Mem Inst Oswaldo Cruz, Rio de Janeiro, 101 (Suppl.I) October 2006
CD4 T cells producing pro-inflammatory interleukin-17 mediate high pathology in schistosomiasi
Department of Pathology, Tufts University School of Medicine, 150 Harrison Ave, Boston, MA 02111, US
In murine schistosomiasis mansoni, pronounced CD4 T cell-mediated, egg-induced, hepato-intestinal immunopathology and death, whether genetically determined or elicited experimentally, are associated with failure to down-regulate a net pro-inflammatory immune response. Important evidence contributing to this notion comes from the observation that immunization with schistosome egg antigens in CFA (SEA/CFA) causes low pathology C57BL/6 mice to develop an exacerbated form of disease and death in a cytokine milieu characterized by elevated interferon (IFN)-g levels. Since such a pro-inflammatory environment presumes a signaling pathway involving interleukin (IL)-12, the SEA/CFA immunization model was used to examine the extent of hepatic immunopathology in the absence of this cytokine. Surprisingly, the IL-12p40 subunit was an absolute requirement for the development of exacerbated disease, whereas the IL-12p35 subunit was not. Moreover, significantly elevated in vitro production of IL-17, but not of IFN-g, correlated with the high pathology, and neutralization of IL-17 in vivo resulted in a significant reduction of hepatic inflammation. Our findings clearly demonstrate the pathogenic potential of the novel IL-17-producing T cell subpopulation (ThIL-17), previously shown to mediate chronic inflammation in autoimmune disease. They also imply that IL-23, but not IL-12, is the critical signal necessary to support the pro-inflammatory ThIL-17 subset involved in high pathology schistosomiasis.
In schistosomiasis caused by the helminth trematode species Schistosoma mansoni, the hepato-intestinal gra-nulomatous and fibrosing inflammation precipitated by parasite eggs is mediated by CD4 T cells. Human patient populations infected with this parasite display a great variation in the magnitude of clinical disease. Likewise, in the murine model, the CBA and C3H strains typically develop a more pronounced hepatic immunopathology characterized by larger egg granulomas with hepatic parenchymal inflammation, whereas in C57BL/6 (BL/6) mice the lesions are uniformly less severe (Cheever et al. 1987, Rutitzky et al. 2005a). Several independent lines of evidence have linked the high pathology to the persistence of a net pro-inflammatory state marked by the increase of Th1-type cytokines such as interferon (IFN)-g and tumor necrosis factor (IFN)-a, the deficiency of anti-inflammatory Th2-type cytokines such as interleukin (IL)-4 and IL-10, or both. For example, schistosome egg antigen (SEA)-stimulated mesenteric lymph node cells from 7 week-infected high pathology CBA mice produce significant amounts of IFN-g, whereas those from low pathology BL/6 mice do not (Rutitzky et al. 2001, 2003a, 2005a). Moreover, mice with impaired costimulatory B7-CD28 (King et al. 1996, Hernandez et al. 1999), CD40-CD154 (MacDonald et al. 2002) and B7RP-1-ICOS (Rutitzky et al. 2003b) systems, or deficient in anti-inflammatory cytokines IL-4 (Brunet et al. 1997) and IL-10 (Hoffmann et al. 2000, Sadler et al. 2003), or in B cells (Hernandez et al. 1997) or in alternatively activated macrophages (Herbert et al. 2004), display a pro-inflammatory cytokine dominance and are prone to pathology exacerbation. Lastly, immunization with SEA in CFA causes the typically low pathology C57BL/6 mice to develop an exacerbated form of disease and death in a cytokine milieu characterized by elevated IFN-g levels (Rutitzky et al. 2001). In essence, a net pro-inflammatory host immune reactivity to egg antigens results in enhanced pathology that manifests itself early after patent infection and often is associated with premature death.
The development of Th1 vs Th2 CD4 cell subtypes endowed with contrasting inflammatory capabilities is the product of distinct activation programs operating in common undifferentiated precursor cells. The signal transducer and activation of transcription factors (STAT) 1 and 4, the transcription factor T-bet and IL-12 are involved in Th1 differentiation (Szabo et al. 2003), whereas STAT 6, GATA-3 and IL-4, respectively, facilitate Th2 development (Zhu et al. 2006).
The work presented here and at the 10th International Symposium on Schistosomiasis, held in Belo Horizonte, Brazil, on September 25-28, 2005, focused specifically on the role of IL-12 in promoting the pro-inflammatory state associated with severe egg-induced immunopathology. IL-12 is a heterodimeric 70 kDa cytokine composed of two subunits of 40 and 35 kDa, termed IL-12p40 and IL-12p35 (Trinchieri 1998, 2003a, Trinchieri et al. 2003). IL-12 is produced by activated innate immunocytes, such as dendritic cells (DC), and is recognized by lymphocytes expressing IL-12Rb1 and IL-12Rb2 receptors for the IL-12p40 and IL-12p35 subunits, respectively, thereby inducing IFN-g and other pro-inflammatory mediator production (Trinchieri 1998, 2003).
Effective examination of the role of IL-12 in the development of schistosome immunopathology requires the use of mice deficient in either of its subunits, IL-12p40 (IL-12p40-/-) and IL-12p35 (IL-12p35-/-). Because such mice are currently only available on a genetic background (BL/6) conducive to low egg-induced pathology, they were concomitantly immunized with SEA in CFA (Rutitzky et al. 2001). This treatment has been demonstrated to cause in infected BL/6 mice a marked enhancement of the hepatic lesions and early death in a pro-inflammatory environment, a situation comparable to that seen in other mouse strains which are naturally prone to high pathology.
Schistosome-infected IL-12p40-/- mice immunized with SEA/CFA indeed displayed markedly reduced hepatic pathology, which was no different from that seen in unimmunized controls. However, in contrast, SEA/CFA-immunized IL-12p35-/- mice exhibited the accelerated death and exacerbated hepatic lesions typically seen in similarly treated BL/6 WT mice (Rutitzky et al. 2005b). Notably, the dramatic dichotomy in disease severity between the IL-12p40-/- and IL-12p35-/- mice was evident despite the fact that both groups had extremely low levels of pro-inflammatory IFN-g (Rutitzky et al. 2005b). These findings clearly demonstrated that only IL-12p40 was essential for the development of high pathology whereas IL-12p35 (and thus the dimeric form IL-12p70) were not, and, additionally, that there was no correlation between the severity of immunopathology and the levels of IFN-g (Rutitzky et al. 2005b). Surprisingly, the high pathology state most closely correlated with elevated levels of IL-17 produced by SEA-stimulated granuloma and mesenteric lymph node T cells (Rutitzky et al. 2005b).
A compelling reason for investigating IL-17 was because this cytokine has recently received considerable attention as a novel pro-inflammatory mediator of chronic inflammation associated with cell-mediated autoimmune disease. Two models in which IL-17 has been clearly shown to play a decisive role in pathogenesis are experimental allergic encephalomyelitis (EAE) (Cua et al. 2003, Langrish et al. 2005) and collagen induced arthritis (CIA) (Murphy et al. 2003). In both EAE and CIA, the IL-17 was produced by a population of CD4 T cells expressing the CD69 activation marker and a CD44high, CD62low memory phenotype (Aggarwal et al. 2003, Langrish et al. 2005). In our system, cells with these characteristics were similarly increased in the large egg granulomas from the SEA/CFA-immunized WT and IL-12p35-/-, but not in those from the IL-12p40-/- mice. Moreover, neutralization of IL-17 did result in an inhibition of immunopathology, denoting a direct role for this cytokine in the pathogenesis of the enhanced granulomatous inflammation. Importantly, elevated levels of IL-17 were also detected in schistosome-infected CBA mice, which are naturally prone to high pathology, and their lesions were similarly reduced following treatment with neutralizing anti-IL-17 antibodies (Rutitzky et al. 2005b). These findings clearly tie the ThIL-17 cells to the development of high-pathology schistosomiasis.
IL-17A and IL-17F are the members of the IL-17 cytokine family responsible for the pathogenic activity of the ThIL-17 cells, as the antibodies resulting in disease amelioration are directed against these species (Lubberts et al. 2004, Rutitzky et al. 2005b). However, other pro-inflammatory mediators produced by the ThIL-17 cells, including TNF-a, IL-6, IL-8 and other chemokines (Kolls & Linden 2004, Langrish et al. 2004), may also play a significant role in the pathology. One apparent mechanism used by ThIL-17 cells to cause pathology is through recruitment of granulocytes, which in the case of the schistosome egg-granulomas, may include the eosinophils. This issue is currently under investigation.
Current models about the development of ThIL-17 cells place this subset directly under the control of IL-23, but not of IL-12 (Trinchieri et al. 2003, McKenzie et al. 2006). IL-23 is a member of the IL-12 family of heterodimeric cytokines which utilizes the same IL-12p40 subunit in association with a distinct p19 subunit (Oppmann et al. 2000). It thus becomes evident that it is the absence of IL-23 formation in SEA/CFA-immunized, schistosome-infected mice lacking the IL-12p40 subunit that explains their low levels of immunopathology and of IL-17. Like IL-12, IL-23 is a product of stimulated innate immunocytes such as DC and macrophages, which activate T cells bearing the IL-12Rb1 receptor for the common IL-12p40 subunit and a specific IL-23 receptor for the p19 subunit (Parham et al. 2002). Unlike IL-12, the binding of IL-23 to its receptors induces phosphorylation of STAT 3 in addition to STAT 4 (Trinchieri et al. 2003, Holscher 2004), with further differences in signal transduction to be elucidated. It has been recently demonstrated that the ThIL-17 cells activated by IL-23 constitute a fully distinct, de novo, pro-inflammatory T cell differentiation line, which does not branch off from pro-inflammatory Th1 cells activated by IL-12, as first surmised to be the case (Harrington et al. 2005, Park et al. 2005).
The Figure illustrates the likely activation pathway and function of the CD4 ThIL-17 cell subset in infection with schistosomiasis. While there is ample evidence that these cells are stimulated by IL-23 and mediate severe immunopathology, multiple questions regarding the pathogenic IL-23/ThIL-17 pathway still remain to be answered. For example, it is not clear if and how schistosomes, particularly the highly immunopathogenic eggs, directly activate innate immunocytes or other potential cell sources of IL-23. A subject of considerable discussion is about these cells possessing dedicated receptors that recognize specific molecular patterns from schistosomes equivalent to the toll-like receptors (TLR), C-type lectins and other molecules that recognize microbial motifs (discussed in Hokke & Yazdanbakhsh 2005). Other questions concern the variation in IL-23 production by different innate immunocytes and the nature of possible additional mediators capable of stimulating ThIL-17 cells; this issue can be addressed by examination of schistosome-infected mice lacking the IL-23-specific p19 subunit. Finally, regarding the ThIL-17 cells themselves, it is not clear whether they all are of the CD4 type; moreover, their kinetics of IL-23R expression, precise costimulation requirements and full range of secreted pro-inflammatory mediators remain to be fully elucidated.
Research into the basis of the dissimilar immunopathology development in murine schistosomiasis has revealed a critical pathogenic role of a novel pro-inflammatory CD4 T cell subset producing IL-17 (ThIL-17), which is specifically detected in situations of high pathology, either induced or genetically determined. Clearly, the ThIL-17 cells are stimulated by the heterodimeric cytokine IL-23, for which they possess specific receptors. The origin of IL-23 and other possible activating signals is ascribed to innate immunocytes, although the precise pathogen-derived stimuli are not known. ThIL-17 cells have been shown to participate in an increasing number of host responses stimulated by a variety of self antigens and pathogens (Infante-Duarte et al. 2000, Cua et al. 2003, Murphy et al. 2003, Lubberts et al. 2004, Happel et al. 2005, Khader et al. 2005, Langrish et al. 2005, Kleinschek et al. 2006, Mensah-Brown et al. 2006). It now is of great interest and importance to ascertain their presence and activity in human diseases including schistosomiasis.
Aggarwal S, Ghilardi N, Xie MH, de Sauvage FJ, Gurney AL 2003. Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17. J Biol Chem 278: 1910-1914.
Brunet LR, Finkelman FD, Cheever AW, Kopf MA, Pearce EJ 1997. IL-4 protects against TNF-alpha-mediated cachexia and death during acute schistosomiasis. J Immunol 159: 777-785.
Cheever A, Duvall R, Hallack Jr T, Minker R, Malley J, Malley K 1987. Variation of hepatic fibrosis and granuloma size among mouse strains infected with Schistosoma mansoni. Am J Trop Med Hyg 37: 85-97.
Cua DJ, Sherlock J, Chen Y, Murphy CA, Joyce B, Seymour B, Lucian L, To W, Kwan S, Churakova T, Zurawski S, Wiekowski M, Lira SA, Gorman D, Kastelein RA, Sedgwick JD 2003. Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421(6924): 744-748.
Happel KI, Dubin PJ, Zheng M, Ghilardi N, Lockhart C, Quinton LJ, Odden AR, Shellito JE, Bagby GJ, Nelson S, Kolls JK 2005. Divergent roles of IL-23 and IL-12 in host defense against Klebsiella pneumoniae. J Exp Med 202: 761-769.
Harrington LE, Hatton RD, Mangan PR, Turner H, Murphy TL, Murphy KM, Weaver CT 2005. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol 6: 1123-1132.
Herbert DR, Holscher C, Mohrs M, Arendse B, Schwegmann A, Radwanska M, Leeto M, Kirsch R, Hall P, Mossmann H, Claussen B, Forster I, Brombacher F 2004. Alternative macrophage activation is essential for survival during schistosomiasis and downmodulates T helper 1 responses and immunopathology. Immunity 20: 623-635.
Hernandez HJ, Sharpe AH, Stadecker MJ 1999. Experimental murine schistosomiasis in the absence of B7 costimulatory molecules: reversal of elicited T cell cytokine profile and partial inhibition of egg granuloma formation. J Immunol 162: 2884-2889.
Hernandez HJ, Wang Y, Stadecker MJ 1997. In infection with Schistosoma mansoni, B cells are required for T helper type 2 cell responses but not for granuloma formation. J Immunol 158: 4832-4837.
Hoffmann KF, Cheever AW, Wynn TA 2000. IL-10 and the dangers of immune polarization: excessive type 1 and type 2 cytokine responses induce distinct forms of lethal immunopathology in murine schistosomiasis. J Immunol 164: 6406-6416.
Hokke CH and Yazdanbakhsh M 2005. Schistosome glycans and innate immunity. Parasite Immunol 27: 257-264.
Holscher C 2004. The power of combinatorial immunology: IL-12 and IL-12-related dimeric cytokines in infectious diseases. Med Microbiol Immunol (Berl) 193: 1-17.
Infante-Duarte C, Horton HF, Byrne MC, Kamradt T 2000. Microbial lipopeptides induce the production of IL-17 in Th cells. J Immunol 165: 6107-6115.
Khader SA, Pearl JE, Sakamoto K, Gilmartin L, Bell GK, Jelley-Gibbs DM, Ghilardi N, deSauvage F, Cooper AM 2005. IL-23 compensates for the absence of IL-12p70 and is essential for the IL-17 response during tuberculosis but is dispensable for protection and antigen-specific IFN-gamma responses if IL-12p70 is available. J Immunol 175: 788-795.
King CL, Xianli J, June CH, Abe R and Lee KP 1996. CD28-deficient mice generate an impaired Th2 response to Schistosoma mansoni infection. Eur J Immunol 26: 2448-2455.
Kleinschek MA, Muller U, Brodie SJ, Stenzel W, Kohler G, Blumenschein WM, Straubinger RK, McClanahan T, Kastelein RA, Alber G 2006. IL-23 enhances the inflammatory cell response in Cryptococcus neoformans infection and induces a cytokine pattern distinct from IL-12. J Immunol 176: 1098-1106.
Kolls JK, Linden A 2004. Interleukin-17 family members and inflammation. Immunity 21: 467-476.
Langrish CL, Chen Y, Blumenschein WM, Mattson J, Basham B, Sedgwick JD, McClanahan T, Kastelein RA, Cua DJ 2005. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J Exp Med 201: 233-240.
Langrish CL, McKenzie BS, Wilson NJ, de Waal Malefyt R, Kastelein RA, Cua DJ 2004. IL-12 and IL-23: master regulators of innate and adaptive immunity. Immunol Rev 202: 96-105.
Lubberts E, Koenders MI, Oppers-Walgreen B, van den Bersselaar L, Coenen-de Roo CJ, Joosten LA, van den Berg WB 2004. Treatment with a neutralizing anti-murine interleukin-17 antibody after the onset of collagen-induced arthritis reduces joint inflammation, cartilage destruction, and bone erosion. Arthritis Rheum 50: 650-659.
MacDonald AS, Patton EA, La Flamme AC, Araujo MI, Huxtable CR, Bauman B, Pearce EJ 2002. Impaired Th2 development and increased mortality during Schistosoma mansoni Infection in the absence of CD40/CD154 interaction. J Immunol 168: 4643-4649.
McKenzie BS, Kastelein RA, Cua DJ 2006. Understanding the IL-23-IL-17 immune pathway. Trends Immunol 27: 17-23.
Mensah-Brown EP, Shahin A, Al-Shamisi M, Wei X and Lukic ML 2006. IL-23 leads to diabetes induction after subdia-betogenic treatment with multiple low doses of streptozotocin. Eur J Immunol 36: 216-223.
Murphy CA, Langrish CL, Chen Y, Blumenschein W, McClanahan T, Kastelein RA, Sedgwick JD, Cua DJ 2003. Divergent pro- and antiinflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation. J Exp Med 198: 1951-1957.
Oppmann B, Lesley R, Blom B, Timans JC, Xu Y, Hunte B, Vega F, Yu N, Wang J, Singh K, Zonin F, Vaisberg E, Churakova T, Liu M, Gorman D, Wagner J, Zurawski S, Liu Y, Abrams JS, Moore KW, Rennick D, de Waal-Malefyt R, Hannum C, Bazan JF, Kastelein RA 2000. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 13: 715-725.
Parham C, Chirica M, Timans J, Vaisberg E, Travis M, Cheung J, Pflanz S, Zhang R, Singh KP, Vega F, To W, Wagner J, O'Farrell AM, McClanahan T, Zurawski S, Hannum C, Gorman D, Rennick DM, Kastelein RA, de Waal Malefyt R, Moore KW 2002. A receptor for the heterodimeric cytokine IL-23 is composed of IL-12Rbeta1 and a novel cytokine receptor subunit, IL-23R. J Immunol 168: 5699-5708.
Park H, Li Z, Yang XO, Chang SH, Nurieva R, Wang YH, Wang Y, Hood L, Zhu Z, Tian Q, Dong C 2005. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol 6: 1133-1141.
Rutitzky LI, Hernandez HJ, Stadecker MJ 2001. Th1-polarizing immunization with egg antigens correlates with severe exacerbation of immunopathology and death in schistosome infection. Proc Natl Acad Sci USA 98: 13243-13248.
Rutitzky LI, Mirkin GA, Stadecker MJ 2003a. Apoptosis by neglect of CD4+ Th cells in granulomas: a novel effector mechanism involved in the control of egg-induced immunopathology in murine schistosomiasis. J Immunol 171: 1859-1867.
Rutitzky LI, Ozkaynak E, Rottman JB, Stadecker MJ 2003b. Disruption of the ICOS-B7RP-1 costimulatory pathway leads to enhanced hepatic immunopathology and increased gamma interferon production by CD4 T cells in murine schistosomiasis. Infect Immun 71: 4040-4044.
Rutitzky LI, Hernandez HJ, Yim YS, Ricklan DE, Finger E, Mohan C, Peter I, Wakeland EK, Stadecker MJ 2005a. Enhanced egg-induced immunopathology correlates with high IFN-gamma in murine schistosomiasis: identification of two epistatic genetic intervals. J Immunol 174: 435-440.
Rutitzky LI, Lopes da Rosa JR, Stadecker MJ 2005b. Severe CD4 T cell-mediated immunopathology in murine schistosomiasis is dependent on IL-12p40 and correlates with high levels of IL-17. J Immunol 175: 3920-3926.
Sadler CH, Rutitzky LI, Stadecker MJ, Wilson RA 2003. IL-10 is crucial for the transition from acute to chronic disease state during infection of mice with Schistosoma mansoni. Eur J Immunol 33: 880-888.
Szabo SJ, Sullivan BM, Peng SL, Glimcher LH 2003. Molecular mechanisms regulating Th1 immune responses. Annu Rev Immunol 21: 713-758.
Trinchieri G 1998. Interleukin-12: a cytokine at the interface of inflammation and immunity. Adv Immunol 70: 83-243.
Trinchieri G 2003. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol 3: 133-146.
Trinchieri G, Pflanz S, Kastelein RA 2003. The IL-12 family of heterodimeric cytokines: new players in the regulation of T cell responses. Immunity 19: 641-644.
Zhu J, Yamane H, Cote-Sierra J, Guo L, Paul WE 2006. GATA-3 promotes Th2 responses through three different mechanisms: induction of Th2 cytokine production, selective growth of Th2 cells and inhibition of Th1 cell-specific factors. Cell Res 16: 3-10.