Spontaneously developing chronic colitis in IL-10/iNOS double-deficient mice

Mice deficient in both inducible nitric oxide synthase (iNOS) and interleukin (IL)-10 (iNOS(-/-)/IL-10(-/-)) were created to examine the role of iNOS in spontaneously developing intestinal inflammation. IL-10(-/-)/iNOS(-/-) mice were compared with IL-10(-/-) (iNOS(+/+)) littermates over 6 mo. RT-PCR, Western blot analysis, and immunohistochemistry were performed to measure iNOS message and protein levels. Plasma nitrate/nitrite (NO(x)) levels were assessed by HPLC. Damage scores (macroscopic and microscopic) and granulocyte infiltration were assessed. At 3-4 wk, IL-10(-/-) and IL-10(-/-)/iNOS(-/-) mice had no signs of colonic inflammation or granulocyte infiltration. Plasma NO(x) levels were not different from controls. By 3-4 mo, IL-10(-/-) mice had increased damage scores and granulocyte infiltration concurrent with increased mRNA and protein synthesis (restricted to the epithelium) for iNOS in intestinal tissues but not other tissues. Plasma NO(x) levels increased fivefold. Interestingly, in the absence of iNOS induction or increased plasma NO(x), iNOS(-/-)/IL-10(-/-) mice had damage and granulocyte infiltration equivalent to those observed in IL-10(-/-) littermates. These data suggest that iNOS does not impact on the development or severity of spontaneous chronic inflammation in IL-10-deficient mice.     

Autoantigen-specific IL-10-transduced T cells suppress chronic arthritis by promoting the endogenous regulatory IL-10 response

Deficient T cell regulation can be mechanistically associated with development of chronic autoimmune diseases. Therefore, combining the regulatory properties of IL-10 and the specificity of autoreactive CD4(+) T cells through adoptive cellular gene transfer of IL-10 via autoantigen-specific CD4(+) T cells seems an attractive approach to correct such deficient T cell regulation that avoids the risks of nonspecific immunosuppressive drugs. In this study, we studied how cartilage proteoglycan-specific CD4(+) T cells transduced with an active IL-10 gene (T(IL-10)) may contribute to the amelioration of chronic and progressive proteoglycan-induced arthritis in BALB/c mice. TCR-transgenic proteoglycan-specific T(IL-10) cells ameliorated arthritis, whereas T(IL-10) cells with specificity for OVA had no effect, showing the impact of Ag-specific targeting of inflammation. Furthermore, proteoglycan-specific T(IL-10) cells suppressed autoreactive proinflammatory T and B cells, as T(IL-10) cells caused a reduced expression of IL-2, TNF-alpha, and IL-17 and a diminished proteoglycan-specific IgG2a Ab response. Moreover, proteoglycan-specific T(IL-10) cells promoted IL-10 expression in recipients but did not ameliorate arthritis in IL-10-deficient mice, indicating that T(IL-10) cells suppress inflammation by propagating the endogenous regulatory IL-10 response in treated recipients. This is the first demonstration that such targeted suppression of proinflammatory lymphocyte responses in chronic autoimmunity by IL-10-transduced T cells specific for a natural Ag can occur via the endogenous regulatory IL-10 response.     

C5a controls TLR-induced IL-10 and IL-12 production independent of phosphoinositide 3-kinase

The complement system is a classic central player in innate immunity. Most pathogens activate both complement and the toll-like receptor (TLR) pathway. Therefore, to provide a more comprehensive understanding of innate immunity, it is important to understand the crosstalk between these two systems. Mouse macrophages produce IL-12 and IL-10 in response to TLR ligands such as LPS, CpG, Poly I:C and Malp2. The TLR-induced IL-12 production was decreased, while that of IL-10 was increased by concurrent stimulation with a complement fragment C5a. Pharmacological studies have suggested that C5a regulates TLR4-induced IL-12 production in a phosphoinositide 3-kinase (PI3K)-dependent mechanism. In the present study, however, we found that the C5a-mediated changes can be observed in macrophages from mice lacking PI3K p85α or PI3K p110γ. The result indicates that the C5a action is PI3K-independent; neither class IA nor class IB PI3K subtype is involved in this regulation. The actions of C5a were sensitive to pertussis toxin and PD98059, suggesting a role of G protein-mediated activation of the Erk1/2 pathway.     

IL-10 and IL-12 are the main regulatory cytokines in visceral leishmaniasis

Visceral leishmaniasis (VL) is characterized by the absence of cytokines such as IFN-gamma and IL-12. Cure of VL is associated with a restoration of the ability to make these cytokines. The aim of the present study was to evaluate the role of IL-12 in the recovery of the ability to produce IFN-gamma and to test whether or not IL-4 IL-10 and/or TGF-beta could suppress IFN-gamma production by PBMC from treated VL patients. High stimulation index (SI) of proliferation was observed in PBMC from subjects stimulated with Leishmania chagasi antigen (181+/-83). Neutralizing IL-12 inhibited lymphoproliferation [stimulation index (SI) of 210+/-114 to 1+/-0.6 (P<0.01)] and/or the production of IFN-gamma [2792+/-402 pg/ml to 407+/-449 pg/ml (P<0.01)]. Recombinant IL-10 abrogated the lymphoproliferation (SI=2+/-3) while recombinant IL-4 or TGF-beta had no effect on this response (147+/-22 and 194+/-12 respectively). IFN-gamma was high when PBMCs were stimulated with L. chagasi (873+/-400 pg/ml) and this was abrogated by the addition of IL-10 (5+/-2 pg/ml). In contrast neither IL-4 or TGF-beta suppressed IFN-gamma production (837+/-244 pg/ml and 759+/-523 pg/ml). These results indicate that IL-12 plays an important role in the ability of treated VL patients to make IFN-gamma and that IL-10 but not IL-4 or TGF-beta inhibits this response.     

IL-4 and IL-10 are both required for the induction of oral tolerance

Protection from the development of experimental autoimmune uveitis (EAU) can be induced by feeding mice interphotoreceptor retinoid binding protein before uveitogenic challenge with the same protein. Two different regimens are equally effective in inducing protective tolerance, although they seem to do so through different mechanisms: one involving regulatory cytokines (IL-4, IL-10, and TGF-beta), and the other with minimal involvement of cytokines. Here we studied the importance of IL-4 and IL-10 for the development of oral tolerance using mice genetically engineered to lack either one or both of these cytokines. In these animals we were able to protect against EAU only through the regimen inducing cytokine-independent tolerance. When these animals were fed a regimen that in the wild-type animal is thought to predominantly induce regulatory cells and is associated with cytokine secretion, they were not protected from EAU. Interestingly, both regimens were associated with reduced IL-2 production and proliferation in response to interphotoreceptor retinoid binding protein. These findings indicate that both IL-4 and IL-10 are required for induction of protective oral tolerance dependent on regulatory cytokines, and that one cytokine cannot substitute for the other in this process. These data also underscore the fact that oral tolerance, manifested as suppression of proliferation and IL-2 production, is not synonymous with protection from disease.     

IL-12 and neutralization of endogenous IL-10 revert the in vitro antigen-specific cellular immunosuppression of paracoccidioidomycosis patients

Treatment of patients with paracoccidioidomycosis is still a challenge. Patients present defective lymphoproliferation and IFN-gamma responses to the main Paracoccidioides brasiliensis antigen (gp43), which correlates with disease severity. Here, we demonstrated that the patients show also a defective synthesis of interleukin (IL)-12. Therefore, we attempted to revert this immune disfunction by adding IL-12 and neutralizing anti-IL-10 antibody to gp-43-stimulated peripheral blood mononuclear cell cultures. Both treatments increased IFN-gamma secretion to levels observed with healthy sensitized individuals, but affected proliferation only modestly. When combined, the treatments further increased IFN-gamma synthesis and cell proliferation. The addition of suboptimal concentrations of IL-2 also further increased the IL-12-mediated secretion of IFN-gamma. Interestingly, the immune modulation was mostly antigen-specific, since the responses to Candida albicans' antigen were not affected. These results suggest that appropriate immune intervention with cytokines and/or anti-cytokines may help in the treatment of PCM.     

IL-27 and IL-21 are associated with T cell IL-10 responses in human visceral leishmaniasis

IL-10 is believed to underlie many of the immunologic defects in human visceral leishmaniasis (VL). We have identified CD4(+)CD25(-)Foxp3(-) T cells as the major source of IL-10 in the VL spleen. IL-27, a member of the IL-6/IL-12 cytokine family, has been shown to promote development of IL-10-producing T cells, in part by upregulating their production of autocrine IL-21. We investigated whether IL-27 and IL-21 are associated with human VL. IL-27 was elevated in VL plasma, and at pretreatment, spleen cells showed significantly elevated mRNA levels of both IL-27 subunits, IL-27p28 and EBI-3, as well as IL-21, compared with posttreatment biopsies. CD14(+) spleen cells were the main source of IL-27 mRNA, whereas CD3(+) T cells were the main source of IL-21. IL-27 mRNA could be strongly upregulated in normal donor macrophages with IFN-γ and IL-1β, conditions consistent with those in the VL spleen. Last, a whole-blood assay revealed that most VL patients could produce Ag-specific IFN-γ and IL-10 and that the IL-10 could be augmented with recombinant human IL-21. Thus, proinflammatory cytokines acting on macrophages in the VL spleen have the potential to upregulate IL-27, which in turn can induce IL-21 to expand IL-10-producing T cells as a mechanism of feedback control.     

IL-1alpha and IL-1beta are endogenous mediators linking cell injury to the adaptive alloimmune response

Preoperative or perioperative ischemic injury of allografts predisposes to graft arteriosclerosis, the major cause of late graft failure. We hypothesize that injured tissues release mediators that increase the production of pathogenic cytokines by alloreactive T cells. We find that freeze-thaw lysates of human endothelial cells (EC) increase both IFN-gamma and IL-17 production by human CD4(+) T cells activated by HLA-DR(+) allogeneic EC. Immunoadsorption of high-mobility group box 1 protein (HMGB1) reduces this activity in the lysates by about one-third, and recombinant HMGB1 increases T cell cytokine production. HMGB1 acts by inducing IL-1beta secretion from contaminating monocytes via TLR4 and CD14. Upon removal of contaminating monocytes, the remaining stimulatory activity of EC lysates is largely attributable to IL-1alpha. Recombinant IL-1 directly augments IFN-gamma and IL-17 production by activated memory CD4(+) T cells, which express IL-1R1. Furthermore, IL-1 increases the frequency of alloreactive memory CD4(+) T cells that produce IL-17, but not those that produce IFN-gamma, in secondary cultures. Our results suggest that IL-1, released by injured EC or by HMGB1-stimulated monocytes, is a key link between injury and enhanced alloimmunity, offering a new therapeutic target for preventing late graft failure.     

Antigen-induced IL-10+ regulatory T cells are independent of CD25+ regulatory cells for their growth, differentiation, and function

Recent studies have emphasized the importance of T cells with regulatory/suppressor properties in controlling autoimmune diseases. A number of different types of regulatory T cells have been described with the best characterized being the CD25(+) population. In addition, it has been shown that regulatory T cells can be induced by specific Ag administration. In this study, we investigate the relationship between peptide-induced, CD4(+) regulatory T cells and naturally occurring CD4(+)CD25(+) cells derived from the Tg4 TCR-transgenic mouse. Peptide-induced cells were FoxP3(-) and responded to Ag by secreting IL-10, whereas CD25(+) cells failed to secrete this cytokine. Both cell types were able to suppress the proliferation of naive lymphocytes in vitro although with distinct activation sensitivities. Depletion of CD25(+) cells did not affect the suppressive properties of peptide-induced regulators. Furthermore, peptide-induced regulatory/suppressor T cells could be generated in RAG(-/-), TCR-transgenic mice that do not spontaneously generate CD25(+) regulatory cells. These results demonstrate that these natural and induced regulatory cells fall into distinct subsets.     

Haplotypes of IL-10 promoter variants are associated with susceptibility to severe malarial anemia and functional changes in IL-10 production

Plasmodium falciparum malaria is one of the leading global causes of morbidity and mortality with African children bearing the highest disease burden. Among the various severe disease sequelae common to falciparum malaria, severe malarial anemia (SMA) in pediatric populations accounts for the greatest degree of mortality. Although the patho-physiological basis of SMA remains unclear, dysregulation in inflammatory mediators, such as interleukin (IL)-10, appear to play an important role in determining disease outcomes. Since polymorphic variability in innate immune response genes conditions susceptibility to malaria, the relationship between common IL-10 promoter variants (−1,082A/G, −819T/C, and −592A/C), SMA (Hb < 6.0 g/dL), and circulating inflammatory mediator levels (i.e., IL-10, TNF-α, IL-6 and IL-12) were investigated in parasitemic Kenyan children (n = 375) in a holoendemic P. falciparum transmission area. Multivariate logistic regression analyses demonstrated that the −1,082G/−819C/−592C (GCC) haplotype was associated with protection against SMA (OR; 0.68, 95% CI, 0.43–1.05; P = 0.044) and increased IL-10 production (P = 0.029). Although none of the other haplotypes were significantly associated with susceptibility to SMA, individuals with the −1,082A/−819T/−592A (ATA) haplotype had an increased risk of SMA and reduced circulating IL-10 levels (P = 0.042). Additional results revealed that the IL-10:TNF-α ratio was higher in the GCC group (P = 0.024) and lower in individuals with the ATA haplotype (P = 0.034), while the IL-10:IL-12 ratio was higher in ATA haplotype (P = 0.006). Results presented here demonstrate that common IL-10 promoter haplotypes condition susceptibility to SMA and functional changes in circulating IL-10, TNF-α, and IL-12 levels in children with falciparum malaria. The study was approved by the ethical and scientific review committees at the Kenya Medical Research Institute and the institutional review board at the University of Pittsburgh.     

Dendritic cells post-maturation are reprogrammed with heightened IFN-gamma and IL-10

Mature dendritic cells (mDCs) undergo "exhaustion" in producing cytokines. Nevertheless, whether this "exhaustion" of mDCs is selective to certain cytokines, or whether mDCs have specific cytokine-producing profiles has yet to be defined. Herein, we investigated the cytokine production in vitro by immature DCs (iDCs) and LPS-induced mDCs. Compared to iDCs, mDCs produced comparable levels of IL-6 and TNF-alpha. Strikingly, mDCs produced significantly higher IFN-gamma and IL-10. IL-12 production of mDCs was suppressed. Kinetic studies of the responses of iDCs and mDCs to LPS or CD40L showed that mDCs acquired progressively heightened activity in producing IFN-gamma and IL-10. TNF-alpha-, IL-6-producing capability of mDCs was maintained. Nevertheless, IL-12 production by mDCs was not recovered at any time point. Mature DCs were potent in priming both Th1 and Th2 cells. In conclusion, upon maturation, DCs are reprogrammed with a distinct cytokine-secreting profile, which may play an important role in regulating T cell functions.     

Antifungal type 1 responses are upregulated in IL-10-deficient mice

C57BL/6 mice are highly resistant to infections caused by Candida albicans and Aspergillus fumigatus. To elucidate the role of IL-10 produced by C57BL/6 mice during these infections, parameters of infection and immunity to it were evaluated in IL-10-deficient and wild-type mice with disseminated or gastrointestinal candidiasis or invasive pulmonary aspergillosis. Unlike parasitic protozoan infection, C. albicans or A. fumigatus infection did not induce significant acute toxicity in IL-10-deficient mice, who, instead, showed reduced fungal burden and fungal-associated inflammatory responses. The increased resistance to infections as compared to wild-type mice was associated with upregulation of innate and acquired antifungal Th1 responses, such as a dramatically higher production of IL-12, nitric oxide (NO) and TNF-alpha as well as IFN-gamma by CD4+ T cells. Pharmacological inhibition of NO production greatly reduced resistance to gastrointestinal candidiasis, thus pointing to the importance of IL-10-dependent NO regulation at mucosal sites in fungal infections. These results are reminiscent of those obtained in genetically susceptible mice, in which IL-10 administration increased, and IL-10 neutralization decreased, susceptibility to C. albicans and A. fumigatus infections. Collectively, these observations indicate that the absence of IL-10 augments innate and acquired antifungal immunity by upregulating type 1 cytokine responses. The resulting protective Th1 responses lead to a prompt reduction of fungal growth, thus preventing tissue destruction and lethal levels of proinflammatory cytokines.     

Mechanisms of peripheral tolerance following intracameral inoculation are independent of IL-13 or STAT6

The peripheral tolerance that is elicited by the anterior chamber-associated immune deviation (ACAID) protocol is characterized by impairment of Th1 responses such as delayed-type hypersensitivity. It has been proposed that suppression of Th1 responses is mediated by a deviation toward Th2 responses. Because NKT cells have a prominent role in ACAID and NKT cell-derived IL-13 is required in a tumor model of tolerance, we postulated that NKT cell-derived Th2 cytokines might have a role in ACAID. However, contrary to the tumor model, in this study we show that NKT cells from IL-13-deficient mice or IL-4/IL-13 double deficient mice were able to reconstitute the capability of J alpha18-deficient mice (lacking invariant NKT) to develop peripheral tolerance postintracameral inoculation of Ag. Also, we were able to induce peripheral tolerance directly in IL-13-deficient, IL-4/IL-13-double deficient, and STAT6-deficient mice by inoculation of Ag into their eye. We conclude that neither IL-4 nor IL-13 cytokines are required for the generation of efferent CD8+ T regulatory cells during eye-induced peripheral tolerance. We propose that Ags inoculated into the anterior chamber of the eye induce the immunoresponse to deviate from producing immune T effector cells to producing efferent T regulatory cells, rather than deviating from Th1- to Th2-type effector cells.