In vivo diabetogenic action of CD4+ T lymphocytes requires Fas expression and is independent of IL-1 and IL-18

CD4(+) T lymphocytes are required to induce spontaneous autoimmune diabetes in the NOD mouse. Since pancreatic β cells upregulate Fas expression upon exposure to pro-inflammatory cytokines, we studied whether the diabetogenic action of CD4(+) T lymphocytes depends on Fas expression on target cells. We assayed the diabetogenic capacity of NOD spleen CD4(+) T lymphocytes when adoptively transferred into a NOD mouse model combining: (i) Fas-deficiency, (ii) FasL-deficiency, and (iii) SCID mutation. We found that CD4(+) T lymphocytes require Fas expression in the recipients' target cells to induce diabetes. IL-1β has been described as a key cytokine involved in Fas upregulation on mouse β cells. We addressed whether CD4(+) T cells require IL-1β to induce diabetes. We also studied spontaneous diabetes onset in NOD/IL-1 converting enzyme-deficient mice, in NOD/IL-1β-deficient mice, and CD4(+) T-cell adoptively transferred diabetes into NOD/SCID IL-1β-deficient mice. Neither IL-1β nor IL-18 are required for either spontaneous or CD4(+) T-cell adoptively transferred diabetes. We conclude that CD4(+) T-cell-mediated β-cell damage in autoimmune diabetes depends on Fas expression, but not on IL-1β unveiling the existing redundancy regarding the cytokines involved in Fas upregulation on NOD β cells in vivo.     

Blockage of tumor necrosis factor prevents intestinal mucosal inflammation through down-regulation of interleukin-23 secretion

Tumor necrosis factor (TNF) plays an important part in the pathogenesis of several inflammatory diseases. Its expression is highly increased in inflamed mucosa of inflammatory bowel disease, and anti-TNF treatment appears to improve mucosal inflammation in these patients. However, the role of TNF in the pathogenesis remains to be investigated. In the present study, an experimental colitis was induced by transfer of syngeneic CD45RB(high)CD4(+) T cells into SCID mice. Quantitative analysis of interleukin (IL)-23p19 and IL-17 mRNA was demonstrated to be markedly increased in inflamed colon 4 and 8 weeks after CD45RB(high)CD4(+) T-cell transfer. These SCID recipients were treated i.p. with anti-TNF mAb starting at the beginning (early treatment) or 4 weeks (delayed treatment) after T-cell transfer to investigate the in vivo relevance of TNF to the pathogenesis. The results demonstrated that early treatment with anti-TNF effectively prevented intestinal mucosal inflammation, as evidenced by gradual increase of body weight and absence of diarrhea. Anti-TNF significantly suppressed leukocyte infiltration in the inflamed colon, and down-regulated IFN-gamma, IL-2 and TNF secretion by lamina propria CD4(+) T cells. Interestingly, anti-TNF also significantly decreased expression of IL-23p19 and IL-17 in inflamed colon. Moreover, delayed anti-TNF treatment demonstrated to markedly improve mucosal inflammation. The data suggest that administration of anti-TNF reverses mucosal inflammation via down-regulated pro-inflammatory cytokines, particularly IL-23p19 and IL-17, and decreased leukocyte infiltration in the bowel, thus providing additional relevance of target therapy against TNF.     

Immunosenescent colitogenic CD4(+) T cells convert to regulatory cells and suppress colitis

Inflammatory bowel diseases progress steadily by the expansion of colitogenic CD4(+) cells. However, it remains unknown whether colitogenic CD4(+) cells are long-living like memory cells or exhausted like effector cells. To assess the longevity of colitogenic lamina propria (LP) CD4(+) cells, we performed sequential transfers of LP CD4(+) cells from colitic CD4(+)CD45RB(high) cell-transferred SCID mice into new SCID mice. Although SCID mice transferred with colitic LP CD4(+) cells stably developed colitis until at least the sixth transfer, the interval to the development of colitis gradually lengthened as the number of transfers increased. The incidence of colitis gradually decreased after the seventh transfer. Furthermore, non-colitic LP CD4(+) cells from mice transferred over seven times expressed significantly higher levels of PD-1 and produced significantly lower amounts of IFN-gamma, TNF-alpha, and IL-17 than colitic LP CD4(+) cells recovered after the first transfer. Most notably, we found that re-transfer of non-colitic LP CD4(+) cells recovered after multiple transfers prevented the development of colitis in SCID mice co-transferred with CD4(+)CD45RB(high) cells. Thus, colitogenic LP CD4(+) cells may be exhausted over time, become non-functional, convert to regulatory cells, and finally suppress colitis in the process of immunosenescence.     

CD8alpha+ dendritic cells enhance the antigen-specific CD4+ T-cell response and accelerate development of collagen-induced arthritis

To investigate the role of CD8alpha(+) DCs in the development of collagen-induced arthritis (CIA). The immunogenic properties of CD8alpha(+) and CD8alpha(-) DC subsets were investigated by mixed-lymphocyte reaction and cytokine enzyme-linked immunoassay. CII-pulsed CD8alpha(+) DCs or CD8alpha(-) DCs with CD4(+) T cells from CIA mice were adoptively transferred onto the hind footpad of DBA mice. The onset of arthritis and the arthritis index were examined for 14 weeks after adoptive transfer. Expression of MHC-II and CD80 but not CD86 and CD40 was higher in CD8alpha(+) DCs than in CD8alpha(-) DCs from the spleens of CIA mice. Culturing CD8alpha(+) DCs with CD4(+) T cells significantly increased the proliferative response of CD4(+) T cells in the presence of CII. The production of interleukin (IL)-12p70, IL-17, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha was slightly increased in CD8alpha(+) DCs than in CD8alpha(-) DCs. DBA/1 mice that were adoptively transferred with CII-pulsed CD8alpha(+) DCs and CD4(+) T cells into the footpads showed accelerated onset of CIA compared to control group. By contrast, CD8alpha(-) DCs showed a partial inhibitory effect on CIA. These findings show that CD8alpha(+) DCs accelerated the onset of CIA when aoptively transferred with CD4(+) T cells and that CD8alpha(+) DCs provoke the development of CIA probably by stimulating the immune responses of CII-reactive CD4(+) T cells and by increasing the production of inflammatory cytokines.     

Serotonin activates dendritic cell function in the context of gut inflammation

Mucosal inflammation in the gut is characterized by infiltration of innate and adaptive immune cells and by an alteration in serotonin-producing enterochromaffin cells. We investigated the role of serotonin in the function of dendritic cells (DCs) and sequential T-cell activation in relation to generation of gut inflammation. DCs isolated from tryptophan hydroxylase-1-deficient (TPH1(-/-)) mice, which have reduced serotonin in the gut, and wild-type (TPH1(+/+)) mice with or without dextran sulfate sodium (DSS)-induced colitis were stimulated with lipopolysaccharide to assess interleukin-12 (IL-12) production. Isolated DCs from TPH1(+/+) and TPH1(-/-) mice were also cocultured with CD4(+) T cells of naive TPH1(+/+) mice to assess the role of serotonin in priming T cells. In addition, serotonin-pulsed DCs were transferred to TPH1(-/-) mice to assess the effect on DSS-induced colitis. Consistent with a reduced severity of colitis, DCs from DSS-induced TPH1(-/-) mice produced less IL-12 compared with the TPH1(+/+) mice. In vitro serotonin stimulation restored the cytokine production from TPH1(-/-) DCs and adoptive transfer of serotonin-pulsed DCs into TPH1(-/-) up-regulated colitis. Furthermore, CD4(+) T cells primed by TPH1(-/-) DCs produce reduced the levels of IL-17 and interferon-γ. This study provides novel information on serotonin-mediated immune signaling and promotion of interactions between innate and adaptive immune responses in the context of gut inflammation, which may ultimately lead to improved strategies to combat gut inflammatory disorders.     

Influence of Mycobacterium avium subsp. paratuberculosis on colitis development and specific immune responses during disease

The granulomatous and intramural inflammation observed in cases of inflammatory bowel diseases (IBD) and veterinary Johne's disease suggests that Mycobacterium avium subsp. paratuberculosis is a causative agent. However, an incomplete understanding of the immunological steps responsible for the pathologies of IBD makes this conclusion uncertain. Sera from interleukin-10-deficient (IL-10(-/-)) mice with spontaneous colitis displayed significantly higher M. avium subsp. paratuberculosis-specific immunoglobulin G2a antibody responses than did sera from similar mice without disease. Pathogen-free IL-10(-/-) mice received control vehicle or the vehicle containing heat-killed or live M. avium subsp. paratuberculosis. Mucosal CD4(+) T cells from the mice that developed colitis proliferated and secreted higher levels of gamma interferon and tumor necrosis factor alpha after ex vivo stimulation with a Vbeta11(+) T-cell receptor-restricted peptide from the MPT59 antigen (Ag85B) than those secreted from cells from mice before the onset of colitis. The data from this study provide important information regarding the mechanisms of colitis in IL-10(-/-) mice, which are driven in part by Ag85B-specific T cells. The data suggest a plausible mechanism of Ag-specific T-cell responses in colitis driven by potent Ags conserved in Mycobacterium species.     

Dendritic cells generated in the presence of interferon-alpha stimulate allogeneic CD4+ T-cell proliferation: modulation by autocrine IL-10, enhanced T-cell apoptosis and T regulatory type 1 cells

Dendritic cells (DCs) generated in the presence of IFN-alpha (IFN-DCs) exhibit high expression of major histocompatibility and co-stimulatory molecules and a potent ability to stimulate CD8(+) T-cell responses. Here, we found that IFN-DCs were more potent stimulators of bulk and purified CD8(+) T-cell proliferation, as compared with IL-4-DCs. In contrast, IFN-DCs were less efficient than IL-4-DCs in stimulating allogeneic CD4(+) T-cell proliferation, due to a weak induction of naive CD4(+)CD45RO(-) T-cell proliferation by these DCs. However, both DC populations induced similar levels of proliferation of memory CD4(+)CD45RO(+) T cells. IFN-DCs and IL-4-DCs exhibited a similar phenotype and production of IL-10 following maturation induced by CD40 ligation. In contrast, IFN-DCs produced higher levels of IL-10 during the first days of differentiation. In addition, neutralization of IL-10 during the differentiation of DCs increased the expression of DC-LAMP and MHC class II by IFN-DCs, and the ability of IFN-DCs to stimulate allogeneic CD4(+) T-cell proliferation at similar levels, than IL-4-DCs. Independently of IL-10 production, IFN-DCs were found to induce higher levels of CD4(+)T-cell apoptosis, this effect being more sticking on naive T cells. Finally, we demonstrated that IFN-DCs induced a differentiation bias of naive CD4(+) T cells towards Th1 and Tr1 cells, compared to IL-4-DCs. Taken together, these results indicate that, despite the induction of Tr1 cells and enhanced apoptosis of naive CD4(+) T cells, IFN-DCs are potent stimulators of CD8(+) and memory CD4(+) T cells, and induce a strong polarization of naive CD4(+) T cells towards Th1 cells, further supporting their use in immune-based therapy.     

Alpha CD 2 mAb treatment safely attenuates adoptive transfer colitis

Increased proliferation, defective apoptosis, and cytokine dysregulation of T lymphocytes are thought to be important for the pathogenesis of inflammatory bowel disease. Since these phenomena can be corrected by alpha CD 2 mAb, we asked whether CD2 directed immunotherapy safely prevents and/or ameliorates adoptive transfer colitis. Colitis was induced by transfer of CD4(+) T cell blasts to syngenic RAG 1(-/-) mice or CD 45 RB(high) CD4(+) T cells to SCID mice. The alpha CD 2 mAb 12-15 or rat IgG was given, starting either initially or upon first signs of colitis. Disease activity was assessed by clinical monitoring, microscopic scoring, hemoccult, endoscopy, and blood count analysis. Cytokine production of stimulated LPL was measured by ELISA and cell proliferation by [(3)H]-thymidine incorporation. Parasite control was analyzed in a murine model of infection with Toxoplasma gondii. The alpha CD 2 mAb significantly increased mean survival time when starting at transfer of blasts (survival >35 days: alpha CD 2 69% vs 0% of controls, P<0.001). In the SCID colitis model hematochezia and macroscopic colitis were delayed. When used in established T-cell blast colitis, the benefit was less pronounced, even in combination with dexamethasone (mean survival+/-s.e.m.: alpha CD 2+dexa: 13.5+/-2.9 vs dexa+IgG: 6.3+/-1.0, P<0.05). In the preventive experiment the alpha CD 2 mAb markedly reduced IL-2 secretion and T-cell proliferation. The immune response towards Toxoplasma gondii was not impaired. These studies show for the first time that CD2 directed immunotherapy can attenuate or delay adoptive transfer colitis and ameliorate established colitis. Most likely inhibition of IL-2 secretion and T-cell proliferation are responsible for these effects. Still, immune defence towards T. gondii is maintained.     

A new model of chronic colitis in SCID mice induced by adoptive transfer of CD62L+ CD4+ T cells: insights into the regulatory role of interleukin-6 on apoptosis.

OBJECTIVE: The proinflammatory cytokine interleukin (IL)-6 is involved in various chronic inflammatory processes. IL-6 is a predominant cytokine produced by lamina propria T cells in Crohn's disease and experimental colitis. This study was designed to examine the effect of a neutralizing IL-6-receptor (IL-6R) antibody on the programmed cell death of mucosal T cells in the CD62L+ CD4+ SCID transfer model of chronic experimental colitis in mice and to gain more insight into the pathogenesis of this transfer colitis model. METHODS: For adoptive transfer, we isolated CD62L+ CD4+ double-positive T cells from wild-type BALB/c mice followed by intraperitoneal application of 1 million cells in CB17 SCID mice. The purity of the transferred T-cell population was tested by FACS analysis. Cytokine secretion was measured by ELISA. Western blot analysis was performed to detect phospho-STAT-3 in protein extracts of splenic cells. Cryo- and paraffin colon cross-sections were used to perform immunochemical or fluorescence TUNEL stainings. RESULTS: We isolated CD62L+ CD4+ and CD62L- CD4+ T cells. In vitro studies showed an increased production of IL-4 by CD62L- CD4+ T cells compared to CD62L+ T cells. 8-10 weeks after transfer of CD62L+ CD4+ T cells in SCID mice, reconstituted mice developed wasting disease, anal prolapse and diarrhea, whereas mice reconstituted with CD62L- CD4+ did not, similar to anti-IL-6R-treated CD62L+ CD4+-reconstituted SCID mice. Anti-IL-6R-treated reconstituted SCID mice showed decreased levels of activated STAT-3. The previously described efficacy of anti-IL-6R antibody treatment on colitis activity appeared to be due to the induction of apoptosis, as many TUNEL-positive cells were detected in the lamina propria. CONCLUSIONS: These results suggest that the activation of the IL-6/STAT-3-signaling pathway plays a pivotal role in the pathogenesis of CD62L+ CD4+ transfer colitis. Moreover, the application of a neutralizing antibody to IL-6R induces apoptosis in transferred T cells. These data implicate the importance of anti-apoptotic pathways in chronic disease and might contribute to future therapies.     

CD4+ Foxp3+ regulatory T cells suppress γδ T-cell effector functions in a model of T-cell-induced mucosal inflammation

CD4(+) CD25(+) Foxp3(+) regulatory T (T(REG) ) cells are critical mediators of peripheral immune tolerance, and abrogation of their function provokes a variety of autoimmune and inflammatory states including inflammatory bowel disease. In this study, we investigate the functional dynamics of T(REG) -cell responses in a CD4(+) T-cell-induced model of intestinal inflammation in αβ T-cell-deficient (TCR-β(-/-) ) hosts to gain insights into the mechanism and cellular targets of suppression in vivo. We show that CD4(+) T effector cell transfer into T-cell-deficient mice rapidly induces mucosal inflammation and colitis development, which is associated with prominent Th1 and Th17 responses. Interestingly, we unveil a prominent role for resident γδ T cells in mucosal inflammation as they promote Th1 and particularly Th17 responses in the early phase of inflammation, thus exacerbating colitis development. We further demonstrate that CD4(+) CD25(+) Foxp3(+) T(REG) cells readily inhibit these responses and mediate disease protection, which correlates with their accumulation in the draining LN and lamina propria. Moreover, T(REG) cells can directly suppress γδ T-cell expansion and cytokine production in vitro and in vivo, suggesting a pathogenic role of γδ T cells in intestinal inflammation. Thus, functional alterations in T(REG) cells provoke dysregulated CD4(+) and γδ T-cell responses to commensal antigens in the intestine.     

Enhanced IL-10 production by CD4+ T cells primed in IL-15Rα-deficient mice

In this study, we investigated the functional outcomes of CD4(+) T cells primed in the absence of IL-15 transpresentation. Compared with their WT counterparts primed in WT mice, IL-15Rα KO CD4(+) T cells primed in KO mice were found to exclusively overproduce IL-10 upon in vitro restimulation(.) The comparable expression of IL-4 and Foxp3 in CD4(+) T cells primed in the WT and IL-15Rα KO mice indicated that this was neither due to T(H) 2- nor Treg cell-differentiation. IL-10 overproduction was also observed when OVA-specific TCR transgenic CD4(+) T (OT-II) cells were primed in KO mice, excluding an intrinsic deficiency of KO CD4(+) T cells. To investigate the WT and KO microenvironment, DCs from both WT and IL-15Rα KO mice were compared. DCs from both backgrounds were indistinguishable in their steady-state survival and in their expression of MHC class II and costimulatory molecules CD80, CD86, and CD40. However, IL-15Rα KO DCs primed OT-II cells in vitro to produce higher levels of IL-10 upon their restimulation. Additionally, IL-15Rα KO DCs produced significantly more IL-10 upon activation, and IL-10 neutralization during DC-mediated in vitro priming abolished IL-10 overproduction by CD4(+) T cells. Thus, IL-15Rα KO DCs provide an IL-10-enriched environment that preferentially primes CD4(+) T cells for more IL-10 production, highlighting a regulatory role for IL-15 transpresentation in CD4(+) T-cell priming.     

Nitric oxide controls an inflammatory-like Ly6C(hi)PDCA1+ DC subset that regulates Th1 immune responses

Using NOS2 KO mice, we investigated the hypothesis that NO modulation of BM-DC contributes to the NO-mediated control of Th1 immune responses. BM-DCs from NOS2 KO mice, compared with WT BM-DCs, have enhanced survival and responsiveness to TLR agonists, develop more Ly6C(hi)PDCA1(+) DCs that resemble inflammatory DCs and produce high levels of inflammatory cytokines. Also, compared with WT-infected mice, NOS2 KO mice infected with WNV showed enhanced expansion of a similar inflammatory Ly6C(hi)PDCA1(+) DC subset. Furthermore, in contrast to WT DCs, OVA-loaded NOS2 KO BM-DCs promoted increased IFN-γ production by OTII CD4(+) T cells in vitro and when adoptively transferred in vivo. The addition of a NO donor to NOS2 KO BM-DCs prior to OTII T cells priming in vivo was sufficient to revert Th1 immune responses to levels induced by WT BM-DCs. Thus, autocrine NO effects on maturation of inflammatory DCs and on DC programming of T cells may contribute to the protective role of NO in autoimmune diseases and infections. Regulating NO levels may be a useful tool to shape beneficial immune responses for DC-based immunotherapy.     

Analysis of cytokine production in the colon of nude mice with experimental colitis induced by adoptive transfer of immunocompetent cells from mice infected with a murine retrovirus

LP-BM5 murine leukemia virus (MuLV) is known to induce murine AIDS (MAIDS). We have shown that Sj?gren's syndrome (SjS)-like exocrinopathy can be induced in mice with MAIDS and that adoptive transfer of spleen cells from MAIDS mice can induce inflammatory bowel disease-like colitis as well as SjS-like exocrinopathy in nude mice. To assess the role of interferon (IFN)-gamma and interleukin (IL)-10 in the pathogenesis of our experimental model, we tried to identify the cells producing these cytokines and their localization in the colitis lesions in situ. Expression of mRNA for IFN-gamma and IL-10 was assessed by RT-PCR, and protein expression of these cytokines was also analyzed in frozen sections of colon by double-color-staining immunofluorescence (IF). An increase of IFN-gamma and IL-10 mRNA was detected in the colon of mice with colitis, but not in that of control mice. Double-color IF showed that Mac-1(+) cells were positive for IFN-gamma or IL-10 and that most CD4(+) T cells were positive for IL-10, although the population of IFN-gamma-positive CD4(+) T cells was low. In our experimental colitis model, Mac-1(+) macrophages that produce both IFN-gamma and IL-10 might play a crucial role in the pathogenesis of colitis in combination with CD4(+) T cells.     

Adhesive substrate-modulation of adaptive immune responses

While it is well-known that adsorbed proteins on implanted biomaterials modulate inflammatory responses, modulation of dendritic cells (DCs) via adhesion-dependent signaling has only been begun to be characterized. In this work, we demonstrate that adhesive substrates elicit differential DC maturation and adaptive immune responses. We find that adhesive substrates support similar levels of DC adhesion and expression of stimulatory and co-stimulatory molecules. Conversely, DC morphology and differential production of pro- and anti-inflammatory cytokines (IL-12p40 and IL-10, respectively) is adhesive substrate-dependent. For example, DCs cultured on collagen and vitronectin substrates generate higher levels of IL-12p40, whereas DCs cultured on albumin and serum-coated tissue culture-treated substrates produce the higher levels of IL-10 compared to other substrates. Additionally, our results suggest substrate-dependent trends in DC-mediated allogeneic CD4(+) T-cell proliferation and T-helper cell type responses. Specifically, we show that substrate-dependent modulation of DC IL-12p40 cytokine production correlates with CD4(+) T-cell proliferation and T(h)1 type response in terms of IFN-gamma producing T-helper cells. Furthermore, our results suggest substrate-dependent trends in DC-mediated stimulation of IL-4 producing T-cells, but this T(h)2 type response is not dependent on DC production of IL-10 cytokine. This work has impact in the rational design of biomaterials for diverse applications such as tissue-engineered constructs, synthetic particle-based vaccines and the ex vivo culture of DCs for immunotherapies.     

CD4 T cells monospecific to ovalbumin produced by Escherichia coli can induce colitis upon transfer to BALB/c and SCID mice.

Although some animal models suggest an involvement of CD4 T cells reactive to luminal microbial antigen(s) for the pathogenesis of inflammatory bowel diseases (IBD), direct linkage between microflora-driven clonal expansion of CD4 T cells and the development of colitis has not been well studied. Here, BALB/c and SCID mice were given CD4 T cells purified from Rag-2(-/-) mice crossed to transgenic mice expressing TCR specific to ovalbumin (OVA) then administered with antibiotic-resistant Escherichia coli producing OVA (ECOVA) or LacZ (ECLacZ) via the rectum. The ECOVA-inoculated BALB/c and SCID mice developed a subacute colitis with microscopic features of distortion of crypt architecture, loss of goblet cells, and focal infiltration by mononuclear cells in the lamina propria (LP) and submucosa. Expanding OVA-specific CD4 T cells were detected in colonic follicles of mice with ECOVA. Early in colitis, OVA-specific CD4 T cells producing IFN-gamma predominate in the LP of the colon, which was followed by an emergence of OVA-specific CD4 T cells producing IL-4 and IL-10 at a later time point. Co-transfer of an IL-10-secreting OVA-specific CD4 T cell line prevented colitis. Thus, an expansion of CD4 T cells monospecific to OVA, an antigen non-cross-reactive to colonic tissue, can mediate both induction and inhibition of the colitis which was associated with hyperplasia of lymph follicles.     

Production of IFN-γ by CD4(+) T cells in response to malaria antigens is IL-2 dependent

T-cell immune responses are critical for protection of the host and for disease pathogenesis during infection with Plasmodium species. We examined the regulation of CD4(+) T-cell cytokine responses during infection with Plasmodium berghei ANKA (PbA). CD4(+) T cells from PbA-infected mice produced IFN-γ, IL-4 and IL-10 in response to TCR stimulation at levels higher than those from uninfected mice. This altered cytokine response was dependent on parasitemia. To examine the specificity of the response, mice were adoptively transferred with CD4(+) T cells from OT-II TCR transgenic mice and were infected with PbA expressing OVA. Unexpectedly, CD4(+) T cells from the OT-II-transferred wild-type PbA-infected mice showed high levels of IFN-γ production after stimulation with OVA and the cells producing IFN-γ were not OT-II but were host CD4(+) T cells. Further investigation revealed that host CD4(+) T cells produced IFN-γ in response to IL-2 produced by activated OT-II cells. This IFN-γ response was completely inhibited by anti-CD25 mAbs, and this effect was not due to the block of the survival signals provided by IL-2. Furthermore, IFN-γ production by CD4(+) T cells in response to PbA antigens was dependent on IL-2. These findings suggest the importance of IL-2 levels during infection with malaria parasites and indicate that CD4(+) T cells can produce IFN-γ without TCR engagement via a bystander mechanism in response to IL-2 produced by other activated CD4(+) T cells.     

TSLP-induced placental DC activation and IL-10(+) NK cell expansion: comparative study based on BALB/c x C57BL/6 and NOD/SCID x C57BL/6 pregnant models

Thymic stromal lymphopoietin (TSLP)-TSLP receptor (TSLP-R) interactions activate CD11c(+) dendritic cells (DCs) and increase epithelial cell Th2-type cytokine production. We detected intracellular TSLP expression on CK7(+) trophoblast cells and TSLP-R expression on placental DCs from pregnant BALB/cxC57BL/6 and NOD/SCIDxC57BL/6 mice on gestational day 12.5. Murine recombinant TSLP activated DCs from BALB/c mice, with increased CD80 and CD83 expressions; TSLP-activated DCs induced IL-10-producing NK cell expansion. This was abrogated by anti-TSLP Ab or by culturing CD49b(+) NK cells alone. No TSLP-DC-induced IL-10(+)CD49b(+) cell expansion occurred when DCs and CD49b(+) cells were cultured separately. Although TSLP-induced DC activation occurred in NOD/SCID mice, the IL-10(+) NK cell percentage was unchanged. CK7(+) trophoblast cells may activate placental DCs via a TSLP-TSLP-R interaction and induce DC-dependent placental NK cell IL-10 production. TSLP-DC and NK cell contact appears necessary for IL-10(+)CD49b(+) cell expansion. Placental NK cells from NOD/SCIDxC57BL/6 mice appear less sensitive to TSLP-DC induction.     

Induction of interleukin-10 in the T helper type 17 effector population by the G protein coupled estrogen receptor (GPER) agonist G-1

Interleukin-10 (IL-10) is a potent suppressor of the immune system, commonly produced by CD4(+) T cells to limit ongoing inflammatory responses minimizing host damage. Many autoimmune diseases are marked by large populations of activated CD4(+) T cells within the setting of chronic inflammation; therefore, drugs capable of inducing IL-10 production in CD4(+) T cells would be of great therapeutic value. Previous reports have shown that the small molecule G-1, an agonist of the membrane-bound G-protein-coupled estrogen receptor GPER, attenuates disease in an animal model of autoimmune encephalomyelitis. However, the direct effects of G-1 on CD4(+) T-cell populations remain unknown. Using ex vivo cultures of purified CD4(+) T cells, we show that G-1 elicits IL-10 expression in T helper type 17 (Th17) -polarized cells, increasing the number of IL-10(+) and IL-10(+) IL-17A(+) cells via de novo induction of IL-10. T-cell cultures differentiated in the presence of G-1 secreted threefold more IL-10, with no change in IL-17A, tumour necrosis factor-α, or interferon-γ. Moreover, inhibition of extracellular signal-regulated kinase (but not p38 or Jun N-terminal kinase) signalling blocked the response, while analysis of Foxp3 and RORγt expression demonstrated increased numbers of IL-10(+) cells in both the Th17 (RORγt(+)) and Foxp3(+) RORγt(+) hybrid T-cell compartments. Our findings translated in vivo as systemic treatment of male mice with G-1 led to increased IL-10 secretion from splenocytes following T-cell receptor cross-linking. These results demonstrate that G-1 acts directly on CD4(+) T cells, and to our knowledge provide the first example of a synthetic small molecule capable of eliciting IL-10 expression in Th17 or hybrid T-cell populations.     

CXCL10-producing mucosal CD4+ T cells, NK cells, and NKT cells are associated with chronic colitis in IL-10(-/-) mice, which can be abrogated by anti-CXCL10 antibody inhibition

We have shown previously that there is a temporal increase in the levels of CXCL10 and CXCR3 mRNA during spontaneous murine colitis. We now show that CXCL10 is significantly expressed by mucosal CD4+ T cells, natural killer (NK) cells, and NKT cells, but not by dendritic cells (DCs), during chronic murine colitis. CXCL10 blockade alleviated chronic colitis and attenuated the associated increase in serum amyloid A (SAA), interleukin-12 (IL-12)p40, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), IL-1 alpha, and IL-1 beta levels as well as in the number of CD4+ T, NKT, and NK cells that express CXCL10 and CXCR3, compared with groups treated with control antibody (Ab). After CXCL10 blockade, the number of CXCR3+ DCs in the mesenteric lymph nodes (MLNs) and Peyer's patches (PPs) were increased to levels found before the onset of colitis. In contrast, the numbers of splenic and intestinal lamina propria (LP) CXCR3+ DCs were reduced after anti-CXCL10 Ab treatment, compared with controls. Ex vivo antigen and CXCL10 stimulation of mucosal cells caused an increase in MHC class II, CD40, and CD86 as well as a decrease in CD30 ligand (CD30L) expression by DCs. This study provides insights into CXCL10 expression during inflammatory bowel disease (IBD) and the cellular and molecular mechanisms of CXCL10-mediated colitis. Our data also support the premise that CXCL10 blockade can attenuate chronic colitis by preventing the activation and recruitment of CXCR3+ leukocytes during IBD.     

Role of Stat4-mediated signal transduction events in the generation of aggressor CD4+ T cells in herpetic stromal keratitis pathogenesis.

Ocular infection with herpes simplex virus (HSV) causes a vision-impairing inflammatory reaction called herpetic stromal keratitis. In murine models, herpetic stromal keratitis lesions appear to be immunopathologic, mediated by CD4(+) T cells of Th1 phenotype. To provide insight about cytokine networks and signaling events involved in the development of aggressor CD4(+) T cells, ocular HSV infection was followed in mice deficient in Stat4 (Stat4(-/-) mice), the signal transducer for the cytokine interleukin-12 (IL-12). After ocular HSV infection of Stat4(-/-) and control BALB/c mice, clinical, histologic, and immunologic analyses were carried out. Further, to evaluate the involvement of Stat4 in the development of this aggressor population, naive CD4(+) T cells from Stat4(-/-) and BALB/c mice were adoptively transferred to C.B-17 SCID mice 1 day after corneal infection. Although Stat4(-/-) mice demonstrated increased susceptibility to lethal encephalitis and facial lesions, interestingly, these mice had less severe stromal keratitis in comparison to control animals. Adoptive transfer of naive CD4(+) T cells from Stat4(-/-) mice failed to produce disease in infected SCID recipients. The data imply a significant role of Stat4-mediated signaling events in the generation of an aggressor CD4(+) T cell population in stromal keratitis pathogenesis.