Interleukin-27R (WSX-1/T-Cell Cytokine Receptor) Gene-Deficient Mice Display Enhanced Resistance to Leishmania donovani Infection but Develop Severe Liver Immunopathology
2006; Elsevier BV; Volume: 168; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2006.050013
ISSN1525-2191
AutoresLucia E. Rosas, Anjali A. Satoskar, Kimberly M. Roth, Tracy L. Keiser, Joseph Barbi, Christopher A. Hunter, Frédéric J. de Sauvage, Abhay R. Satoskar,
Tópico(s)Urticaria and Related Conditions
ResumoThe interleukin-27 (IL-27)/T-cell cytokine receptor (TCCR) pathway plays an important role in development of protective immunity against cutaneous leishmaniasis caused by Leishmania major. In this study, we analyzed the role of IL-27/TCCR pathway in the host defense against visceral leishmaniasis (VL) by monitoring the course of L. donovani infection in TCCR-deficient C57BL/6 (TCCR−/−) mice. TCCR−/− mice mounted a robust inflammatory response, produced high levels of pro-inflammatory cytokines, and developed severe liver pathology after L. donovani infection that eventually resolved. Interestingly, L. donovani-infected TCCR−/− mice controlled the parasite growth in their organs significantly faster than similarly infected TCCR+/+ mice. Adoptive cell transfer and cell depletion studies revealed that CD4+ T cells were involved in mediating liver immunopathology and controlling L. donovani growth in TCCR−/− mice. These results indicate that the IL-27/TCCR pathway is not essential for the induction of protective Th1 response during VL but is involved in mediating susceptibility to L. donovani. Additionally, the data demonstrate that although the IL-27/TCCR interaction limits the severity of liver inflammation during VL by controlling CD4+ T-cell activity, it is not required for the resolution of hepatic immunopathology. The interleukin-27 (IL-27)/T-cell cytokine receptor (TCCR) pathway plays an important role in development of protective immunity against cutaneous leishmaniasis caused by Leishmania major. In this study, we analyzed the role of IL-27/TCCR pathway in the host defense against visceral leishmaniasis (VL) by monitoring the course of L. donovani infection in TCCR-deficient C57BL/6 (TCCR−/−) mice. TCCR−/− mice mounted a robust inflammatory response, produced high levels of pro-inflammatory cytokines, and developed severe liver pathology after L. donovani infection that eventually resolved. Interestingly, L. donovani-infected TCCR−/− mice controlled the parasite growth in their organs significantly faster than similarly infected TCCR+/+ mice. Adoptive cell transfer and cell depletion studies revealed that CD4+ T cells were involved in mediating liver immunopathology and controlling L. donovani growth in TCCR−/− mice. These results indicate that the IL-27/TCCR pathway is not essential for the induction of protective Th1 response during VL but is involved in mediating susceptibility to L. donovani. Additionally, the data demonstrate that although the IL-27/TCCR interaction limits the severity of liver inflammation during VL by controlling CD4+ T-cell activity, it is not required for the resolution of hepatic immunopathology. Visceral leishmaniasis (VL) is the most severe form of Leishmania infection caused by Leishmania donovani and Leishmania chagasi.1Alexander J Satoskar AR Russell DG Leishmania species: models of intracellular parasitism.J Cell Sci. 1999; 112: 2993-3002Crossref PubMed Google Scholar VL is characterized by hepatosplenomegaly, fever, abdominal pain, and weight loss associated with widespread dissemination of parasites throughout the reticuloendothelial system. Complications such as secondary infection, anemia, and malnutrition due to hepatosplenomegaly are primarily responsible for the mortality associated with this disease. Interleukin (IL)-27 is a heterodimeric cytokine produced by endothelial cells, dendritic cells, and monocytes during inflammation.2Villarino AV Huang E Hunter CA Understanding the pro- and anti-inflammatory properties of IL-27.J Immunol. 2004; 173: 715-720PubMed Google Scholar It is composed of the interleukin-12 p40- and p35-related proteins EBI3 and p28, respectively.3Pflanz S Timans JC Cheung J Rosales R Kanzler H Gilbert J Hibbert L Churakova T Travis M Vaisberg E Blumenschein WM Mattson JD Wagner JL To W Zurawski S McClanahan TK Gorman DM Bazan JF de Waal MR Rennick D Kastelein RA IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4(+) T cells.Immunity. 2002; 16: 779-790Abstract Full Text Full Text PDF PubMed Scopus (1156) Google Scholar IL-27 mediates its functions by signaling through the IL-27R, which is composed of the T-cell cytokine receptor (TCCR/WSX-1) and the signal transducer gp130.4Sprecher CA Grant FJ Baumgartner JW Presnell SR Schrader SK Yamagiwa T Whitmore TE O'Hara PJ Foster DF Cloning and characterization of a novel class I cytokine receptor.Biochem Biophys Res Commun. 1998; 246: 82-90Crossref PubMed Scopus (117) Google Scholar, 5Pflanz S Hibbert L Mattson J Rosales R Vaisberg E Bazan JF Phillips JH McClanahan TK de Waal MR Kastelein RA WSX-1 and glycoprotein 130 constitute a signal-transducing receptor for IL-27.J Immunol. 2004; 172: 2225-2231PubMed Google Scholar, 6Yoshida H Hamano S Senaldi G Covey T Faggioni R Mu S Xia M Wakeham AC Nishina H Potter J Saris CJ Mak TW WSX-1 is required for the initiation of Th1 responses and resistance to L. major infection.Immunity. 2001; 15: 569-578Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar Both immune and nonimmune cells express gp130, which is a shared receptor signaling chain used by several related cytokines.7Taga T Kishimoto T Gp130 and the interleukin-6 family of cytokines.Annu Rev Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1291) Google Scholar However, the expression of WSX-1 is restricted to cells of the immune system such as monocytes, Langerhans cells, dendritic cells, NK cells, T cells, and B cells.4Sprecher CA Grant FJ Baumgartner JW Presnell SR Schrader SK Yamagiwa T Whitmore TE O'Hara PJ Foster DF Cloning and characterization of a novel class I cytokine receptor.Biochem Biophys Res Commun. 1998; 246: 82-90Crossref PubMed Scopus (117) Google Scholar, 5Pflanz S Hibbert L Mattson J Rosales R Vaisberg E Bazan JF Phillips JH McClanahan TK de Waal MR Kastelein RA WSX-1 and glycoprotein 130 constitute a signal-transducing receptor for IL-27.J Immunol. 2004; 172: 2225-2231PubMed Google Scholar, 6Yoshida H Hamano S Senaldi G Covey T Faggioni R Mu S Xia M Wakeham AC Nishina H Potter J Saris CJ Mak TW WSX-1 is required for the initiation of Th1 responses and resistance to L. major infection.Immunity. 2001; 15: 569-578Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 8Chen Q Ghilardi N Wang H Baker T Xie MH Gurney A Grewal IS de Sauvage FJ Development of Th1-type immune responses requires the type I cytokine receptor TCCR.Nature. 2000; 407: 916-920Crossref PubMed Scopus (336) Google Scholar The role of IL-27 in regulating the host immune response is complex because this cytokine has been shown to exert both pro- and anti-inflammatory activities.2Villarino AV Huang E Hunter CA Understanding the pro- and anti-inflammatory properties of IL-27.J Immunol. 2004; 173: 715-720PubMed Google Scholar For example, like IL-12, IL-27 induces interferon (IFN)-γ production from NK and CD4+ T cells.3Pflanz S Timans JC Cheung J Rosales R Kanzler H Gilbert J Hibbert L Churakova T Travis M Vaisberg E Blumenschein WM Mattson JD Wagner JL To W Zurawski S McClanahan TK Gorman DM Bazan JF de Waal MR Rennick D Kastelein RA IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4(+) T cells.Immunity. 2002; 16: 779-790Abstract Full Text Full Text PDF PubMed Scopus (1156) Google Scholar TCCR (WSX-1)−/− mice, which completely lack IL-27 signaling, fail to mount an efficient Th1-type response and thus show increased susceptibility to intracellular pathogens such as Listeria monocytogenes and Leishmania major.6Yoshida H Hamano S Senaldi G Covey T Faggioni R Mu S Xia M Wakeham AC Nishina H Potter J Saris CJ Mak TW WSX-1 is required for the initiation of Th1 responses and resistance to L. major infection.Immunity. 2001; 15: 569-578Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 9Artis D Johnson LM Joyce K Saris C Villarino A Hunter CA Scott P Cutting edge: early IL-4 production governs the requirement for IL-27-WSX-1 signaling in the development of protective Th1 cytokine responses following Leishmania major infection.J Immunol. 2004; 172: 4672-4675PubMed Google Scholar Furthermore, IL-27 is also expressed at high levels in human Th1-associated granulomatous diseases such as tuberculosis, sarcoidosis, and Crohn's disease.10Larousserie F Pflanz S Coulomb-L'Hermine A Brousse N Kastelein R Devergne O Expression of IL-27 in human Th1-associated granulomatous diseases.J Pathol. 2004; 202: 164-171Crossref PubMed Scopus (115) Google Scholar On the other hand, WSX-1−/− mice produce high levels of IFN-γ and develop significant immunopathology when infected with Toxoplasma gondii and Trypanosoma cruzi, indicating that IL-27 is also involved in antagonizing or modulating inflammatory responses in these diseases.11Villarino A Hibbert L Lieberman L Wilson E Mak T Yoshida H Kastelein RA Saris C Hunter CA The IL-27R (WSX-1) is required to suppress T cell hyperactivity during infection.Immunity. 2003; 19: 645-655Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 12Hamano S Himeno K Miyazaki Y Ishii K Yamanaka A Takeda A Zhang M Hisaeda H Mak TW Yoshimura A Yoshida H WSX-1 is required for resistance to Trypanosoma cruzi infection by regulation of proinflammatory cytokine production.Immunity. 2003; 19: 657-667Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar The protective role of IL-27 in control of cutaneous L. major infection has been attributed to its ability to enhance IFN-γ production from both NK cells and CD4+ T cells during the early phase of infection and subsequently to generate disease protective Th1 response.6Yoshida H Hamano S Senaldi G Covey T Faggioni R Mu S Xia M Wakeham AC Nishina H Potter J Saris CJ Mak TW WSX-1 is required for the initiation of Th1 responses and resistance to L. major infection.Immunity. 2001; 15: 569-578Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 9Artis D Johnson LM Joyce K Saris C Villarino A Hunter CA Scott P Cutting edge: early IL-4 production governs the requirement for IL-27-WSX-1 signaling in the development of protective Th1 cytokine responses following Leishmania major infection.J Immunol. 2004; 172: 4672-4675PubMed Google Scholar However, a recent study has shown that early IL-4 production governs the requirement for IL-27 in the induction of Th1 responses in cutaneous leishmaniasis caused by L. major.9Artis D Johnson LM Joyce K Saris C Villarino A Hunter CA Scott P Cutting edge: early IL-4 production governs the requirement for IL-27-WSX-1 signaling in the development of protective Th1 cytokine responses following Leishmania major infection.J Immunol. 2004; 172: 4672-4675PubMed Google Scholar Interestingly, IL-27 is not required to main-tain Th1 response during L. major infection because TCCR−/− mice develop large lesions during the early phase of infection but eventually resolve the infection and control parasite loads.6Yoshida H Hamano S Senaldi G Covey T Faggioni R Mu S Xia M Wakeham AC Nishina H Potter J Saris CJ Mak TW WSX-1 is required for the initiation of Th1 responses and resistance to L. major infection.Immunity. 2001; 15: 569-578Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 9Artis D Johnson LM Joyce K Saris C Villarino A Hunter CA Scott P Cutting edge: early IL-4 production governs the requirement for IL-27-WSX-1 signaling in the development of protective Th1 cytokine responses following Leishmania major infection.J Immunol. 2004; 172: 4672-4675PubMed Google Scholar To determine the role of the IL-27/TCCR (WSX-1) pathway in the outcome of VL, we examined the course of L. donovani infection in TCCR−/− C57BL/6 mice and compared it with that in the wild-type (TCCR+/+) C57BL/6 mice. TCCR−/− mice mounted a robust Th1 response after L. donovani infection and controlled parasite burdens significantly faster when compared with TCCR+/+ mice. However, rapid clearance of L. donovani in TCCR−/− mice was associated with the development of significant liver pathology accompanied by diffuse inflammation and aberrant granuloma formation. Depletion of CD4+ T cells in TCCR−/− mice dramatically reduced liver pathology after L. donovani infection but rendered them more susceptible to the disease. Conversely, syngenic RAG2−/− recipient mice reconstituted with TCCR−/− T cells developed severe liver inflammation and rapidly controlled parasite growth in their organs. Finally, simultaneous neutralization of both tumor necrosis factor (TNF)-α and IFN-γ also reduced severity of hepatic pathology in TCCR−/− mice during VL but enhanced their susceptibility to infection. These findings demonstrate that the IL-27/TCCR pathway, in contrast to its protective role in L. major infection, is involved in the pathogenesis of L. donovani infection. The results also indicate that the IL-27/TCCR pathway suppresses the tissue inflammation associated with VL by regulating CD4+ T-cell function. TCCR (WSX-1) gene-deficient C57BL/6 mice were generated as described previously8Chen Q Ghilardi N Wang H Baker T Xie MH Gurney A Grewal IS de Sauvage FJ Development of Th1-type immune responses requires the type I cytokine receptor TCCR.Nature. 2000; 407: 916-920Crossref PubMed Scopus (336) Google Scholar and bred by mating homozygous parents. The deletion of TCCR in offspring was confirmed by genotypic analysis using PCR and by phenotypic analysis using flow cytometry. The wild-type C57BL/6 mice were purchased from Jackson Laboratories. The experiments were performed using 8- to 10-week-old sex-matched mice in a facility at The Ohio State University according to the guidelines for animal research as required by National Institutes of Health regulations. Under these guidelines, mice were bred and maintained in ventilated microisolator cages in a specific pathogen-free facility. Any mice showing signs of distress were immediately sacrificed. Animal care facilities at the Ohio State University are fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care-International and follow the National Research Council's Guide for the Care and Use of Laboratory Animals. Deletion of TCCR gene in the offspring was confirmed by genotypic analysis using PCR. Primer sequences used for amplifying the wild-type allele were sense, 5′GTTGAGATGCAGAACCTGGA-3′, and antisense, 5′-GCTGCTGATAAGTTCCCAAG-3′, and for mutated allele were sense, 5′-GCTTTCGTCTCCCGTGTGCT-3′, and antisense, 5′-TGAGCCCAGAAAGCGAAGGA-3′. The primers for detecting wild-type mice were designed to amplify a fragment in targeted region (exon 3 to exon 8) as described previously and hence were specific for the wild-type allele.8Chen Q Ghilardi N Wang H Baker T Xie MH Gurney A Grewal IS de Sauvage FJ Development of Th1-type immune responses requires the type I cytokine receptor TCCR.Nature. 2000; 407: 916-920Crossref PubMed Scopus (336) Google Scholar A primer set specific for Neo gene in the targeting vector was used for detecting mutated allele.8Chen Q Ghilardi N Wang H Baker T Xie MH Gurney A Grewal IS de Sauvage FJ Development of Th1-type immune responses requires the type I cytokine receptor TCCR.Nature. 2000; 407: 916-920Crossref PubMed Scopus (336) Google Scholar Additionally, flow cytometric analysis using rat anti-mouse TCCR monoclonal antibody (mAb) was performed to demonstrate the absence of TCCR on T cells from TCCR−/− mice. Anti-mouse TCCR mAb was kindly provided by Dr. Fred de Sauvage (Genentech, San Francisco, CA). L. donovani (1 Sudan strain) was maintained by serial passage of amastigotes in Syrian golden hamster. Amastigotes were isolated from the spleen of an infected hamster, and experimental mice were infected with 1 × 107 L. donovani amastigotes by intravenous injection into the tail vein. Parasite loads in the liver and spleen of L. donovani-infected TCCR−/− and TCCR+/+ mice were measured on 15, 30, and 60 days after infection as described previously.13Satoskar A Bluethmann H Alexander J Disruption of the murine interleukin-4 gene inhibits disease progression during Leishmania mexicana infection but does not increase control of Leishmania donovani infection.Infect Immun. 1995; 63: 4894-4899PubMed Google Scholar Briefly, livers and spleens were weighed and sectioned to prepare impression smears that were Giemsa stained to enumerate the amastigotes per 1000 nucleated cells. The parasite loads were expressed as Leishman-Donovan units (LDU):LDU=Number of amastigotes per1000nucleated cells×Organ weight(g) At this time, tissue sections from livers and spleens of L. donovani-infected TCCR−/− and TCCR+/+ mice were excised and fixed in 10% buffered formalin for 48 hours. The tissues were processed and embedded in paraffin, and 5-μm sections were cut. The sections were stained by routine hematoxylin and eosin staining. The slides were coded and analyzed to assess the inflammation. Special staining was performed to analyze collagen deposition (Masson Trichrome) and reticulin fibers (reticulin). For immunofluroscence microscopy, liver tissue was snap frozen in Tissue Tek OCT and cut into 5-μm sections using a cryostat (Leica Systems). Tissue sections were fixed in ice-cold acetone for 1 minute and blocked at room temperature for 30 minutes using protein-free block (DAKO). Subsequently, sections were incubated with rat anti-mouse CD11b (clone M1/70; BD Pharmingen, San Diego, CA) or rat anti-mouse CD4 (clone L3T4; BD Pharmingen, San Diego, CA) antibody at room temperature for 2 hours. Bound primary antibody was detected using biotin-labeled secondary Ab (BD Pharmingen, San Diego, CA) and streptavidin conjugated to appropriate fluorescent dye (e. g. AlexaFluor 594 (red) and AlexaFluor 488 (green); Molecular Probes). The spleens were removed from L. donovani-infected TCCR+/+ and TCCR−/− mice on days 15, 30, and 60 after infection. Single cell suspensions were prepared by gentle teasing of the spleen, and live cells were enumerated by trypan blue exclusion after lysing the red blood cells using Boyle's solution. Cells (5 × 105) were added in quadruplicate to the wells of sterile 96-well flat-bottom tissue culture plates and stimulated with freeze-thaw Leishmania antigen (LdAg) (20 μg/ml). LdAg was prepared from stationary phase promastigotes of L. donovani. Promastigotes were washed three times in ice-cold phosphate-buffered saline and subjected to six cycles of freezing −70°C and thawing at 37°C. Freeze-thawed preparations were centrifuged to remove the particulate material, and protein concentration was determined using Bradford's assay as per manufacturer's instructions (Pierce Lab, Rockford, IL). The proliferation responses were measured by Alamar Blue assay as described previously.14Ahmed SA Gogal Jr, RM Walsh JE A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay.J Immunol Methods. 1994; 170: 211-224Crossref PubMed Scopus (1171) Google Scholar, 15Rosas LE Barbi J Lu B Fujiwara Y Gerard C Sanders VM Satoskar AR CXCR3−/− mice mount an efficient Th1 response but fail to control Leishmania major infection.Eur J Immunol. 2005; 35: 515-523Crossref PubMed Scopus (51) Google Scholar Supernatants were collected after 72 hours of incubation at 37°C and analyzed for the production of IFN-γ, IL-12p70, IL-4, and IL-10 by enzyme-linked immunosorbent assay (ELISA). Reagents used for cytokine ELISA were all purchased from BD PharMingen, San Diego, CA. Spleens were removed aseptically from naïve TCCR+/+ and TCCR−/− donor mice, and single cell suspensions were prepared by gentle teasing in cold RPMI1640 supplemented with 10% fetal calf serum. Red blood cells were lysed using Boyle's solution, and cells were run through a nylon wool column twice to remove B cells. Depletion of B cells was confirmed by staining for B220+ cells using flow cytometry. For depletion of NK and natural killer T cells (NKT cells) B cell-depleted spleen cells were labeled using fluorescein isothiocyanate-conjugated anti-NK1.1 Ab (BD Pharmingen, San Diego, CA) and depleted by immunomagnetic separation using anti-fluorescein isothiocyanate Ab-coupled magnetic beads according to manufacturer's instruction (Miltyni Inc., Auburn, CA). Efficiency of depletion and purity of T-cell population was confirmed by flow cytometry. No NK1.1+ cells were detectable by flow cytometry after depletion, and 97% of the cells were T cells (data not shown). Subsequently, cells were washed twice and resuspended in sterile saline. Cells were enumerated by trypan blue exclusion, and 107 cells were injected intravenously via tail vein injections into recipient RAG2 knockout mice. The mice rested for 3 weeks after reconstitution before infection with parasites. TCCR−/− mice were administered 400 μg of anti-CD4 (clone GK1.5) or anti-CD8 (clone H35) or control IgG intraperitoneally 2 days before L. donovani infection followed by weekly single injection of the same dose thereafter. Efficiency of CD4+- and CD8+-cell depletion was nearly 90 to 92%, as determined by flow cytometry at the time of sacrifice (data not shown). TCCR−/− mice were administered intraperitoneally with 400 μg of anti-IFN-γ mAb (clone XMG.6) or 250 μg of affinity-purified anti-TNF-α rabbit polyclonal Ab (Calbiochem, EMD Biosciences Inc., La Jolla, CA) or a combination of both 24 hours before L. donovani infection and weekly thereafter. A group of TCCR−/− mice received control antibodies in the same doses. Student's unpaired t-test was used to determine statistical significance of differences in the values. A value of P < 0.05 was considered significant. The statistical significance of antibody titers was determined by nonparametric tests using the Mann-Whitney U-Wilcoxon rank test. As anticipated, PCR analysis of tail DNA revealed the presence of the TCCR gene in TCCR+/+ but not TCCR−/− mice (Figure 1A). Furthermore, flow cytometric analysis of T cells from TCCR−/− mice confirmed the absence of TCCR on these cells (Figure 1, B and C). On day 15 after intravenous inoculation with 1 × 107 L. donovani amastigotes, livers and spleens from TCCR−/− mice contained less parasites compared with those from TCCR+/+; however, the difference in parasite loads between the groups was only statistically significant for the liver (Figure 2). Liver and spleen parasite burdens increased in L. donovani-infected TCCR+/+ mice as the infection progressed and peaked at day 30 after infection (Figure 2). However, at 30 days, L. donovani-infected TCCR−/− mice showed a significant decrease in their liver parasite loads (Figure 2A). At this time, the spleens from TCCR−/− mice also contained fewer parasites compared with TCCR+/+ mice, but the difference was less profound when compared with the liver. Three replicate experiments showed that differences in spleen parasites loads were radically less profound compared with those in the liver (data not shown). By day 60 after infection, both TCCR+/+ and TCCR−/− mice efficiently controlled the growth of L. donovani in their liver and spleen, but parasite burdens in the liver were significantly lower in TCCR−/− mice (Figure 2A). These results demonstrate that the IL-27/TCCR pathway is not required for the development of host immunity to L. donovani but show that the IL-27/TCCR pathway delays the resolution of L. donovani infection and plays a role in pathogenesis of visceral leishmaniasis. On gross examination, livers from L. donovani-infected TCCR−/− mice were larger and more fragile when compared with those from TCCR+/+ mice (data not shown). On days 15 and 30 after infection, a microscopic examination of livers from L. donovani-infected TCCR+/+ showed tissue inflammation associated with the formation of mature granulomas comprised of macrophages and T cells, with clusters of amastigotes within individual macrophages (Figure 3). On the other hand, L. donovani-infected TCCR−/− mice showed extensive destruction of the liver parenchyma associated with the presence of large granulomas and many foci of a diffuse inflammatory infiltrate comprised of macrophages, neutrophils, and lymphocytes (Figure 3). Livers from TCCR−/− mice also displayed foci of necrosis and an extensive perivascular inflammatory infiltrate composed of macrophages, lymphocytes, and few plasma cells in both portal and peri-sinusoidal spaces. Immunohistochemical analysis revealed that diffuse inflammatory foci in the livers of TCCR−/− mice predominantly contained CD11b+ macrophages and CD4+ T cells (Figure 4). Both groups showed significant amounts of collagen in the liver granulomas on days 15 and 30 (Figure 5), but collagen deposition was more extensive in the livers from TCCR−/− mice (Figure 5, C and G). Similarly, livers from TCCR−/− mice had significantly more deposition of reticulin fibers at 15 and 30 days (Figure 5, D and H). Interestingly, L. donovani-infected TCCR−/− mice resolved liver inflammation more efficiently than wild-type mice and contained fewer granulomas in their livers on day 60 (Figure 3, I and K). These results demonstrate that the IL-27/TCCR pathway suppresses liver inflammation during L. donovani infection.Figure 4Immunohistochemical analysis of cell populations in livers from TCCR+/+ and TCCR−/− mice on day 30 after L. donovani infection. Cryostat cut sections from livers were stained using rat anti-mouse CD11b (A and B) or anti-mouse CD4 antibodies (C and D), and bound primary Ab was detected by appropriate secondary biotinylated Ab and streptavidin AlexaFluor 488 (green) or AlexaFluor 594 (red). Livers from TCCR+/+ mice showed well-organized granulomas composed of CD11b+ macrophages (A) and CD4+ T cells (C), whereas those from TCCR−/− mice displayed diffuse parenchymal infiltration by these cell populations (B and D).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Analysis of collagen and reticulin fiber deposition in livers from TCCR+/+ and TCCR−/− mice on days 15 (A, B, C, and D), 30 (E, F, G, and H), and 60 (I, J, K, and L) after L. donovani infection. Livers from TCCR−/− mice showed significantly more deposition of collagen (C and G) and reticulin fiber (D and H) on days 15 and 30 after infection compared with those from the wild-type mice (A and E (collagen); B and F (reticulin fibers)). At day 60 after infection, livers from TCCR−/− mice displayed less collagen and reticulin fiber deposition compared with TCCR+/+ mice (I, J, K, and L). Note that collagen is stained blue and that reticulin fibers, which stain black, are denoted by white arrows. Magnification, ×10.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The absence of IL-27/TCCR (WSX-1) pathway increases production of pro-inflammatory cytokines and induces systemic inflammation in TCCR−/− mice during certain infections.11Villarino A Hibbert L Lieberman L Wilson E Mak T Yoshida H Kastelein RA Saris C Hunter CA The IL-27R (WSX-1) is required to suppress T cell hyperactivity during infection.Immunity. 2003; 19: 645-655Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 12Hamano S Himeno K Miyazaki Y Ishii K Yamanaka A Takeda A Zhang M Hisaeda H Mak TW Yoshimura A Yoshida H WSX-1 is required for resistance to Trypanosoma cruzi infection by regulation of proinflammatory cytokine production.Immunity. 2003; 19: 657-667Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar Therefore, we measured levels of IL-12p70, TNF-α, and IFN-γ in sera of TCCR+/+ and TCCR−/− mice at different intervals after L. donovani infection. Because TCCR−/− mice efficiently resolve liver inflammation, we also determined the levels of anti-inflammatory cytokine IL-10 at these intervals. On day 15, L. donovani-infected TCCR−/− displayed significantly higher levels of IFN-γ and TNF-α in their serum compared with TCCR+/+ mice (Figure 6). Although both groups produced the same amounts of IL-12 on day 15 after infection, WSX-1−/− mice produced significantly more IL-12 on day 30. However, at this time point, no significant difference was noted in the serum levels of IFN-γ between the groups (Figure 6). By day 60, levels of IL-12, TNF-α, and IFN-γ dropped significantly in L. donovani-infected TCCR−/− mice. However, at the same time, sera from L. donovani-infected TCCR+/+ mice contained significantly more TNF-α. Throughout the course of infection, L. donovani-infected TCCR+/+ mice produced more IL-10 when compared with similarly infected TCCR−/− mice, but these differences were not statistically significant. None of these cytokines were detectable in sera from naïve TCCR+/+ or TCCR−/− mice. Together, these findings show that the IL-27/TCCR pathway controls the severity of VL-associated inflammation by negatively regulating production of pro-inflammatory cytokines. Because TCCR has been shown to suppress T-cell hyperactivity during infection with certain intracellular pathogens such as T. gondii, we compared LdAg-specific proliferation and cytokine production by spleen cells from L. donovani-infected TCCR+/+ and TCCR−/− mice. The spleen cells from L. donovani-infected TCCR+/+ and TCCR−/− mice proliferated efficiently in response to in vitro stimulation with LdAg on days 15 and 30 after infection, although the magnitude of proliferation was marginally higher in TCCR+/+ mice at day 30. Interestingly, on day 60 after infection, LdAg-stimulated spleen cells from TCCR−/− mice mounted a robust proliferation response (10- to 15-fold higher) compared with those from TCCR+/+ mice (data not shown). TCCR+/+ and TCCR−/− mice displayed significant levels of Leishmania-specific IgG1 and IgG2a antibo
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