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T helper type 17 (Th17) cells are a distinct lineage of T cells that produce the effector molecules IL-17, IL-17F, IL-21 and IL-22. Although the role of Th17 cells in autoimmunity is well documented, there is growing evidence that the Th17 lineage and other IL-17 producing cells are critical for host defense against bacterial, fungal and viral infections at mucosal surfaces. Here we summarize recent progress in our understanding of the function of IL-17 producing cells as a bridge between innate and adaptive immunity against infectious diseases at the mucosa.
CD4+ T helper cells are important mediators of adaptive immune responses. Following interaction with antigen presenting cells, T cells receive signals by engagement of the T cell receptor (signal 1), costimulatory molecules (signal 2) and a complex network of cytokine signals (signal 3) and undergo activation and differentiation into effector CD4+ T cells. Their critical role in host defense against infections is clearly manifested in humans with either congenital deficiencies or acquired deficiency (most commonly through HIV infection) of the T-cell lineage. It was recognized shortly after the initial description of AIDS that the primary immunodeficiency was loss of circulating CD4+ T-cells and that the risk of opportunistic infection with many pathogens was directly related to the severity of the CD4+ T-cell deficiency. Four years after the initial clinical description of AIDS, Mossmann and Coffman described the first two CD4+ T-cells subsets based on discrete cytokine profiles1. Th1 effectors produce Interferon-gamma (IFN-γ) and regulate cellular immunity against intracellular infections whereas Th2 cells produce Interleukin (IL)-4, IL-5 and IL-13 and mediate humoral immunity against parasite infections (Figure 1). However this dichotomy of T-cell subsets could not fully explain the infections seen in congenital or acquired absence of CD4+ T-cells such as mucosal Candidiasis, Pneumocystis carinii pneumonia, or some bacterial pneumonias. For example mice deficient in Th1, Th2 responses (or both) are not permissive for P. carinii pneumonia2, a hallmark infection in AIDS patients with low CD4+ T-cell counts. These data suggested other CD4+ T-cell lineages are critical for host defenses against opportunistic infections. Recent compelling evidence has clearly changed this traditional paradigm of Th1/Th2 cell dichotomy to include a third subset of T cells referred to as Th17 cells3-6. Th17 cells produce the cytokines IL-17A (IL-17) 4,5 and IL-17F3, as well as the cytokines IL-217,8 and IL-22 9 10 (Figure 1). This new Th17 cell lineage fills in some of the missing gaps in host immunity not fully explained by the Th1/Th2 paradigm.
IL-17 is the prototype of Th17 cytokines and is the best studied of the Th17 cytokines. The receptor for IL-17 (IL-17R) is a Type I transmembrane protein and is ubiquitously expressed on various organs including lung, kidney and spleen11. Cells that express the receptor for IL-17 are leucocytes, epithelial cells, mesothial cells, vascular endothelial cells, keratinocytes and fibroblasts and respond to IL-17R-mediated signaling by production of granulocyte colony-stimulating factor (G-CSF), IL-6, IL-8 and mediate granulopoiesis, neutrophil recruitment and inflammatory responses (reviewed in12). IL-17 was initially identified as a key factor in the induction of inflammation and tissue destruction associated with animal models of autoimmune disease like Multiple Sclerosis, Collagen Induced Arthritis and experimental colitis (reviewed in13,14). Based on the efficacy of targeting IL-17 in pre-clinical models of these chronic inflammatory conditions, IL-17 is currently a promising therapeutic target15. Initial work implicating IL-17 to be protective against extracellular bacterial infections at the lung mucosal surface was through effective recruitment of neutrophils mediated by chemokine induction16-18. More recent advances in our understanding of the complexity of the Th17 cytokines suggests broader effects for these cytokines in protecting against a variety of pathogenic bacteria, fungi and viruses at different mucosal surfaces. In this review we have summarized recent progress in our understanding of the role of Th17 cells and effector cytokines and their function as a bridge between innate and adaptive immunity against infectious diseases at the mucosa.
The differentiation of Th17 cells is determined by the exposure to TGF-β, IL-6, IL-21, while IL-23 further stabilizes the commitment of Th17 cells to this lineage (reviewed in19). These polarizing cytokines act on newly primed cells to induce the expression of the transcription factor RORγt and RORα which induces Th17 differentiation20,21. RORγτ also control the expression of IL-23 inducible receptors on newly primed T cells, further expanding their responsiveness to IL-23 to sustain the T lineage specific responses. The gp-130-Stat3 pathway is essential for expression of RORγτ and Th17 development22-24. Recently it has been shown that IL-21 acts downstream of these events to further amplify Th17 cell generation in an autocrine manner7. Additional co factors for the differentiation of Th17 are IL-1β and TNF-α25,26. Although in vitro studies have shown that IL-23 is redundant in the generation of Th17 responses27, in vivo studies in the mouse model 18,28,29 and in humans 30 have shown that IL-23 is critical for maintenance of Th17 responses in vivo. Since over-induction of Th17 cells can impact tissue damage due to induction of inflammatory pathways, the generation of Th17 cells is strictly regulated. For example, cytokines such as IL-2731,32, those belonging to Th1 (IFNγ) and Th2 (IL-4) 4,5 and IL-2 33 tightly regulate the induction of Th17 cells. Endogenous lipid mediators like prostaglandin E2 (PGE2) released under inflammatory conditions promotes Th17 cells differentiation 34 35 36-39 suggesting that external infection-induced mediators can also influence the decision between a Th1/Th2/Th17 and T regulatory cell responses.
Several of these Th17 polarizing cytokines such as IL-23, TGF-β, IL-6 and IL-1β are induced in dendritic cells activated by components of microbes. Several bacteria and its products such as Kleibsiella pneumoniae18,40, Mycobacterium tuberculosis26,41, Helicobacter pylori42, Francisella tularensis43Salmonella enterica44 and Bordetella pertusis45 induce these cytokines through TLR signaling. Further bacterial peptidoglycans can induce the generation of Th17 cells through nucleotide oligomerization domain 2 (NOD2) receptor signaling in dendritic cells25. Viruses such as Herpes simplex virus 46 and fungus and fungal components such as B–glucans26,47, Cryptococcus48, Candida albicans47 and Aspergillus fumigatus49 can all induce some or all of these polarizing cytokines from dendritic cells and play a role in differentiation of Th17 cells. These studies suggest that relative amounts of the polarizing cytokines induced by the pathogen may define the final outcome of differentiation of naïve T cells to Th1, Th2, Th17 or T regulatory cells during infection.
Although most of the recent focus has been on IL-17 produced by CD4αβ T cells, γδ T cells in some cases are more potent producers of IL-17 during the early immune response at mucosal sites following infections. In a pulmonary murine model of Mycobacterium tuberculosis50 and M.bovis BCG51 the major producers of IL-17 in T cells isolated from lungs of infected mice were γδ T cells. This finding was also highlighted in a recent study where γδ T cells were the major source of IL-17 in human tuberculosis patients52. In addition to γδ T cells, subsets of NKT cells 53 and NK cells expressing RORγτ+NKp46+ 54,55 can produce IL-17 and may impact the innate response to infections. Further, the detection of IL-17 mRNA in neutrophils 56 as well as production of IL-17 and IL-22 by Lymphoid-tissue inducer like cells(Lti)57 54,55,58-60 suggests that the role of IL-17 during the innate immune response is largely under-explored. It is likely that the production of IL-17 by innate cells at mucosal sites can serve as a mechanism to provide defense mechanism until the adaptive immune cells are recruited to control the infection.
Early work in establishing a critical role for IL-17 in protective immunity against extracellular bacterial infections was using K.pneumoniae in a respiratory infection model17. IL-17R knockout (KO) mice displayed significant delays in neutrophil recruitment and had greater dissemination of K. pneumoniae. These studies provided early evidence that IL-17R signaling was important for neutrophil-mediated control of pulmonary K. pneumoniae infection by optimal induction of chemokines such as macrophage inflammatory protein-2(MIP-2) and granulocyte colony-stimulating factor (G-CSF) (Figure 2). IL-17 induces granulopoietic factors (G-CSF and stem cell factor) and CXC chemokines CXCL1, CXCL2, and CXCL-5 in fibroblasts and epithelial cells61-63. Furthermore, IL-17 mediated induction of G-CSF also results in differentiation of CD34+ progenitors into neutrophil progentiors61. Therefore, over expression of a recombinant adenovirus encoding IL-17 (AdIL-17) reversed the disease phenotype in mice challenged with K. pneumoniae by induction of chemokines, augmented polymorphonuclear leukocyte recruitment and enhanced bacterial clearance and survival16. Further that IL-17 can induce the expression of human beta defensin-264,65, S100 proteins and chemokines CXCL5, CXCL9, CCL3 65 and CCL-20 66 in lung epithelial cells, suggests that IL-17 can also enhance host defense against pathogens by induction of anti-microbials and chemokines for recruitment of immune cells. The cellular source of IL-17 was soon identified to be CD4 and CD8 T cells40, was dependent on IL-23 and mediated by TLR4 signaling18. More recently IL-22, another effector cytokine produced by Th17 cells has been implicated in induction of host cell antimicrobials and defensins in the respiratory epithelium and is required for early control of K. pneumoniae, in part by regulating the expression of the antimicrobial protein lipocalin-265. Further that IL-17 and IL-22 function synergistically in induction of antimicrobials such as human beta defensin-2 and S100 proteins66, suggests that the Th17 cytokines may have evolved to generate effective host defense mechanisms against extracellular pathogens at mucosal sites. Further studies also show that Th17 cells play protective roles against extracellular bacterial infections in the gut mucosa. Citrobacter rodentum, a naturally occurring mouse pathogen requires the generation of IL-23-dependent protective Th17 cells in the lamina propria for protection67,68. Also, IL-22 contributes to the early host defense against C.rodentium through the direct induction of the Reg family of antimicrobial proteins in colonic epithelial cells29. IL-17 signaling also appears to be host-protective in the oral mucosa, as IL-17R-deficient mice are highly susceptible to infection by the gram-negative anaerobic periodontal pathogen, Porphyromonas gingivalis, due to reduced neutrophil mobilization and recruitment69,70.
These studies provide clear evidence that Th17 cytokines are critical for protection against a variety of extracellular bacteria and are perfectly poised to control infection either by recruitment of protective cells or induction of anti-microbials during the early immune responses at mucosal sites. However, the Th17 response in some cases can also be a double-edged sword and the balance between protection and pathology may define the outcome of the infection. For example, whooping cough caused by B.pertusis, a gram negative extracellular bacterial infection in the respiratory tract results in persistent cough as one of the hallmarks of the clinical disease. Accumulating evidence suggests that the B.pertusis infection may bias the host response towards the production of Th17 cytokines 35,45 by preferentially inhibiting IL-12 and inducing IL-2345. B.pertusis causes severe respiratory pathology including bronchiectasis while B.parapertusis causes less severe disease pathology71. Interestingly, B.pertusis lipooligosacharide induces potent production of IL-23, IL-1β and IL-6 from DCs and drives a more robust differentiation of naïve CD4 T cells to Th17 cells when compared to DCs activated with LPS from B.parapertusis45. The current emerging hypothesis is that the host bias towards Th17 results following B.pertusis infection results in inflammation and destruction of the airways leading to bronchiectasis and causes persistent cough. This hypothesis is further supported by another cause of bronchiectasis, cystic fibrosis which is highly associated with bronchiectasis due to chronic biofilm infection with P. aeruginosa and elevated IL-17 and IL-22 responses in draining lung lymph node cells65. These disease models may serve as an example for the role of IL-17 in mediating pathology while conferring protection against extracellular bacterial infections in the respiratory mucosa. Another fine example of how a protective Th17 response may culminate in a pathogenic inflammatory response is evident in H.pylori infections in the gut mucosa. H.pyroli is a gram negative bacteria that colonizes the gastric mucosa and induces an inflammatory infiltration of neutrophils associated with robust IL-17 production by CD4 and CD8 T cells72,73. These data suggests that IL-23/IL-17 pathway play a crucial role in defining the ongoing gastric inflammation in H pylori-infected patients.
While a clear protective role for Th17 cytokines in extracellular bacterial infections is emerging, Th17 effector cytokines appear to be dispensable for protection against most studied intracellular bacterial infections at the mucosal sites. The absence of the IL-23/IL-17 axis impacts the formation of granulomas 51 and inflammation following mycobacterial pulmonary infection41. However, IL-23KO mice and IL-17RKO mice are not more susceptible than wild type control mice to M.tuberculosis41,65 and M.bovis BCG pulmonary infections51. Interestingly, pulmonary but not intradermal route of infection with a intracellular bacteria F.tularensis LVS, induces Th17 cells and inhibits Th1 cell generation36. However, whether IL-17 is required for protection against F. tularensis respiratory infection is not known. These studies implicate that the host Th17 effector cytokines have evolved as protective immune mechanisms against extracellular bacteria and are dispensable for primary protection against most intracellular pathogens that require a Th1 pathway for protection.
In contrast, intracellular pathogens that may require both CD4 Th1 cells and neutrophils for protection at mucosal sites may be dependent on IL-23/IL-17 axis for pathogen control (Figure 1). For example, the induction of IL-17 and IL-17F production following acute Mycoplasma pneumonia pulmonary infection is IL-23-dependent and contributes to neutrophil recruitment and mediates protection against the infection74. Also, infection with Salmonella typhimurium, which can exist as an intracellular pathogen, results in induction of IL-17 and IL-22 in the ileal mucosa and the absence of IL-17R signaling results in increased translocation of the bacteria to the mesenteric lymph nodes, reduced induction of chemokines, anti-microbials and reduced neutrophilic recruitment to the ileal mucosa75. These studies show that IL-17 may be critical for the full induction of immune responses that lead to neutrophil influx and contribute to control of bacteria in some intracellular infections.
Using a macaque model of Simian Immunodeficiency Virus (SIV) to study HIV human disease and related complications arising due to bacterial coinfections such as S.typhimurium, it was found that SIV coinfection selectively inhibits Th17 responses elicited by S. typhimurium, probably due to depletion of CD4+ T cells in the ileal mucosa75. This results in an inability of SIV-infected macaques to mount normal mucosal inflammatory response to S. typhimurium infection and allows dissemination of bacteria to the mesenteric lymph node. This data may provides a basis for the observation that people with HIV are at a increased risk of developing bacteremia due to dissemination of bacteria resulting from reduced CD4 Th17 responses.
Inflammation is a critical component of the protective host response to fungal infections. However resolution of inflammation is essential for reducing the immunopathology resulting from the infection. Candida albicans infection models appear to reflect this dichotomy. C. albicans is a commensal organism of the oral cavity and gastrointestinal(GI) tract, but can become pathogenic in settings of immunodeficiency. The most commonly used experimental model of Candida infection represents the disseminated form of disease, which in humans occurs as a nosocomial infection with a 40% mortality rate. In mice, IL-17 receptor signaling is highly protective in this setting, acting through recruitment and expansion of neutrophils76. In the oral cavity, Candida causes thrush (oropharyngeal candidiasis, OPC), which occurs in > 90% of HIV+ individuals77. In a mouse model of OPC, Th17 cells and IL-17 receptor signalling but not Th1 cells and IFNγ are necessary for host protection78,79. New data show that Th17-based immunity appears to be mediated primarily through the macrophage mannose receptor80. Patients with Chronic mucocutaneous candidiasis (CMC) produced significantly lower amounts of IL-17 and IL-22 mRNA and protein in vitro following antigen stimulation when compared to healthy individuals, suggesting that Th17 cytokine production correlates with protection81. Even more compelling, patients with autosomal dominant hyper-IgE syndrome (HIES, Job's Syndrome) resulting from a mutation in the STAT-3 gene are extremely susceptible to bacterial infections such as S.aureus and mucocutaneous fungal infections caused by Candida species. In line with a critical role for STAT-3 in driving Th17 cellular responses, these patients do not generate C.albicans- and S.aureus- specific Th17 cellular responses24. Therefore, the mechanism underlying the susceptibility to recurrent fungal infections commonly seen in these patients is likely an inability to mount Th17 responses23,24,82. In contrast, a gastric model of C. albicans infection stimulates severe gut pathology, which is exacerbated by IL-23 and IL-1749. Finally, C. albicans also causes vaginal yeast infections. Interestingly, female HIV+ patients do not experience a high incidence of vaginal Candida infections, despite their high incidence of thrush77. It is believed that the vaginal epithelial cells play a far more critical role in this setting, but the role of IL-17 is not well defined. Therefore, route of entry and site of infection dictate the consequence of Th17-mediated immunity in fungal infections.
In respiratory tract models of fungal infections using Pneumocytis carnii83 and Aspergillus fumigatus84, induction of IL-23 and IL-17 following pathogen challenge is protective, since IL-23KO mice or neutralization of the IL-23/IL-17 axis resulted in impaired clearance of the pathogen. Although these studies suggest that the production of IL-23 and IL-17 is protective during fungal infections, it has been suggested that heightened IL-23 dependent Th17 responses against two major fungal pathogens C. albicans and A.fumigatus mediates neutrophilic inflammation and severe tissue pathology49. Further, phagocytes from patients with Chronic Granulomatous Disease (CGD) lack NADPH oxidase activity and hence the ability to generate reactive oxygen species, resulting in recurrent bacterial and fungal infections. In a mouse model of CGD, pulmonary aspergillosis infection is lethal due to heightened Th17 response and inflammatory lung pathology85. Although lL-17 neutralization was moderately beneficial, complete cure and reversal of the phenotype was only achieved with replacement therapy with a natural kynurenine85. In the context of CGD, the inflammatory pathology mediated by IL-17 is suggested to be pathogenic rather than protective. Therefore it appears that the early induction of IL-23 and IL-17 during fungal infections is protective. However, heightened Th17 responses that follow may result in the generation of pathological rather than protective outcomes during fungal infections.
CD4 T cells can regulate the adaptive immune responses to viruses by providing help to CD8 and B cells and also by direct anti-viral activity. Documentation of the presence of viral specific CD4 Th17 cells in HIV infection86, cytomegalovirus (CMV) specific IL-17 producing cells in humans86, and in mice 87 suggests that Th17 cells may have a role to play in viral infections. Herpes simplex virus (HSV-1) infection of the cornea results in early induction of IL-2346 and IL-1788 and mice that lack IL-17R showed reduced early infiltration of neutrophils and corneal opacity following HSV infection88. On the contrary, IL-23KO mice showed a more severe disease phenotype to HSV infection, probably as a consequence of increased IL-12 responses and higher numbers of IFNγ producing cells46. Human rhinovirus (HRV) infections are associated with exacerbations of asthma and chronic obstructive pulmonary infiltration and IL-17 was shown to function synergistically with HRV to induce IL-8 from epithelial cells and may contribute to the recruitment of neutrophils, immature DCs and memory T cells to the lung contributing to severe inflammatory profiles seen during viral exacerbations of airway disease89. More recently, neutralization of IL-17 responses during a influenza challenge in mice resulted in increased weight loss and reduced survival of mice90. These studies suggest that the fine balance between protection and pathological manifestations of Th17 responses will define the outcome of viral infections at the mucosa and further research is required to clarify the Th17 cellular balance at the mucosa.
Convincing data show a protective role for IL-17 in immunity to primary infections against extracellular pathogens and fungal infections. However, the role of IL-17 in memory immune responses to infections is less well studied and understood. IL-23 was initially reported to act on memory or activated T cells that express the IL-23 receptor and produce IL-17 91 and therefore it is likely that these cells may have a role to play in vaccine-induced immunity. Although IL-17 responses are induced during primary M.tuberculosis pulmonary infection, it is dispensable for protection against primary challenge with M.tuberculosis41. However, induction of IL-17 during a memory response to M.tuberculosis challenge correlates with protection. Following challenge with M. tuberculosis, protection is associated with the recruitment of protective Th1 cells and production of IFNγ which results in macrophage activation and mycobacterial killing. In this vaccine-induced protection model, Th17 cells upregulate CXCR3 ligating chemokines and accelerate the recruitment of Th1 cells that express the receptor CXCR3 28(Figure 1). Further, peripheral blood of mycobacteria-exposed healthy adults express M.tuberculosis specific memory Th17 cells producing IL-17 or the related cytokine IL-22 92 suggesting that the IL-23/IL-17 axis contribute to protective immunity during tuberculosis. In support for a protective role for IL-23 and IL-17 in immunotherapy against M.tuberculosis, over expression of an recombinant adenovirus expressing IL-23 when delivered prior to M.tuberculosis pulmonary infection results in increased IFN-γ and IL-17 responses in the lung and mediates improved protection compared to control adenovirus treated mice93. In the absence of effective current vaccines against tuberculosis, the protective role for Th17 cells in generation of memory immunity will play a critical role in design of new vaccines against tuberculosis. Further, vaccine-induced protection against another pulmonary pathogen B.pertussis is mediated by IL-17. Th17 cells are induced by vaccination with whole cell pertussis vaccines (Pw) and neutralization of IL-17 reduces protection following a pulmonary challenge with B. pertussis. Contrary to the M.tuberculosis model, in this model IL-17 was suggested to have a protective role via direct activation of macrophages and B. pertussis killing94. In a CD4 T cell dependent, antibody-independent model of vaccine-induced protection following S. pneumoniae challenge, treatment with antiserum to IL-17 resulted in reduced immunity to pneumococcal colonization compared to the control serum treated mice95. In viral vaccine-induced responses, mucosal immunization of mice with rotavirus V6 protein reduces rotavirus fecal shedding and was associated with the presence of memory Th1 and Th17 cells in the intestine96,97. Although it is thought that the Th1 response is the major protective mechanism in this model, it is suggested that the Th17 response may play an indirect protective role. These data suggest a protective role for IL-17 in vaccine-induced immune responses against bacterial, fungal and viral infections. However further studies are required to dissect the direct and indirect pathways and molecular mechanisms involved in protection.
A protective versus pathogenic role for IL-17 in settings of chronic infections that occur in diseases such as cystic fibrosis (CF), recurrent fungal infections or CGD is currently an area of extensive research. IL-17 induction associated with these diseases may often be pathologic rather than protective, as a result of severe defects in downstream effector functions of responding cells. For example, bacterial infections such as Klebsiella and Pseudomonas that are often associated with CF disease, induce IL-17-dependent neutrophil recruitment and induction of anti-microbial proteins65,98, and under normal conditions this IL-17 dependent response is protective. However in the context of chronic biofilm infection in CF patients, the resulting induction of IL-17 and neutrophil recruitment may not result in bacterial clearance but in inflammation, possibly due to the defective mucociliary clearance mechanisms in CF respiratory epithelial cells98. Similarly, in a mouse model of CGD, A.fumigatus induces IL-17 and recruits high numbers of neutrophils85. However, the lack of NAPDPH oxidase activity and the inability to generate reactive oxygen species by the neutrophils renders this downstream effector IL-17 ineffective, resulting in acute inflammatory responses and inability to clear the pathogen. Likewise in the oral cavity, chronic periodontal infection has been associated with elevated IL-17 levels70. Taken together these data suggest that Th17 mucosal immunity requires functional downstream effector cells, either epithelial cells or neutrophils for effective clearance of the pathogen.
Evidence suggests that Th17 cells have evolved to mediate protective immunity against a variety of pathogens at different mucosal sites. Moreover the lack of Th17 responses appears to explain in part some of the immunodeficiency observed in both AIDS and in patients with Job's syndrome, particularly in terms of mucosal infection with C. albicans and some cases of bacterial pneumonia. These data may lead to new avenues of immunotherapy to treat or prevent these infections in these patient groups. Also, the emerging evidence that Th17 cells are crucial players in generation of vaccine-induced protective responses against a variety of pathogens suggests that the incorporation of this knowledge into the design of current and future vaccines against infectious diseases should be an active area of research. It is also becoming apparent that the fine balance between protection and pathological manifestations of Th17 responses will define the outcome of infections and further research is required to define the Th17 cellular balance at mucosal sites.
Funding for this project was provided, in part, by the Pennsylvania Department of Health, Tobacco Formula Funding and the National Institutes of Health to JKK, Pennsylvania Department of Health, Tobacco Formula Funding, R00AI075106 from the National Institute Of Allergy And Infectious Diseases and Children's Hospital of Pittsburgh to SAK. SLG was supported by AR054389 and DE018822.