Human retinal vascular endothelial cells are more sensitive to infection than other endothelial cell types, suggesting a potential mechanism of entry into the eye ( 151 ). T. gondii cysts have been observed in retinal pigmented epithelial cells ( 152 ). In a mouse model of ocular toxoplasmosis, retinal pigment epithelial cells and infiltrating immune cells expressed the T cell inhibitory ligand PD-L1 ( 153 ). This may be an important mechanism to limit tissue pathology, although the parasites may exploit this axis for persistence. IFN-γ and IL-6, which are both critical in restricting systematic parasitemia ( 79 , 100 ), were elevated in the vitreous humor of mice with ocular lesions. However, intraocular injection of an IFN-γ–blocking antibody impaired parasite control and worsened tissue damage, while, perhaps counterintuitively, injection of an IL-6–blocking antibody improved parasite control and minimized ocular damage ( 154 – 156 ). Patients infected with virulent South American haplotypes of T. gondii, which have been associated with aggressive chorioretinitis, had less IFN-γ and IL-17 but higher IL-13 and IL-6 levels in the eye compared with European patients infected with virulent type I ( 157 ). However, it is currently not clear whether these differences in immune regulation control ocular disease severity.

    In mice and rats, infection with T. gondii leads to a well-established loss of innate aversion behavior to felines, which has been proposed to benefit the parasite by facilitating transmission via predation ( 11 , 158 , 159 ). Whether these behavioral phenotypes are driven by specific changes in neural activity or a more general effect of inflammation is an open question. The observation that T. gondii expresses two aromatic amino acid hydrolases that produce l -DOPA, AAH1, and AAH2 led to the hypothesis that the parasite could modulate dopaminergic neuron function. However, deletion of AAH2 failed to alter brain dopamine levels, neuroinflammation, or behavioral alterations in T. gondii–infected mice ( 160 , 161 ), although these genes are necessary for oocyst development in the cat ( 162 ). Notably, mice infected with an avirulent mutant of type I T. gondii or the related organism Neospora caninum, which are cleared before establishing chronic infection, exhibit loss of aversion behavior even though chronic infection is not sustained ( 163 ). These data suggest that acute inflammation may be sufficient to trigger sustained behavioral changes, although the molecular bases for behavioral changes in T. gondii infection are unclear. Recently, the olfactory GPCR trace amine-associated receptor 4 (TAAR4) was shown to recognize 2-phenylethylamine, a metabolite enriched in urine of predators, including feline species. There is no homolog of TAAR4 in humans, but mice deficient in TAAR4 do not engage in avoidance behavior to bobcat and mountain lion urine ( 164 ). That the olfactory neurons expressing TAAR4 are altered or damaged during T. gondii infection is a compelling hypothesis that remains to be tested.

    Sustained interaction with the immune system is a hallmark of T. gondii infection: throughout chronic infection humans and mice have high titers of T. gondii–specific IgG and sera cytokines. There is growing evidence that T. gondii infection is associated with cachexia in mice, an immune-metabolic disease of sustained muscle wasting. Cachexia positively correlates with parasite load and inflammation severity however, hypermetabolic weight loss cannot be rescued by diet supplementation ( 13 , 165 – 167 ). In oral infection, intestinal barrier inflammation resolves during chronic infection, but commensal dysbiosis does not ( 14 , 168 ) however, dysbiosis is not sufficient for cachexia, as uninfected cage mates experienced a similar microbial shift but did not develop cachexia ( 14 ). Chronically infected mice have sustained changes in splenic and lymph node architecture and are more susceptible to acute viral challenge ( 169 ). Moreover, cachectic mice were more susceptible to LPS challenge than mice that recovered weight ( 170 ). Recently, mice deficient in the IL-1R axis were shown to recover from acute cachectic weight loss, although chronic parasite burden was similar to that in wild-type mice ( 171 ). A study from the Wohlfert laboratory showed that infection-induced myositis could be reversed by depletion of regulatory T cells, which were enriched in skeletal muscle ( 172 ). Parasite biology that promotes behavior modification and cachexia in rodent hosts may provide a selective advantage to T. gondii by increasing the likelihood of predation and transmission to feline hosts. It is important to note that there is currently no evidence of cachexia in immune-competent humans with chronic T. gondii infection. However, cachexia is a predictor of mortality in almost every chronic human disease with limited experimental tools to probe sustained disease. The interaction between T. gondii and mice is proving an informative model to understand the pathophysiology of cachexia, which can be applied to understand other disease settings.

    T.gondii’s ability to establish a persistent chronic infection is essential for parasite transmission. However, there is much to learn about this stage of infection in animal and human hosts. Deep sequencing has unraveled a far greater diversity in T. gondii gene assortment than originally thought, which has opened the door to understanding how parasite genetics influences pathology associated with chronic infection. CRISPR/Cas9 tools are expanding our ability to manipulate the T. gondii genome to understand how gene expression in bradyzoites controls differentiation, cyst stability, and oral infectivity of the parasite. Bradyzoite biology is intimately linked to the immune response during chronic infection. The coming decades will likely reveal mechanisms of cell-autonomous immunity to chronic T. gondii infection in the brain and other chronically infected tissues, as well as reveal the costs of the chronic inflammatory response for host homeostasis. A better understanding of this biology is needed to develop therapeutic strategies that effectively target bradyzoite cysts. Given the long evolutionary relationship between mammalian hosts and T. gondii, such studies are likely to discover important information about the regulation of immune functions during chronic inflammation more broadly.

    Conflict of interest: The authors have declared that no conflict of interest exists.


    Cytotoxic T cells mediate one arm of the cellular immune response

    There are two main types of T cells: helper T lymphocytes (TH) and the cytotoxic T lymphocytes (TC). The TH lymphocytes function indirectly to tell other immune cells about potential pathogens, while cytotoxic T cells (TC) are the key component of the cell-mediated part of the adaptive immune system which attacks and destroys infected cells. TC cells are particularly important in protecting against viral infections because viruses replicate within cells where they are shielded from extracellular contact with circulating antibodies. Once activated, the TC creates a large clone of cells with one specific set of cell-surface receptors, similar to the proliferation of activated B cells. As with B cells, the clone includes active TC cells and inactive memory TC cells. The resulting active TC cells then identify infected host cells.

    TC cells attempt to identify and destroy infected cells by triggering apoptosis (programmed cell death) before the pathogen can replicate and escape, thereby halting the progression of intracellular infections. To recognize which cells to pursue, TC recognize antigens presented on MHC I complexes, which are present on all nucleated cells. MHC I complexes display a current readout of intracellular proteins inside a cell and will present pathogen antigens if the pathogen is present in the cell. TC cells also support NK lymphocytes to destroy early cancers.


    Mechanisms of T Cell-mediated Immune Responses

    Mature T cells become activated by recognizing processed foreign antigen in association with a self-MHC molecule and begin dividing rapidly by mitosis. This proliferation of T cells is called clonal expansion and is necessary to make the immune response strong enough to effectively control a pathogen. How does the body select only those T cells that are needed against a specific pathogen? Again, the specificity of a T cell is based on the amino acid sequence and the three-dimensional shape of the antigen-binding site formed by the variable regions of the two chains of the T cell receptor. Clonal selection is the process of antigen binding only to those T cells that have receptors specific to that antigen. Each T cell that is activated has a specific receptor &ldquohard-wired&rdquo into its DNA, and all of its progeny will have identical DNA and T cell receptors, forming clones of the original T cell.

    Figure 5. Stem cells differentiate into T cells with specific receptors, called clones. The clones with receptors specific for antigens on the pathogen are selected for and expanded.


    Tc and Th1 interaction and viral immune response - Biology

    Until now, we have concerned ourselves with the molecular details of how viruses replicate in our cells to produce hundreds of progeny per cell. Now we will broaden our view to take account of the fact that these repeated rounds of virus replication are occurring in a body made up of about a hundred trillion cells, including an elaborate immune system that tries to fight off the infection.

    Virus infection in vertebrates results in two general types of immune response. The first is a rapid-onset "innate" response against the virus, which involves the synthesis of proteins called interferons and the stimulation of "natural killer" lymphocytes. In some cases, the innate response may be enough to prevent a large scale infection. However, if the infection proceeds beyond the first few rounds of viral replication, the "adaptive immune response", kicks into high gear. The adaptive immune response itself has two components, the humoral response (the synthesis of virus-specific antibodies by B lymphocytes) and the cell-mediated response (the synthesis of specific cytotoxic T lymphocytes that kill infected cells). Both of these components of the adaptive immune response result also in the production of long-lived "memory cells" that allow for a much more rapid response (i.e., immunity) to a subsequent infection with the same virus.

    1. How can we classify viral infections by 'outcome of the immune response'?

    We can roughly establish four categories of virus infection based on what happens to the host (not including sub-clinical, inapparent infections).


    Category 1 includes all cases for which the person gets sick and then either dies or recovers completely, with the elimination of all virus from the body. For categories 2-4, the initial infection may be acute or inapparent, but the body's immune response does not clear the virus completely, and things proceed to one of these situations, where there may be very little ("latent"), some ("chronic/persistent"), or abundant ("progressive") virus replication going on during the rest of the person's life. There is some overlap and ambiguity in these terms, but they are useful categorizations nonetheless.

    2. After recovery from an acute viral infection, a person is usually "immune" to getting the same viral disease for years (perhaps a lifetime). What, then, is the explanation for the fact that we remain susceptible to getting a rhinovirus-caused "cold" throughout our lifetime?

    There are over 100 different "serotypes" of rhinoviruses (and a smaller number of coronaviruses and adenoviruses) that all cause upper respiratory infections. When we get infected with one of these, our immune system responds and clears the virus from the body within a couple of weeks. The immune response also leaves us with a supply of specific "memory cells" that prevent that same virus from causing a clinically significant infection in us for years. But. there are all of those other related viruses that can still infect us. For example, although all of the rhinoviruses are similar, their surface proteins are different enough such that the memory cells made during a previous immune response against, say, rhinovirus #36 will most likely not provide immunity to rhinovirus #24. So, at any time we are somewhat susceptible to whatever rhinoviruses we have not been infected by previously. (And, for older people, it's probably possible to get a cold from the same rhinovirus a couple decades apart.)

    An update on the 100 or so rhinovirus serotypes was provided in a 2006 article in Journal of General Virology by Laine et al titled " Alignment of capsid protein VP1 sequences of all human rhinovirus prototype strains: conserved motifs and functional domains."

    3. What is the basic understanding of the human adaptive immune response we need in order to progress with our study of virology?

    The basics of B and T cell clonal selection and the various cellular interactions involved in the humoral and cell-mediated immune responses are the foundation knowledge of immunology needed for proceeding further in a study of virology. It is during the early stages of the clonal selection process that immunoglobulin gene DNA rearrangements occur. (Remember this? We covered it in BIOS 115 ) .


    Infection Immunity

    The development of an infectious disease in an individual involves complex interactions between the microbe and the host. The key events during infection include entry of the microbe, invasion and colonization of host tissues, evasion of host immunity, and tissue injury or functional impairment. Microbes produce disease by directly killing the host cells they infect, or by liberating toxins that can cause tissue damage and functional derangements in neighboring or distant cells and tissues that are not infected.

    The interaction of the immune system with infectious organisms is a dynamic interplay of host mechanisms aimed at eliminating infections and microbial strategies designed to permit survival in the face of powerful defenses. Different types of infectious agents stimulate distinct types of immune responses and have evolved unique mechanisms for evading immunity. In some infections, the immune response is the cause of tissue injury and disease.

    Here we will consider the main features of immunity to four major categories of pathogenic microorganisms: extracellular and intracellular bacteria, fungi, viruses, and protozoan as well as multicellular parasites.

    Immunity to Bacteria

    Extracellular bacteria are capable of replicating outside host cells, for example, in the blood, in connective tissues, and in tissue spaces such as the lumens of the airways and gastrointestinal tract. Many different species of extracellular bacteria are pathogenic, and disease is caused by two principal mechanisms. First, these bacteria induce inflammation, which results in tissue destruction at the site of infection. Second, bacteria produce toxins, which have diverse pathologic effects. The toxins may be endotoxins, which are components of bacterial cell walls, or exotoxins, which are secreted by the bacteria. The endotoxin of gram-negative bacteria, also called lipopolysaccharide (LPS), is a potent activator of macrophages, dendritic cells, and endothelial cells. Many exotoxins are cytotoxic, and others cause disease by various mechanisms. For instance, diphtheria toxin shuts down protein synthesis in infected cells, cholera toxin interferes with ion and water transport, tetanus toxin inhibits neuromuscular transmission, and anthrax toxin disrupts several critical biochemical signaling pathways in infected cells. Other exotoxins interfere with normal cellular functions without killing cells, and yet other exotoxins stimulate the production of cytokines that cause disease.

    The principal mechanisms of innate immunity to extracellular bacteria are complement activation, phagocytosis, and the inflammatory response.

    Complement activation: Peptidoglycans in the cell walls of Gram-positive bacteria and LPS in Gram-negative bacteria activate complement by the alternative pathway. Bacteria that express mannose on their surface may bind mannose-binding lectin, which activates complement by the lectin pathway. One result of complement activation is opsonization and enhanced phagocytosis of the bacteria. In addition, the membrane attack complex generated by complement activation lyses bacteria, Phagocytes and inflammation: Phagocytes (neutrophils and macrophages) use surface receptors, including mannose receptors and scavenger receptors, to recognize extracellular bacteria, and they use Fc receptors and complement receptors to recognize bacteria opsonized with antibodies and complement proteins, respectively. In addition, dendritic cells and phagocytes that are activated by the microbes secrete cytokines, which induce leukocyte infiltration into sites of infection (inflammation). The recruited leukocytes ingest and destroy the bacteria.

    Humoral immunity is a major protective immune response against extracellular bacteria, and it functions to block infection, to eliminate the microbes, and to neutralize their toxins. Antibody responses against extracellular bacteria are directed against cell wall antigens and secreted and cell-associated toxins, which may be polysaccharides or proteins. The polysaccharides are prototypic T-independent antigens, and humoral immunity is the principal mechanism of defense against polysaccharide-rich encapsulated bacteria. The effector mechanisms used by antibodies to combat these infections include neutralization, opsonization and phagocytosis, and activation of complement by the classical pathway. The protein antigens of extracellular bacteria also activate CD4+ helper T cells, which produce cytokines that induce local inflammation, enhance the phagocytic and microbicidal activities of macrophages and neutrophils, and stimulate antibody production. (Figure 1)

    Figure 1. Adaptive immune responses to extracellular microbes. Adaptive immune responses to extracellular microbes such as bacteria and their toxins consist of antibody production and the activation of CD4+ helper T cells.

    A characteristic of facultative intracellular bacteria is their ability to survive and even to replicate within phagocytes. Because these microbes are able to find a niche where they are inaccessible to circulating antibodies, their elimination requires the mechanisms of cell-mediated immunity.

    The innate immune response to intracellular bacteria is mediated mainly by phagocytes and natural killer (NK) cells. Intracellular bacteria activate NK cells by inducing expression of NK cell–activating ligands on infected cells and by stimulating dendritic cell and macrophage production of IL-12 and IL-15, both of which are NK cell activating cytokines. The NK cells produce IFN-γ, which in turn activates macrophages and promotes killing of the phagocytosed bacteria. Thus, the NK cells provide an early defense against these microbes, before the development of adaptive immunity. (Figure 2a)

    The major protective immune response against intracellular bacteria is T cell–mediated recruitment and activation of phagocytes (cell-mediated immunity). Phagocytosed bacteria stimulate CD8+ T cell responses if bacterial antigens are transported from phagosomes into the cytosol or if the bacteria escape from phagosomes and enter the cytoplasm of infected cells. In the cytosol, the microbes are no longer susceptible to the microbicidal mechanisms of phagocytes, and for eradication of the infection, the infected cells have to be killed by CTLs. Thus, the effectors of cell-mediated immunity, namely, CD4+ T cells that activate macrophages and CD8+ CTLs, function cooperatively in defense against intracellular bacteria. (Figure 2b)

    Figure 2 Innate and adaptive immunity to intracellular bacteria.

    Immunity to Virus

    Viruses are obligatory intracellular microorganisms that use components of the nucleic acid and protein synthetic machinery of the host to replicate and spread. Viruses typically infect various cell types by using normal cell surface molecules as receptors to enter the cells. After entering cells, viruses can cause tissue injury and disease by any of several mechanisms. Innate and adaptive immune responses to viruses are aimed at blocking infection and eliminating infected cells. Infection is prevented by type I interferons as part of innate immunity and neutralizing antibodies contributing to adaptive immunity. Once infection is established, infected cells are eliminated by NK cells in the innate response and CTLs in the adaptive response.

    1. Innate Immunity to Virus

    The principal mechanisms of innate immunity against viruses are inhibition of infection by type I interferons and NK cell–mediated killing of infected cells. NK cells kill other cells infected with a variety of viruses and are an important mechanism of immunity against viruses early in the course of infection, before adaptive immune responses have developed (Figure 3a).

    2. Adapted Immunity to Virus

    Adaptive immunity against viral infections is mediated by antibodies, which block virus binding and entry into host cells, and by CTLs, which eliminate the infection by killing infected cells. Antibodies are effective against viruses only during the extracellular stage of the lives of these microbes. Viruses may be extracellular early in the course of infection, before they infect host cells, or when they are released from infected cells by virus budding or if the infected cells die. Antiviral antibodies bind to viral envelope or capsid antigens and function mainly as neutralizing antibodies to prevent virus attachment and entry into host cells. Elimination of viruses that reside within cells is mediated by CTLs, which kill the infected cells. The principal physiologic function of CTLs is surveillance against viral infection. Most virus-specific CTLs are CD8+ T cells that recognize cytosolic, usually endogenously synthesized, viral peptides presented by class I MHC molecules (Figure 3b).

    Figure 3. Innate and adaptive immune responses against viruses.

    Immunity to Parasites

    In infectious disease terminology, parasitic infection refers to infection with animal parasites such as protozoa, helminths, and ectoparasites (e.g., ticks and mites). Most parasites go through complex life cycles, part of which occurs in humans (or other vertebrates) and part of which occurs in intermediate hosts, such as flies, ticks, and snails. Humans are usually infected by bites from infected intermediate hosts or by sharing a particular habitat with an intermediate host. Most parasitic infections are chronic because of weak innate immunity and the ability of parasites to evade or resist elimination by adaptive immune responses. Furthermore, many anti-parasitic drugs are not effective at killing the organisms.

    1. Innate Immunity to Parasites

    Although different protozoan and helminthic parasites have been shown to activate different mechanisms of innate immunity, these organisms are often able to survive and replicate in their hosts because they are well adapted to resist host defenses. The principal innate immune response to protozoa is phagocytosis, but many of these parasites are resistant to phagocytic killing and may even replicate within macrophages. Phagocytes may also attack helminthic parasites and secrete microbicidal substances to kill organisms that are too large to be phagocytosed. However, many helminths have thick teguments that make them resistant to the cytocidal mechanisms of neutrophils and macrophages, and they are too large to be ingested by phagocytes.

    2. Adapted Immunity to Parasites

    Different protozoa and helminths vary greatly in their structural and biochemical properties, life cycles, and pathogenic mechanisms. It is therefore not surprising that different parasites elicit distinct adaptive immune responses. Some pathogenic protozoa have evolved to survive within host cells, so protective immunity against these organisms is mediated by mechanisms similar to those that eliminate intracellular bacteria and viruses. In contrast, metazoa such as helminths to survive in extracellular tissues, and their elimination is often dependent on special types of antibody responses. The principal defense mechanism against protozoa that survive within macrophages is cell-mediated immunity, particularly macrophage activation by TH1 cell–derived cytokines. Defense against many helminthic infections is mediated by the activation of TH2 cells, which result in production of IgE antibodies and activation of eosinophils.

    Immunity to Fungus

    Fungal infections, also called mycoses, are important causes of morbidity and mortality in humans. Some fungal infections are endemic, and these infections are usually caused by fungi that are present in the environment and whose spores enter humans. Other fungal infections are said to be opportunistic because the causative agents cause mild or no disease in healthy individuals but may infect and cause severe disease in immunodeficient persons. Compromised immunity is the most important predisposing factor for clinically significant fungal infections. Different fungi infect humans and may live in extracellular tissues and within phagocytes. Therefore, the immune responses to these microbes are often combinations of the responses to extracellular and intracellular bacteria. However, less is known about antifungal immunity than about immunity against bacteria and viruses.

    Detection and Diagnosis of Infectious Diseases

    Infectious diseases are caused by pathogenic microorganisms, such as bacteria, viruses, parasites or fungi. Some infectious diseases can be passed from person to person. Some are transmitted by bites of insects or animals. And others are acquired by ingesting contaminated food or water or being exposed to organisms in the environment. Infectious diseases resulted in about 9 million deaths every year. The symptoms of infection depend on the type of disease. Some signs of infection affect the whole body generally, such as fatigue, loss of appetite, weight loss, fevers, night sweats, chills, aches and pains. Others are specific to individual body parts, such as skin rashes, coughing, or a runny nose. Source organism diagnosis and detection of infectious disease is the key to its prevention and treatment.

    Diagnosis of infectious disease sometimes involves identifying an infectious agent either directly or indirectly. Microscopy and microbial culture is probably the most direct method and the golden standard to identify the cause organism of an infectious disease, but it usually needs days or weeks before the results available. Furthermore, not all pathogens can be cultured, that make indirectly method very useful in diagnostic and detection of infectious disease.

    Both nucleic acid and proteins as well as small molecular chemicals can be used to detect pathogens. Nucleic acid as genetic material is ubiquity within all kinds of pathogens that make it an ideal marker of pathogen detection and identification. Proteins as the structure and functional material of microbial is also important for diagnostic and detection. Cell wall and pili of bacterial, capsid of the virus, spores of the fungus, surface antigen of parasites and even the metabolic product of these pathogen are all biomarkers for diagnosis and detection of infectious disease (Figure 4).

    Figure 4. Pathogenic factors of infectious diseases.

    Divers biotechnology or immunoassay such as PCR (detect nucleic acid), ELISA (detect pathogenic antigen or antibody) are widely applied in this area. Many of them are commercialized products. Creative diagnostic offers a wide variety of enzyme-linked immunosorbent assay (ELISA) kits, Rapid diagnostic kits as well as the related antigens and antibodies for the diagnostic and detection of infectious disease.

    ELISA Products Related to Infectious Disease
    ELISA Products Related to Toxin Detection (Food Safety)
    ELISA Products Related to Drugs and Chemicals
    RDT Products Related to Infectious Disease
    RDT Production Related to Toxin Detection (Food Safety)


    Lymphocytes

    The 2 main types of lymphocytes are

    B cells (which mature in bone marrow)

    T cells (which mature in the thymus)

    The main types of lymphocytes are morphologically indistinguishable but have different immune functions. They can be distinguished by antigen-specific surface receptors and molecules called clusters of differentiation (CDs), whose presence or absence define some subsets. More than 300 CDs have been identified(for further information on CD antigens, see the Human Cell Differentiation Molecules web site). Each lymphocyte recognizes a specific antigen via surface receptors.

    There are two major classes of lymphocytes involved with specific defenses: B cells and T cells.

    Immature T cells are produced in the bone marrow, but they subsequently migrate to the thymus, where they mature and develop the ability to recognize specific antigens. T cells are responsible for cell-mediated immunity.

    B cells, which mature in the bone marrow, are responsible for antibody-mediated immunity.

    The cell-mediated response begins when a pathogen is engulfed by an antigen-presenting cell, in this case, a macrophage. After the microbe is broken down by lysosomal enzymes, antigenic fragments are displayed with MHC molecules on the surface of the macrophage.

    T cells recognize the combination of the MHC molecule and an antigenic fragment and are activated to multiply rapidly into an army of specialized T cells.

    One member of this army is the cytotoxic T cell. Cytotoxic T cells recognize and destroy foreign cells and tissues or virus-infected cells. Another T cell is the memory cytotoxic T lymphocyte, which remains in reserve in the body. If, sometime in the future, these T cells re-encounter this specific antigen, they will rapidly differentiate into cytotoxic T cells, providing a speedy and effective defense.

    Helper T cells coordinate specific and nonspecific defenses, in large part by releasing chemicals that stimulate T cell and B cell growth and differentiation.

    Suppressor T cells inhibit the immune response so that it ends when the infection has been controlled. Whereas the number of helper T cells increases almost at once, the number of suppressor T cells increases slowly, allowing time for an effective first response.

    B cells

    About 5 to 15% of lymphocytes in the blood are B cells they are also present in the bone marrow, spleen, lymph nodes, and mucosa-associated lymphoid tissues.

    B cells can present antigen to T cells and release cytokines, but their primary function is to develop into plasma cells, which manufacture and secrete antibodies.

    Patients with B-cell immunodeficiencies (eg, X-linked agammaglobulinemia) are especially susceptible to recurrent bacterial infections.

    After random rearrangement of the genes that encode immunoglobulin (Ig), B cells collectively have the potential to recognize an almost limitless number of unique antigens. Gene rearrangement occurs in programmed steps in the bone marrow during B-cell development. The process starts with a committed stem cell, continues through pro‒B and pre‒B cell stages, and results in an immature B cell. At this point, any cells that interact with self antigen (autoimmune cells) are removed from the immature B cell population via inactivation or apoptosis. Elimination of these cells ensures that the immune system is less likely to recognize these antigens as foreign (immune tolerance). Cells that are not removed (ie, those that recognize nonself antigen) continue to develop into mature naive B cells, leave the marrow, and enter peripheral lymphoid organs, where they may encounter antigens.

    Their response to antigen has 2 stages:

    Primary immune response: When mature naive B cells first encounter antigen, they become lymphoblasts, undergo clonal proliferation, and differentiate into memory cells, which can respond to the same antigen in the future, or into mature antibody-secreting plasma cells. After first exposure, there is a latent period of days before antibody is produced. Then, only IgM is produced. After that, with the help of T cells, B cells can further rearrange their Ig genes and switch to production of IgG, IgA, or IgE. Thus, after first exposure, the response is slow and initially provides limited protective immunity.

    Secondary (anamnestic or booster) immune response: When memory B and Th cells are reexposed to the antigen, the memory B cells rapidly proliferate, differentiate into mature plasma cells, and promptly produce large amounts of antibody (chiefly IgG because of a T cell–induced isotype switch). The antibody is released into the blood and other tissues, where it can react with antigen. Thus, after reexposure, the immune response is faster and more effective.

    T cells

    T cells develop from bone marrow stem cells that travel to the thymus, where they go through rigorous selection. There are 3 main types of T cell:

    In selection, T cells that react to self antigen presented by self MHC molecules or to self MHC molecules (regardless of the antigen presented) are eliminated by apoptosis, limiting the likelihood of autoimmunity. Only T cells that can recognize nonself antigen complexed to self MHC molecules survive they leave the thymus for peripheral blood and lymphoid tissues.

    Most mature T cells express either CD4 or CD8 and have an antigen-binding, Ig-like surface receptor called the T-cell receptor (TCR). There are 2 types of TCR:

    Alpha-beta TCR: Composed of TCR alpha and beta chains present on most T cells

    Gamma-delta TCR: Composed of TCR gamma and delta chains present on a small population of T cells

    Genes that encode the TCR, like Ig genes, are rearranged, resulting in defined specificity and affinity for antigen. Most T cells (those with an alpha-beta TCR) recognize antigen-derived peptide displayed in the MHC molecule of an antigen-presenting cell. Gamma-delta T cells recognize protein antigen directly or recognize lipid antigen displayed by an MHC-like molecule called CD1. As for B cells, the number of T-cell specificities is almost limitless.

    For alpha-beta T cells to be activated, the TCR must engage with antigen-MHC (see figure Two-signal model for T cell activation). Costimulatory accessory molecules must also interact (eg, CD28 on the T cell interacts with CD80 and CD86 on the antigen-presenting cell) otherwise, the T cell becomes anergic or dies by apoptosis. Some accessory molecules (eg, CTLA-4 [cytotoxic T-lymphocyte antigen 4] on the T cell, which also interacts with CD80 and CD86 on the antigen-presenting cell, PD-1 [programmed cell death protein 1] on the T cell, which interacts with PD-L1 [programmed cell death protein ligand 1] on the antigen-presenting cell) inhibit previously activated T cells and thus dampen the immune response. Molecules such as CTLA-4 and PD-1, and their ligands, are termed checkpoint molecules because they signal that the T cell needs to be restrained from continuing its activity. Cancer cells that express checkpoint molecules may thus be protected from the immune system by restraining the activity of tumor-specific T cells.

    Monoclonal antibodies that target checkpoint molecules on either T cells or on tumor cells (termed checkpoint inhibitors, see table Some Immunotherapeutic Agents in Clinical Use) are used to prevent downregulation of antitumor responses and effectively treat some heretofore resistant cancers. However, because checkpoint molecules are also involved in other types of immune response, checkpoint inhibitors can cause severe immune-related inflammatory and autoimmune reactions (both systemic and organ specific).

    Polymorphisms in the CTLA-4 gene are associated with certain autoimmune disorders, including Graves disease and type I diabetes.

    Two-signal model for T-cell activation

    The alpha (α ) and beta (β) chains of the T-cell receptor (TCR) bind to antigen (Ag)–major histocompatibility complex (MHC) on an antigen-presenting cell (APC), and CD4 or CD8 interacts with the MHC. Both actions stimulate the T cell (1st signal) through the accessory CD3 chains. However, without a 2nd (coactivation) signal, the T cell is anergic or tolerant.

    The TCR is structurally homologous to the B-cell receptor the α and β (or gamma [γ] and delta [δ] ) chains have constant (C) and variable (V) regions. (1) = 1st signal (2) = 2nd signal.

    Helper T (Th) cells are usually CD4 but may be CD8. They differentiate from Th0 cells into one of the following:

    Th1 cells: In general, Th1 cells promote cell-mediated immunity via cytotoxic T cells and macrophages and are thus particularly involved in defense against intracellular pathogens (eg, viruses). They can also promote the production of some antibody classes.

    Th2 cells: Th2 cells are particularly adept at promoting antibody production by B cells (humoral immunity) and thus are particularly involved in directing responses aimed at extracellular pathogens (eg, bacteria, parasites).

    Th17 cells: Th17 cells promote tissue inflammation.

    Each cell type secretes several cytokines (see table Functions of T Cells). Different patterns of cytokine production identify other Th-cell functional phenotypes. Depending on the stimulating pathogen, Th1 and Th2 cells can, to a certain extent, downregulate each other's activity, leading to dominance of a Th1 or a Th2 response.


    Watch the video: Top 3 Tests For Autoimmune Conditions (July 2022).


Comments:

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