MAP3K2 augments Th1 cell differentiation via IL-18 to promote T cell-mediated colitis

T cell-mediated immunity in the intestine is stringently controlled to ensure proper immunity against pathogenic microbes and to prevent autoimmunity, a known cause of inflammatory bowel disease. However, precisely how T cells regulate intestine immunity remains to be fully understood. In this study, we found that mitogen-activated protein kinase kinase kinase 2(MAP3K2) is required for the CD4~+T cell-mediated inflammation in the intestine. Using a T cell transfer colitis model, we found that MAP3K2-deficient na?ve CD4~+T cells had a dramatically reduced ability to induce colitis compared to wild type T cells. In addition, significantly fewer IFN-γ-but more IL-17A-producing CD4~+T cells in the intestines of mice receiving MAP3K2-deficient T cells than in those from mice receiving wild type T cells was observed. Interestingly, under well-defined in vitro differentiation conditions, MAP3K2-deficient na?ve T cells were not impaired in their ability to differentiate into Th1, Th17 and Treg. Furthermore, the MAP3K2-regulated colitis severity was mediated by Th1 but not Th17 cells in the intestine. At the molecular level, we showed that MAP3K2-mediated Th1 cell differentiation in the intestine was regulated by IL-18 and required specific JNK activation. Together, our study reveals a novel regulatory role of MAP3K2 in intestinal T cell immunity via the IL-18-MAP3K2-JNK axis and may provide a novel target for intervention in T cell-mediated colitis.     

Combined chemotherapy of platinum and fluorouracil promotes T cell–mediated antitumor immunity

The two-drug combined chemotherapy of platinum and fluorouracil has been reported to efficiently kill tumor cells as the first-line treatment for advanced gastric cancer.However,the effect of these drugs on T cells remains unclear.Here,we showed that T cells including CD4+T cells and CD8+T cells of the patients with advanced gastric cancer after platinum and fluorouracil chemotherapy exhibited enhanced ex vivo proliferation ability as compared to that before chemotherapy.In addition,platinum and fluorouracil also promoted the differentiation of human T cells into Th1 and Th9 subtypes and cytotoxic T lymphocytes(CTLs)in vitro and in vivo.Accordingly,the combination therapy greatly suppressed tumor growth with increased tumor infiltration of Th1,Th9,and CTL cells in a mouse tumor model.Moreover,in activated T cells,long-term treatment with these two drugs further facilitates T cell activation along with promoted nuclear factor-κB(NF-κB)activation.Our findings demonstrate a previously unidentified function of platinum and fluorouracil combination chemotherapy in promoting T cell–mediated antitumor immunity.     

Protective immunity against Rickettsia heilongjiangensis in a C3H/HeN mouse model mediated by outer membrane protein B-pulsed dendritic cells

Rickettsia heilongjiangensis is an obligate intracellular bacterium that causes Far-Eastern tick-borne spotted fever. Outer membrane protein B(Omp B) is an important surface protein antigen of rickettsiae. In the present study, the omp B gene of R. heilongjiangensis was divided into four fragments, resulting in four recombinant proteins(OmpB-p1, Omp B-p2, Omp B-p3, and Omp B-p4). Each Omp B was used in vitro to stimulate murine bone marrow-derived dendritic cells(BMDCs) of C3H/He N mice, and the Omp B-pulsed BMDCs were transferred to naive C3H/He N mice. On day 14 post-transfer of BMDCs, the mice were challenged with R. heilongjiangensis and the rickettsial loads in the mice were quantitatively determined on day 7 post-challenge. Mice receiving BMDCs pulsed with Omp B-p2, Omp B-p3, or Omp B-p4 exhibited significantly lower bacterial load compared with mice receiving Omp B-p1-pulsed BMDCs. CD4+ and CD8+ T cells isolated from the spleen of C3H/He N mice receiving BMDCs pulsed with each OmpB were co-cultured with BMDCs pulsed with the respective cognate protein. In flow cytometric analysis, the expression level of CD69 on CD4+ or CD8+ T cells from mice receiving BMDCs pulsed with Omp B-p2, OmpB-p3, or Omp B-p4 was higher than that on cells from mice receiving Omp B-p1-pulsed BMDCs, while the expression level of tumor necrosis factor(TNF)-α on CD8+ T cells and interferon(IFN)-γ on the CD4+ and CD8+ T cells from mice receiving Omp B-p2,-p3, or-p4 was significantly higher than on cells from mice receiving Omp B-p1-pulsed BMDCs. Our results suggest that the protective Omp Bs could activate CD4+ and CD8+ T cells and drive their differentiation toward CD4+ Th1 and CD8+ Tcl cells, respectively, which produce greater amounts of TNF-α and, in particular, IFN-γ, to enhance rickettsicidal activity of host cells.     

Reciprocal expression of TRAIL and CD95L in Th1 and Th2 cells: role of apoptosis in T helper subset differentiation

Upon activation, naive T-helper cells can differentiate into two major distinct subsets, T helper 1 (Th1) and T helper 2 (Th2), as defined by their effector functions and cytokine secretion patterns. Cytokine milieu and costimulatory molecules have been shown to play an essential role in determining T helper differentiation. However, it is still unclear how the effects of signals of co-stimulatory molecules and cytokines are exerted during T helper differentiation. We show evidence suggesting that while cytokine signals initiate differentiation program, the selective action of death effectors determines the endpoint balance of differenti-     

T Cell Repertoire Diversity Is Decreased in Type 1 Diabetes Patients

Type 1 diabetes mellitus (T1D) is an immune-mediated disease. The autoreactive T cells in T1D patients attack and destroy their own pancreatic cells. In order to systematically investigate the potential autoreactive T cell receptors (TCRs), we used a high-throughput immune repertoire sequencing technique to profile the spectrum of TCRs in individual T1D patients and controls. We sequenced the T cell repertoire of nine T1D patients, four type 2 diabetes (T2D) patients and six nondiabetic controls. The diversity of the T cell repertoire in T1D patients was significantly decreased in comparison with T2D patients (P = 7.0E
08 for CD4+ T cells, P = 1.4E
04 for CD8+ T cells) and nondiabetic controls (P = 2.7E
09 for CD4+ T cells, P = 7.6E
06 for CD8+ T cells). Moreover, T1D patients had significantly more highly-expanded T cell clones than T2D patients (P = 5.2E
06 for CD4+ T cells, P = 1.9E
07 for CD8+ T cells) and nondiabetic controls (P =1.7E
07 for CD4+ T cells, P= 3.3E
03 for CD8+ T cells). Furthermore, we identified a group of highly-expanded T cell receptor clones that are shared by more than two T1D patients. Although further validation in larger cohorts is needed, our data suggest that T cell receptor diversity measurements may become a valuable tool in investigating diabetes, such as using the diversity as an index to distinguish different types of diabetes.     

Hippocampal 8-[3H]Hydroxy-2-(Di-n-Propylamino)Tetralin Binding Site Densities, Serotonin Receptor (5-HT1A) Messenger Ribonucleic Acid Abundance, and Serotonin Levels Parallel the Activity of the Hypothalamopituitary-Adrenal Axis in Rat

We have previously demonstrated that susceptibility of the Lewis rat to inflammatory disease, compared with the relatively resistant Fischer F344/N rat, is related to a hyporesponsive hypothalamopituitary-adrenal axis to inflammatory and other stress mediators. Because serotonin (5-HT) and the 5-HT1A receptor are important stimulators of this axis, we have investigated the levels of 8-[3H]-hydroxy-2,3-(di-n-propylamino)tetralin binding sites, 5-HT1A mRNA, 5-HT, and 5-hydroxyindoleacetic acid in various brain regions of Lewis, outbred Harlan Sprague Dawley, and Fischer F344/N rats. Lewis rats expressed significantly fewer hippocampal and frontal cortical 8-[3H]-hydroxy-2,3-(di-n-propylamino)tetralin binding sites and less 5-HT1A mRNA than Harlan Sprague Dawley and Fischer F344/N rats. Adrenalectomy increased the number of 8-[3H]hydroxy-2,3-(di-n-propylamino)tetralin binding sites and 5-HT1A mRNA expression in the hippocampus of all three strains. Levels of hippocampal 5-HT in Fischer F344/N rats were significantly greater than levels detected in the same regions from Lewis and Harlan Sprague Dawley rats. Hypothalamic 5-HT and 5-hydroxyindoleacetic acid levels in Harlan Sprague Dawley rats were higher than the same area from the other two strains. Adrenalectomy increased the levels of 5-hydroxyindoleacetic acid in the hypothalamus of all three strains. We conclude that hippocampal 5-HT1A receptor densities and 5-HT levels in the rat parallel the activity and responsiveness of the hypothalamopituitary-adrenal axis.     

Environmental modulation of autoimmune arthritis involves the spontaneous microbial induction of T cell responses to regulatory determinants within heat shock protein 65

Both genetic and environmental factors are believed to be involved in the induction of autoimmune diseases. Adjuvant arthritis (AA) is inducible in susceptible rat strains by injection of Mycobacterium tuberculosis, and arthritic rats raise T cell responses to the 65-kDa mycobacterial heat-shock protein (Bhsp65). We observed that Fischer 344 (F344) rats raised in a barrier facility (BF-F344) are susceptible to AA, whereas F344 rats maintained in a conventional facility (CV-F344) show significantly reduced incidence and severity of AA, despite responding well to the arthritogenic determinant within Bhsp65. The acquisition of protection from AA can be circumvented if rats are maintained on neomycin/acidified water. Strikingly, naive unimmunized CV-F344 rats but not BF-F344 rats raised T cell responses to Bhsp65 C-terminal determinants (BCTD) (we have previously shown that BCTD are involved in regulation of acute AA in the Lewis rat); however, T cells of naive CV-F344 and BF-F344 gave a comparable level of proliferative response to a mitogen, but no response at all to an irrelevant Ag. Furthermore, adoptive transfer into naive BF-F344 rats of splenic cells of naive CV-F344 rats (restimulated with BCTD in vitro) before induction of AA resulted in a considerably reduced severity of AA. These results suggest that spontaneous (inadvertent) priming of BCTD-reactive T cells, owing to determinant mimicry between Bhsp65 and its homologues in microbial agents in the conventional environment, is involved in modulating the severity of AA in CV-F344 rats. These results have important implications in broadening understanding of the host-microbe interaction in human autoimmune diseases.     

Pertussis toxin enhances Th1 responses by stimulation of dendritic cells

Pertussis toxin (PTX) has been widely used as an adjuvant to induce Th1-mediated organ-specific autoimmune diseases in animal models. However, the cellular and molecular mechanisms remain to be defined. In this study, we showed that dendritic cells (DC) stimulated with PTX (PTX-DC) were able to substitute for PTX to promote experimental autoimmune uveitis (EAU). EAU induced by PTX-DC revealed a typical Th1 response, characterized by high uveitogenic retinal Ag interphotoreceptor retinoid-binding protein (IRBP)-specific IFN-gamma and IL-12 production in the draining lymph nodes, as well as increased levels of anti-IRBP IgG2a and decreased levels of anti-IRBP IgG1 in the serum of IRBP-immunized mice. Furthermore, PTX-DC preferentially induced T cells to produce the Th1 cytokine, IFN-gamma. After being stimulated with PTX, DC exhibited up-regulation of MHC class II, CD80, CD86, CD40, and DEC205. PTX-DC had also increased allostimulatory capacity and IL-12 and TNF-alpha production. Serum IL-12 was increased in naive mice that received PTX-DC i.p. In addition, PTX activated extracellular signal-regulated kinase in DC. Following the inhibition of extracellular signal-regulated kinase signaling, the maturation of PTX-DC was reduced. Subsequently, the ability of PTX-DC to promote IFN-gamma production by T cells in vitro and to induce EAU in vivo was blocked. The results suggest that PTX might exert an adjuvant effect on DC to promote their maturation and the production of proinflammatory cytokines, thereby eliciting a Th1 response.     

Pertussis toxin inhibits induction of tissue-specific autoimmune disease by disrupting G protein-coupled signals.

Pertussis toxin (PTX) has been used for many years as an adjuvant that promotes development of tissue-specific experimental autoimmune diseases such as experimental autoimmune encephalomyelitis, experimental autoimmune uveitis (EAU), and others. Enhancement of vascular permeability and of Th1 responses have been implicated in this effect. Here we report a surprising observation that, in a primed system, PTX can completely block the development of EAU. Disease was induced in B10.RIII mice by adoptive transfer of uveitogenic T cells, or by immunization with a uveitogenic peptide. A single injection of PTX concurrently with infusion of the uveitogenic T cells, or two injections 7 and 10 days after active immunization, completely blocked development of EAU. EAU also was prevented by a 1-h incubation in vitro of the uveitogenic T cells with PTX before infusing them into recipients. Uveitogenic T cells treated with PTX in vitro and lymphoid cells from mice treated with PTX in vivo failed to migrate to chemokines in a standard chemotaxis assay. Neither the isolated B-oligomer subunit of PTX that lacks ADP ribosyltransferase activity nor the related cholera toxin that ADP-ribosylates G(s) (but not G(i)) proteins blocked EAU induction or migration to chemokines. We conclude that PTX present at the time of cell migration to the target organ prevents EAU, and propose that it does so at least in part by disrupting signaling through G(i) protein-coupled receptors. Thus, the net effect of PTX on autoimmune disease would represent an integration of enhancing and inhibitory effects.     

Exogenous nitric oxide inhibits experimental autoimmune uveoretinitis development in Lewis rats by modulation of the Th1-dependent immune response.

Experimental autoimmune uveoretinitis (EAU) is a T cell-mediated autoimmune disease of posterior uvea that closely resembles a human disease. The uveitogenic effector T cell has a Th1-like phenotype [high interferon-gamma (IFN-gamma), low interleukin-4 (IL-4)], and genetic susceptibility to EAU that is associated with an elevated Th1 response. Suppression of CD4+ Th1 cells for the treatment of autoimmune disease is an attractive potential therapeutic approach. Nitric oxide (NO) has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. In this study, we investigated the potential role of NO as an immunoregulator to alter Th1/Th2 cytokine production, as well as to inhibit the interphotoreceptor retinoid binding protein (IRBP)-induced EAU, a CD4+ Th1 cell-mediated autoimmune disease. Injection of IRBP (100 microg) into two footpads resulted in severe EAU. The beginning peak of the disease was days 12 to 15 after immunization. Oral treatment with molsidomine (MSDM), a NO donor, began 24 h before IRBP immunization to the end of the experiments, which resulted in a significant inhibition of the disease by clinical and histopathological criteria. When MSDM was administered until day 21, a complete reduction of incidence and severity of EAU was observed. To investigate the cytokine alterations from Th1 to Th2 cytokines by MSDM, the cytokines were assayed in a culture medium of IRBP-stimulated inguinal lymphocytes. IRBP-immunized rats secreted a high concentration of IFN-gamma and a low concentration of IL-10. In contrast, MSDM treatment enhanced IL-10 secretion and tended to decrease IFN-gamma secretion. In conclusion, we show that the administration of NO suppresses EAU by altering the Th1/Th2 balance of inflammatory immune responses. We suggest that NO may be useful in the therapeutic control of autoimmune uveitis.     

IRBP-specific Th1 cells from peripheral blood were predominant in the experimental autoimmune uveitis

Experimental autoimmune uveitis (EAU) is a Th1-cell-mediated autoimmune disease. In this study, the correlation between IRBP-specific Th1 cells in PBLs and the histological grading in the eyes was evaluated kinetically during EAU induction. EAU was induced in B10.A mice with IRBP immunization and the eyes were enucleated for histological examination on days 0, 3, 7, 15, and 21 after immunization. To determine the Th1-cell-mediated immune response, Th1 cytokines (IL-12p40 and IFN-gamma) were measured by RT-PCR in inflamed eyes. At mean time, CD4(+) and IFN-gamma(+) double positive T cells (Th1 cells) from PBLs were analyzed by flow cytometry. The level of the IRBP-specific Th1 cells was significantly increased and kinetically changed during EAU induction, but the cells reached peak time early before the disease was onset. Those IRBP-specific Th1 cells in the PBLs were evidence for EAU disease, but its peak time was different from EAU disease in the eyes. Our data suggested that it is very important to collect blood from patients at a suitable time point and the Th1 cells measured by flow cytometry are good marker for disease diagnosis.     

Response acquisition with delayed reinforcement in Lewis and Fischer 344 rats

Previous work has shown neurochemical and behavioral differences between Lewis rats and Fischer 344 rats. Some of this work suggests that there might be differential sensitivity to delayed reinforcement between the two strains. To further explore this possibility, Lewis (n=8) and Fischer 344 (n=8) rats were exposed to a response-acquisition task with a non-resetting 20s delay to reinforcement. A tandem fixed-ratio 1, fixed-time 20s schedule of reinforcement was programmed for one of two levers; presses on the alternate lever had no programmed consequences. A greater number of Lewis rats (5/8) acquired lever pressing compared to the Fischer 344 rats (2/8). Future work with these strains may lead to a better understanding of the genetic and/or neurochemical factors involved in temporal control of behavior.     

Experimental autoimmune myasthenia gravis may occur in the context of a polarized Th1- or Th2-type immune response in rats.

Experimental autoimmune myasthenia gravis (EAMG) is a T cell-dependent, Ab-mediated autoimmune disease induced in rats by a single immunization with acetylcholine receptor (AChR). Although polarized Th1 responses have been shown to be crucial for the development of mouse EAMG, the role of Th cell subsets in rat EAMG is not well established. In the present work we show that while the incidence and severity of EAMG are similar in Lewis (LEW) and Brown-Norway (BN) rats, strong differences are revealed in the immune response generated. Ag-specific lymph node cells from LEW rats produced higher amounts of IL-2 and IFN-gamma than BN lymph node cells, but expressed less IL-4 mRNA. IgG1 and IgG2b anti-AChR isotype predominated in BN and LEW rats, respectively, confirming the dichotomy of the immune response observed between the two strains. Furthermore, although IL-12 administration or IFN-gamma neutralization strongly influenced the Th1/Th2 balance in BN rats, it did not affect the disease outcome. These data demonstrate that a Th1-dominated immune response is not necessarily associated with disease severity in EAMG, not only in rats with disparate MHC haplotype but also in the same rat strain, and suggest that in a situation where complement-fixing Ab can be generated as a consequence of either Th1- or Th2-mediated T cell help, deviation of the immune response will not be an adequate strategy to prevent this Ab-mediated autoimmune disease.     

Th1 and Th2 cytokine immunomodulation by gangliosides in experimental autoimmune encephalomyelitis

In experimental autoimmune encephalomyelitis, a classical model for multiple sclerosis, the cytokines provide the necessary signals to activate specific T cells for self-antigens. Gangliosides have multiple immunomodulatory activities, decreasing the lymphoproliferative responses and modulating cytokine production. Here, we tested the effects of gangliosides on the switching of Th1 to Th2 cytokine expression, in spleen cells obtained from Lewis rats during the acute phase of EAE, and after recovery from the disease. For this purpose, total RNA from spleen cells was isolated and submitted to RT-PCR to investigate Th1 (IL-2, TNF-alpha, and IFN-gamma) and Th2/Th3 (IL-10 and TGF-beta) cytokine gene expression. Results demonstrate that the group treated with gangliosides displays mild disease, with low expression of IFN-gamma mRNA and high TGF-beta mRNA expression. We conclude that the gangliosides may modulate Th1 cells by the synthesis of cytokines shifting the profile to the Th2/Th3 phenotype.