The Role And Mechanism Of Th17/IL-17in The Development Of Hepatic Granuloma Of Mice Infected With Schistosoma Japonicum

Schistosomiasis is a serious public health and social development burden. China is oneof the countries most affected by schistosomiasis japonica, with more than360thousandpeople currently infected by the end of2009. Thus, it remains a major parasitic disease,which seriously affects our people's health and the economic and social development.The main pathogenesis of schistosomiasis is primarily attributed to egg-induced granulo-matous inflammatory response. CD4~+T-helper (Th) cells are essential for granulomaformation, both T helper cell type1(Th1) and T helper cell type2(Th2) are importantimmune cells involved in the development of granuloma. Schistosomiasis manifestationin the initial stages of infection is generally considered to be a Th1dominant response.But there is a shift from a moderate Th1to a robust Th2-dominated response with theonset of egg laying. And Th1cell-associated cytokines are closely related with vigorousgranuloma, whereas, Th2response play an important role in chronic granulomatousinflammation and immune modulation.Th17lineage discovered recently is a new subset of effector CD4~+T cells, also referredto inflammatory T help cell (Thi), which plays an active role in various chronic tissueinflammation and cell-mediated autoimmune disease. A combination of the immuno -regulatory cytokine transforming growth factorβ (TGF-β) and the proinflammatory andpleiotropic cytokine interleukin-6(IL-6) is required to drive the initial commitment ofnaive T cells to the Th17phenotype. The key transcription factors involved in thedevelopment of Th17were shown to be signal trasducer and activator of transcription3(STAT3), retinoic acid-related orphan receptor γt (RORγt) and RORα. The interleukin-12(IL-12) family member interleukin-23(IL-23) was not involved in the initialdifferentiation of Th17cells, IL-23signaling, however, is essential for their survival andexpansion. IL-17is the signature cytokine of Th17cells; it has been classified as aproinflammatory cytokine because of its ability to induce the expression of manymediators of inflammation, most strikingly, those involved in the proliferation,maturation and chemotaxis of neutrophils. IL-17can stimulated the production ofadditional cytokines (TNF-α, IL-1, IL-6, G-CSF and GM-CSF), and chemokines(CXCL1, CXCL2, IL-8, CCL2and CCL7) associated with inflammation, throughengagement of IL-17receptor nearly ubiquitously expressed. Furthermore, IL-17canacts in synergy with either TNF-α or IL-6to enhance inflammation.Researchers found that either interferon gamma (IFN-γ) or interleukin-4(IL-4)negatively regulates TH17differentiation. Given the elevated levels of IFN-γ and IL-4at different times, IL-17production thus is likely under tight controled during infectionwith S. japonicum. Maybe, this is the reason why the contribution of Th17/IL-17duringschistosoma infection only recently was uncovered.Rutitzky et al. reported that Schistosoma mansoni-infected C57BL/6mice developedsmaller granulomas than the C3H strains. After immunization with soluble egg antigen(SEA) in complete Freund's adjuvant (CFA), the C57BL/6mice displayed a dramaticincrease in hepatic granuloma size and exacerbation of inflammation. Analysis ofgranuloma and MLN lymphocytes from the SEA/CFA-immunized mice revealed thatlarger granulomas and extensive pathology correlating with elevated IL-17levels. Neutralization of IL-17in vivo resulted in a significant reduction of hepaticinflammation. The same group showed that IL-23p19/mice immunized with SEA/CFA have reduced severe immunopathology, which is associated with a reduction ofIL-17in granulomas and with a decrease in neutrophil recruitment due to reducedchemokine levels. Thus, IL-23critically contributes to the exacerbated immunopathology.The pathogenic mechanisms of S. japonicum are generally considered to be similar tothose of S. mansoni. But greater pathological intensity ascribed to S. japonicuminfection, as S. japonicum egg output is approximately10times that of S. mansonia,often clustered deposition in the organization. In the experimental C57BL/6murinemodel of schistosomiasis, S. japonicum infected-mice naturally develop vigoroushepatic granulomatous inflammation without immunization, whereas in S. mansoniinfected-mice the lesions are significantly smaller. Studying the role of Th17/IL-17innatural infection with S. japonicum larvae model of C57BL/6mice, the impact ofimmunization can be excluded, and more truly reflect the contribution of Th17/IL-17toegg-induced granuloma. Furthermore, previous studies showed that there weredifferences in host response to the same cytokine regulation after infection with S.japonicum or S. mansoni respectively. To date, the role of Th17/IL-17in S. japonicumegg granuloma has been little defined. Therefore, investigation of the role ofTh17/IL-17in granuloma response will highlight our understanding of the immunemechanisms involved in granuloma lesions and may lead to novel treatments for controlgranulom immunopathology.Our study included the following three parts: Fist, enhanced expression of IL-17parallelled the development of granulomatous inflammation in the livers of S.japonicum-infected mice; secondly, the cell sources of IL-17and distribution of IL-17receptor in S. japonicum-infected C57BL/6mice; thirdly, the effect and mechanism ofIL-17-neutralizing in S. japonicum-infected C57BL/6mic. Enhanced expression of IL-17parallelled the development of granulomatousinflammation in the livers of S. japonicum-infected mice.C57BL/6mice were infected with20cercariae of S. japonicum and sacrificed biweeklyafter infection for a total of12weeks. Mesenteric lymph node (MLN) and spleen cellswere stimulated with SEA (50μg/ml) for48h. Cytokines IL-17, IFN-γand IL-4levelswere measured in the culture supernatants by enzyme-linked immunosorbent assay(ELISA). IL-17, IFN-γ, IL-4, IL-21, IL-6, IL-23and TGF-β mRNA and chemokinessuch as CXCL1, CXCL2, CCL11and MCP-1in the livers were detected by Real-timereverse transcription quantitave polymerase chain reaction (RT-qPCR). Histopathologic-al presentation of livers were examined dynamicly by HE staining. Livers injury wasassessed by serum aspartate aminotransferase (AST) and alanine aminotransferase(ALT) levels detected by Lai's method.IL-17protein can be detected in the local granulomatous inflammation, and draininglymph nodes and spleen cell culture supernatant. IL-17mRNA were increased from4weeks postinfection (the time of onset of egg deposition), peaked at6weeks andmaintained relatively at high levels till8weeks. The overlapping of IL-17productionand vigorous granuloma development during the infection may suggest a possiblecorrelation between the two phenomena. Egg deposition may be a major factorresponsible for the increased IL-17production. Simultaneously, the dynamic expressionof the factors associated with Th17and effector molecules downstream of IL-17in thelivers also paralleled granuloma inflammation, which provided potential evidence forTh17/IL-17involved in regulating granuloma.The cell sources of IL-17and distribution of IL-17receptor in S. japonicum-infected C57BL/6mice C57BL/6mice were infected with20cercariae of S. japonicum and sacrificed at6-8week after infection. Granuloma cells, MLN cells and spleen cells were isolatedaseptically. Bulk cell suspensions from hepatic granulomas and MLNs, or spleens wereincubated in the presence of SEA, PMA, ionomycin and BFA. CD4~+T producing IL-17were detected by flow cytometry. Our results showed that IL-17-expressing CD4~+Tcells rarely in spleen cells (<0.1%), with low frequency in MLN cells and granulomcells (0.12±0.03%,0.21±0.036%, respectively), far lower than the expression ofIFN-γ and IL-4in CD4~+T cells. However, IL-17-expressing CD4~+T cells from MLNcells and liver granuloma cells increased significantly (0.41±0.05%,4.79±0.55%),when the medium supplemented with IL-23(100ng/ml), which had no effect on spleencells. These data showed that IL-23was essential for Th17cells survival and thesecretion of IL-17, and that Th17cells are a minor fraction of infiltrating CD4~+T withinthe granuloma.Isolated liver granuloma cells were labeled by FITC-anti-Ly-6G/PE-anti-IL-17andFITC-anti-F4/80/PE-anti-IL-17. A small amount of IL-17-expressing macrophages andneutrophils were detected by laser scanning confocal microscope.Immunostaining with anti-IL-17recepotor (IL-17R) antibodies, IL-17R positive cellswere detected using liver tissue sections. The positive cells were observed around thegranulomatous lesions, most of which may be fibroblasts according to the spindlephenotype. The precise cell sources of IL-17R remain to be identified.The effect and mechanism of IL-17-neutralizing in S. japonicum-infected C57BL/6mice.Many reports have identified the key role of Th17/IL-17in mediating autoimmunediseases. Increased levels of IL-17have been detected both in inflamed tissue and in mononuclear cells isolated from peripheral blood from patients with Multiple Sclerosis(MS), rheumatoid arthritis (RA) as well as in animal models, respectively. Furtheermore,the levels of IL-17had a significant correlation with the extent of disease.Neutralization of endogenous IL-17after the onset of EAE, with IL-17monoclonalantibody or immunization of ovalbumin connected autologous significantly reduces theseverity and shorted the course of disease. To further ascertain the role of Th17/IL-17inthe development of egg-induced granulomas, we treated mice with IL-17-neutralizingmAb or isotype control mAb starting on day24post infection, and continuing everyfour days. After a total of5injections, the mice were sacrificed on day42p.i.. Theeffect on granulomatous inflammation and liver injury were observed after beingneutralized of IL-17. TO investigate the cellular and molecular mechanisms underlyingthese effects, HE staining, RT-qPCR, ELISA, FACS and immunohistochemistry wereused.Our results showed that anti-IL-17mAb, but not the control mAb, significantlyimproved hepatic gross appearance. Morphometric analysis showed that the averagegranuloma size in neutralizing anti-IL-17treated mice reduced significantly comparedwith the control mice. Accordingly, anti-IL-17mAb-treated mice displayed attenuatedliver injury, as indicated by a considerable decrease in serum aspartate aminotransferase(AST) and alanine aminotransferase (ALT) levels. We analyzed egg burden in theindividual livers. No relevant differences could be observed between the two groups.Insignificant differences were also observed in organ weights and parasite burden. IL-6,CXCL1and CXCL2mRNA and protein were significant lower in anti-IL-17treatedmice, and no difference in the production of TNF-α, MCP-1and CCL11in the livers ofbetween the two group mice. Conversely, there was a higher amount of IL-10producedin most neutralizing anti-IL-17treated mice. Flow cytometry was conducted ongranuloma cells from both anti-IL-17mAb and control IgG treated mice. Our data didnot reveal obvious differences in the percentages of CD19+(B cells), CD3+(T cells), butthe accumulation of CD11b+Ly-6G+cells in granuloma of the neutralizing anti-IL-17 treat-ed mice were significantly impaired (7.69±0.77×10~6vs11.19±1.76×10~6, P<0.05), F4/80+(macrophages) increased markedly (6.59±0.38×10~6vs5.60±0.39×10~6,P <0.01). Immunohistochemically, quantitation of MPO positive area, which isexpressed in neutrophils and secreted during their activation, revealed significantlylower numbers of neutrophils in anti IL17-treated mice compared with similarlychallenged control IgG-treated littermates (6.96±2.0%vs3.86±1.08%, P<0.05),which agreed with the FACS results. IFN-γ and IL-4produced by SEA stimulated cellsfrom hepatic granulomas, mesenteric lymph nodes (MLN) and spleens were determined.Granuloma cells from anti-IL-17treated mice secreted significantly lower amounts ofIFN-γ and IL-4compared to the mice which were provided the isotype control IgG. Thelower amount of IFN-γ was more pronounced than that of IL-4. The same change ofcytokine production by SEA-stimulated MLN and spleen cells were also found, but thedifference was not statistically significant. Thus, antibody-mediated IL-17neutraliz-ation attenuated the acute egg-induced hepatic immunopathology and hepatocytenecrosis, which accompanied by reduced release of pro-inflammatory cytokines,chemokines and impaired neutrophiles infiltration. Inhibiting the Th1/Th2response byincreased IL-10may be another mechanism responsible for alleviated granulomatousinflammation.In conclusion, we first delined the dynamic expression of IL-17and IL-17associatedmolecules during the course of hepatic granulomatous inflammation in S. japonicum-infected C57BL/6mice. The levels of IL-17had a significant correlation with theseverity of the liver granulomatous inflammation. CD4~+T cells, macrophages andneutrophils produce IL-17in mice infected with S. japonicum,thereby inducinginflammatory cytokines and chemokines for recruitment of inflammatory cells toinflamed sites, which contribute to granulomatous inflammation. IL-17receptor wasexpressed mainly by fibroblasts surrounding granuloma.Our data suggested that Th17cells can be induced specificly in S. japonicum-infected C57BL/6mice, in addition to Th1, Th2cells. Th17, as well as Th1and Th2cells arealso important effector CD4~+T cells, involved in pathogenesis of egg-inducedgranulomatous inflammation. Although Th17cells are a minor fraction of infiltratingCD4~+T within the granuloma, IL-17might be a potential player in the cytokine networkregulating egg-induced immunopathology in S. japonicum infected C57BL/6mice. Inaddition to inducing proinflammatory cytokines/chemokines production and recruitingneutrophiles, IL-17, maybe, promotes egg-induced granuloma inflammation via anIFN-γ and/or IL-4-dependent mechanism, just as in autoimmune diabetes and acutekidney injury (ischemia-reperfusion injury). Dynamic description of IL-17, expandedthe law of Th cytokine in murine schistosomiasis, and enriched the mechanism ofimmune regulation in schistosome granuloma development. Targeting Th17/IL-17mayrepresent a powerful new approach to curtailing and treating egg-induced immunopath-ology.