Regulatory effects of IFN-β on the development of experimental autoimmune uveoretinitis in B10RIII mice

Background

Experimental autoimmune uveoretinitis (EAU) serves as a model for human intraocular inflammation. IFN-β has been used in the treatment of certain autoimmune diseases. Earlier studies showed that it ameliorated EAU; however, the mechanisms involved in this inhibition are still largely unknown.

Methodology/Principal Findings

B10RIII mice were immunized with interphotoreceptor retinoid-binding protein (IRBP) peptide 161–180 in Complete Freund''s adjuvant. Splenocytes from different time points after immunization were used to evaluate the expression of IFN-β. An increased expression of IFN-β was observed during EAU and its highest expression was observed on day 16, 3 days after the peak of intraocular inflammation. Splenocytes and draining lymph node cells from mice immunized with IRBP_161-180 on day 13 and control mice were activated with anti-CD3/anti-CD28 antibodies or IRBP_161-180 to evaluate the production of IFN-γ and IL-17. The results showed that IFN-γ and IL-17 were significantly higher in immunized mice as compared to the control mice when exposed to anti-CD3/anti-CD28 antibodies. However, the production of IFN-γ and IL-17 was detected only in immunized mice, but not in the control mice when stimulated with IRBP_161-180. Multiple subcutaneous injections of IFN-β significantly inhibited EAU activity in association with a down-regulated expression of IFN-γ, IL-17 and an enhanced IL-10 production. In an in vitro system using cells from mice, IFN-β suppressed IFN-γ production by CD4⁺CD62L⁻ T cells, IL-17 production by CD4⁺CD62L^+/- T cells and proliferation of CD4⁺CD62L^+/- T cells. IFN-β inhibited the secretion of IL-6, but promoted the secretion of IL-10 by monocytes. IFN-β-treated monocytes inhibited IL-17 secretion by CD4⁺CD62L^+/- T cells, but did not influence IFN-γ expression and T cell proliferation.

Conclusions/Significance

IFN-β may exert its inhibitory effect on EAU by inhibiting Th1, Th17 cells and modulating relevant cytokines. IFN-β may provide a potential treatment for diseases mediated by Th1 and Th17 cells.     

The enteric nervous system of P2Y13 receptor null mice is resistant against high-fat-diet- and palmitic-acid-induced neuronal loss

Gastrointestinal symptoms have a major impact on the quality of life and are becoming more prevalent in the western population. The enteric nervous system (ENS) is pivotal in regulating gastrointestinal functions. Purinergic neurotransmission conveys a range of short and long-term cellular effects. This study investigated the role of the ADP-sensitive P2Y₁₃ receptor in lipid-induced enteric neuropathy. Littermate P2Y₁₃^+/+ and P2Y₁₃^−/− mice were fed with either a normal diet (ND) or high-fat diet (HFD) for 6 months. The intestines were analysed for morphological changes as well as neuronal numbers and relative numbers of vasoactive intestinal peptide (VIP)- and neuronal nitric oxide synthase (nNOS)-containing neurons. Primary cultures of myenteric neurons from the small intestine of P2Y₁₃^+/+ or P2Y₁₃^−/− mice were exposed to palmitic acid (PA), the P2Y₁₃ receptor agonist 2meSADP and the antagonist MRS2211. Neuronal survival and relative number of VIP-containing neurons were analysed. In P2Y₁₃^+/+, but not in P2Y₁₃^−/− mice, HFD caused a significant loss of myenteric neurons in both ileum and colon. In colon, the relative numbers of VIP-containing submucous neurons were significantly lower in the P2Y₁₃^−/− mice compared with P2Y₁₃^+/+ mice. The relative numbers of nNOS-containing submucous colonic neurons increased in P2Y₁₃^+/+ HFD mice. HFD also caused ileal mucosal thinning in P2Y₁₃^+/+ and P2Y₁₃^−/− mice, compared to ND fed mice. In vitro PA exposure caused loss of myenteric neurons from P2Y₁₃^+/+ mice while neurons from P2Y₁₃^−/− mice were unaffected. Presence of MRS2211 prevented PA-induced neuronal loss in cultures from P2Y₁₃^+/+ mice. 2meSADP caused no change in survival of cultured neurons. P2Y₁₃ receptor activation is of crucial importance in mediating the HFD- and PA-induced myenteric neuronal loss in mice. In addition, the results indicate a constitutive activation of enteric neuronal apoptosis by way of P2Y₁₃ receptor stimulation.     

Leucine-Rich Repeat Kinase 2 (Lrrk2) Deficiency Diminishes the Development of Experimental Autoimmune Uveitis (EAU) and the Adaptive Immune Response

Background

Mutations in LRRK2 are related to certain forms of Parkinson’s disease and, possibly, to the pathogenesis of Crohn’s disease. In both these diseases inflammatory processes participate in the pathogenic process. LRRK2 is expressed in lymphoid cells and, interestingly, Lrrk2 (-/-) mice were reported to develop more severe experimental colitis than their wild type (WT) controls. Here, we examined the possible involvement of LRRK2 in the pathogenesis of experimental autoimmune uveitis (EAU), an animal model for human uveitis, by testing Lrrk2 (-/-) mice for their capacity to develop this experimental eye disease and related immune responses.

Methods

Lrrk2 (-/-) mice and their WT controls (C57Bl/6) were immunized with interphotoreceptor retinoid-binding protein (IRBP) and compared for their development of EAU, delayed type hypersensitivity (DTH) by skin tests, production of cytokines in culture, and expression of interferon (IFN)-γ, interleukin (IL)-17 and FoxP3 by spleen cells, using flow cytometry. Peritoneal macrophages were examined for their production of cytokines/chemokines in culture following stimulation with LPS or the oligodeoxynucleotide CpG. The Lrrk2 (-/-) and WT mice were also compared for their response to bovine serum albumin (BSA).

Results

The Lrrk2 (-/-) mice developed lower levels of EAU, DTH responses and cytokine production by lymphocytes than did their WT controls. Intracellular expression of IFN-γ and IL-17, by spleen cells, and secretion of cytokines/chemokines by activated peritoneal macrophages of Lrrk2 (-/-) mice trended toward diminished levels, although variabilities were noted. The expression levels of FoxP3 by Lrrk2 (-/-) spleen cells, however, were similar to those seen in WT controls. Consistent with their low response to IRBP, Lrrk2 (-/-) mice responded to BSA less vigorously than their WT controls.

Conclusions

Lrrk2 deficiency in mice diminished the development of EAU and the related adaptive immune responses to IRBP as compared to the WT controls.     

Protective Role of P2Y2 Receptor against Lung Infection Induced by Pneumonia Virus of Mice

ATP released in the early inflammatory processes acts as a danger signal by binding to purinergic receptors expressed on immune cells. A major contribution of the P2Y₂ receptor of ATP/UTP to dendritic cell function and Th2 lymphocyte recruitment during asthmatic airway inflammation was previously reported. We investigated here the involvement of P2Y₂ receptor in lung inflammation initiated by pneumonia virus of mice infection. We demonstrated that P2Y₂ ^−/− mice display a severe increase in morbidity and mortality rate in response to the virus. Lower survival of P2Y₂ ^−/− mice was not significantly correlated with excessive inflammation despite the higher level of neutrophil recruiters in their bronchoalveolar fluids. Interestingly, we observed reduced ATP level and lower numbers of dendritic cells, CD4⁺ T cells and CD8⁺ T cells in P2Y₂ ^−/− compared to P2Y₂ ^+/+ infected lungs. Lower level of IL-12 and higher level of IL-6 in bronchoalveolar fluid support an inhibition of Th1 response in P2Y₂ ^−/− infected mice. Quantification of DC recruiter expression revealed comparable IP-10 and MIP-3α levels but a reduced BRAK level in P2Y₂ ^−/− compared to P2Y₂ ^+/+ bronchoalveolar fluids. The increased morbidity and mortality of P2Y₂ ^−/− mice could be the consequence of a lower viral clearance leading to a more persistent viral load correlated with the observed higher viral titer. The decreased viral clearance could result from the defective Th1 response to PVM with a lack of DC and T cell infiltration. In conclusion, P2Y₂ receptor, previously described as a target in cystic fibrosis therapy and as a mediator of Th2 response in asthma, may also regulate Th1 response protecting mice against lung viral infection.     

ICOS-Expressing Lymphocytes Promote Resolution of CD8-Mediated Lung Injury in a Mouse Model of Lung Rejection

Acute rejection, a common complication of lung transplantation, may promote obliterative bronchiolitis leading to graft failure in lung transplant recipients. During acute rejection episodes, CD8⁺ T cells can contribute to lung epithelial injury but the mechanisms promoting and controlling CD8-mediated injury in the lung are not well understood. To study the mechanisms regulating CD8⁺ T cell–mediated lung rejection, we used a transgenic model in which adoptively transferred ovalbumin (OVA)-specific cytotoxic T lymphocytes (CTL) induce lung injury in mice expressing an ovalbumin transgene in the small airway epithelium of the lungs (CC10-OVA mice). The lung pathology is similar to findings in humans with acute lung transplant. In the presence of an intact immune response the inflammation resolves by day 30. Using CC10-OVA.RAG^-/- mice, we found that CD4⁺ T cells and ICOS^+/+ T cells were required for protection against lethal lung injury, while neutrophil depletion was not protective. In addition, CD4⁺Foxp3 ⁺ ICOS⁺ T cells were enriched in the lungs of animals surviving lung injury and ICOS^+/+ Tregs promoted survival in animals that received ICOS^-/- T cells. Direct comparison of ICOS^-/- Tregs to ICOS^+/+ Tregs found defects in vitro but no differences in the ability of ICOS^-/- Tregs to protect from lethal lung injury. These data suggest that ICOS affects Treg development but is not necessarily required for Treg effector function.     

TLR2 mediates immunity to experimental cysticercosis

Information concerning TLR-mediated antigen recognition and regulation of immune responses during helminth infections is scarce. TLR2 is a key molecule required for innate immunity and is involved in the recognition of a wide range of viruses, bacteria, fungi and parasites. Here, we evaluated the role of TLR2 in a Taenia crassiceps cysticercosis model. We compared the course of T. crassiceps infection in C57BL/6 TLR2 knockout mice (TLR2^-/-) with that in wild type C57BL/6 (TLR2^+/+) mice. In addition, we assessed serum antibody and cytokine profiles, splenic cellular responses and cytokine profiles and the recruitment of alternatively activated macrophages (AAMφs) to the site of the infection. Unlike wild type mice, TLR2^-/- mice failed to produce significant levels of inflammatory cytokines in either the serum or the spleen during the first two weeks of Taenia infection. TLR2^-/- mice developed a Th2-dominant immune response, whereas TLR2^+/+ mice developed a Th1-dominant immune response after Taenia infection. The insufficient production of inflammatory cytokines at early time points and the lack of Th1-dominant adaptive immunity in TLR2^-/- mice were associated with significantly elevated parasite burdens; in contrast, TLR2^+/+ mice were resistant to infection. Furthermore, increased recruitment of AAMφs expressing PD-L1, PD-L2, OX40L and mannose receptor was observed in TLR2^-/- mice. Collectively, these findings indicate that TLR2-dependent signaling pathways are involved in the recognition of T. crassiceps and in the subsequent activation of the innate immune system and production of inflammatory cytokines, which appear to be essential to limit infection during experimental cysticercosis.     

Exacerbated intestinal inflammation in P2Y6 deficient mice is associated with Th17 activation

Extracellular nucleotides are released as constitutive danger signals by various cell types and activate nucleotide (P2) receptors such as P2Y₆ receptor. P2Y₆ activation on monocytes induces the secretion of the chemokine CXCL8 which may propagate intestinal inflammation. Also, P2Y₆ expression is increased in infiltrating T cells of Crohn's disease patients. As inflammatory bowel disease (IBD) is associated with immune cell recruitment, we hypothesised that P2Y₆ would participate to the establishment of inflammation in this disease. To address this, we used P2Y₆ deficient (P2ry6^‐−/−) mice in the dextran sodium sulfate (DSS) murine model of IBD. In disagreement with our hypothesis, P2Y₆ deficient mice were more susceptible to inflammation induced by DSS than WT mice. DSS treated-P2ry6^−/− mice showed increased histological damage and increased neutrophil and macrophage infiltration that correlated with increased mRNA levels of the chemokines KC and MCP-1. DSS treated-P2ry6^−/− mice exhibited also higher levels of Th17/Th1 lymphocytes in their colon which correlated with increased levels of IFN-γ and IL-17A in the sera as well as increased mRNA levels of IFN-γ, IL-17A, IL-6, IL-23 and IL-1β in P2ry6^−/− colons. This inflammation was also accompanied by a decreased cell proliferation and goblet cell number. Importantly, injection of anti-IL-17 intraperitoneally partially protected P2ry6^−/− mice from DSS-induced colitis. Taken together, in the absence of P2Y₆, an exacerbated intestinal inflammation to DSS was observed which correlated with increased recruitment of Th17/Th1 lymphocytes. These data suggest a protective role of P2Y₆ expressed on leukocytes in intestinal inflammation.     

Deletion of IL-33R (ST2) Abrogates Resistance to EAE in BALB/C Mice by Enhancing Polarization of APC to Inflammatory Phenotype

The administration of interleukin 33 and deletion of IL-33 receptor, ST2 molecule, affects the induction of autoimmunity in different experimental models of human autoimmune diseases. The aim of this study was to analyze the effect of ST2 deletion on the induction of experimental autoimmune encephalomyelitis (EAE) in resistant BALB/c mice. Mice were immunized with MOG_35–55 peptide or disease was induced by passive transfer of encephalitogenic singenic cells and EAE was clinically and histologically evaluated. Expression of intracellular inflammatory cytokines, markers of activation and chemokine receptors on lymphoid tissue and CNS infiltrating mononuclear cells was analyzed by flow cytometry. We report here that deletion of ST2^−/− molecule abrogates resistance of BALB/c mice to EAE induction based on clinical and histopathological findings. Brain and spinal cord infiltrates of ST2^−/− mice had significantly higher number of CD4⁺ T lymphocytes containing inflammatory cytokines compared to BALB/c WT mice. Adoptive transfer of ST2^−/− primed lymphocytes induced clinical signs of the disease in ST2^−/− as well as in WT mice. MOG_35–55 restimulated ST2^−/− CD4⁺ cells as well as ex vivo analyzed lymph node cells had higher expression of T-bet and IL-17, IFN-γ, TNF-α and GM-CSF in comparison with WT CD4⁺ cells. ST2^−/− mice had higher percentages of CD4⁺ cells expressing chemokine receptors important for migration to CNS in comparison with WT CD4⁺ cells. Draining lymph nodes of ST2^−/− mice contained higher percentage of CD11c⁺CD11b⁺CD8⁻ cells containing inflammatory cytokines IL-6 and IL-12 with higher expression of activation markers. Transfer of ST2^−/− but not WT dendritic cells induced EAE in MOG_35–55 immunized WT mice. Our results indicate that ST2 deficiency attenuates inherent resistance of BALB/c mice to EAE induction by enhancing differentiation of proinflammatory antigen presenting cells and consecutive differentiation of encephalitogenic T cells in the draining lymph node rather than affecting their action in the target tissue.     

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.     

Cyclooxygenase-2 in newborn hyperoxic lung injury

Supraphysiological O₂ concentrations, mechanical ventilation, and inflammation significantly contribute to the development of bronchopulmonary dysplasia (BPD).Exposure of newborn mice to hyperoxia causes inflammation and impaired alveolarization similar to that seen in infants with BPD.Previously, we demonstrated that pulmonary cyclooxygenase-2 (COX-2) protein expression is increased in hyperoxia-exposed newborn mice.The present studies were designed to define the role of COX-2 in newborn hyperoxic lung injury.We tested the hypothesis that attenuation of COX-2 activity would reduce hyperoxia-induced inflammation and improve alveolarization.Newborn C3H/HeN micewere injected daily with vehicle, aspirin (nonselective COX-2 inhibitor), or celecoxib (selective COX-2 inhibitor) for the first 7 days of life.Additional studies utilized wild-type (C57Bl/6, COX-2^+/+), heterozygous (COX-2^+/-), and homozygous (COX-2^-/-) transgenic mice.Micewere exposed to room air (21% O₂) or hyperoxia (85% O₂) for 14 days.Aspirin-injected and COX-2^-/- pups had reduced levels of monocyte chemoattractant protein (MCP-1) in bronchoalveolar lavage fluid (BAL).Both aspirin and celecoxib treatment reduced macrophage numbers in the alveolar walls and air spaces.Aspirin and celecoxib treatment attenuated hyperoxia-induced COX activity, including altered levels of prostaglandin (PG)D₂ metabolites.Decreased COX activity, however, did not prevent hyperoxia-induced lung developmental deficits.Our data suggest thatincreased COX-2 activity may contribute to proinflammatory responses, including macrophage chemotaxis, during exposure to hyperoxia.Modulation of COX-2 activity may be a useful therapeutic target to limit hyperoxia-induced inflammation in preterm infants at risk of developing BPD.     

Pregnancy ameliorates induction and expression of experimental autoimmune uveitis

Female patients suffering from autoimmune uveitis are reported to experience a temporary remission during pregnancy. Experimental autoimmune uveitis (EAU) is a model for human uveitis. Here we examine the effect of pregnancy on the development of EAU and its associated immunological responses. Susceptible C57BL/6 mice were immunized with interphotoreceptor retinoid-binding protein (IRBP). EAU scores and Ag-specific responses were evaluated 21 days later. Mice immunized during pregnancy developed significantly less EAU than nonpregnant controls. Their lymph node cells and splenocytes produced a distinct pattern of cytokines in response to IRBP: reduced IFN-gamma and IL-12 p40, but unchanged levels of TNF-alpha, IL-4, IL-5, and IL-10. Anti-IRBP Ab isotypes revealed an up-regulation of IgG1, indicating a possible Th2 bias at the humoral level. Ag-specific proliferation and delayed hypersensitivity, as well as mitogen-induced IFN-gamma production, remained undiminished, arguing against an overall immune deficit. Interestingly, pregnant mice that received an infusion of IRBP-primed lymphoid cells from nonpregnant donors also developed reduced EAU, suggesting that pregnancy suppresses not only the generation, but also the function of mature uveitogenic effector T cells. Pregnant mice at the time of immunization exhibited elevated levels of TGF-beta, but not of IL-10, in the serum. We suggest that protection from EAU during pregnancy is due primarily to a selective reduction of Ag-specific Th1 responses with only marginal enhancement of Th2 function, and that these effects may in part be secondary to elevated systemic levels of TGF-beta.     

Uveitogenic potential of lymphocytes sensitized to interphotoreceptor retinoid-binding protein

In our previous study rats immunized with bovine retinal interphotoreceptor retinoid-binding protein (IRBP) were found to develop inflammation in the eye and the pineal gland. This inflammatory disease was distinct in several aspects from experimental autoimmune uveoretinitis (EAU) induced by the retinal S-antigen (S-Ag). The current study examined the adoptive transfer of IRBP-induced EAU. We established that lymphocytes from IRBP immunized donor rats were capable of transferring EAU after in vitro stimulation with either IRBP (lymph node or spleen cells) or concanavalin A (spleen cells only). Recipients of these cells developed uveoretinitis and pinealitis identical to the actively induced disease. As compared with the S-Ag system, recipients of IRBP sensitized cells developed disease earlier, and smaller numbers of cells were needed to transfer EAU. Development of inflammation was directly related to a cellular response to the specific retinal antigen used for sensitization. Moreover, the unique nature of ocular inflammation was reestablished in the IRBP system: high proportions of polymorphonuclear leukocytes were found in the inflamed tissue of certain recipients despite a lack of a humoral response to the specific antigen. In contrast to the eye, only mononuclear leukocytes comprised the inflammation in the pineal gland.     

H-2 I alloantigens and recall of memory cytotoxic responses

AQR mice were immunized with H-2K and H-2 I encoded alloantigens presented by (Ax6R)F₁ splenocytes. Spleen cells from these alloimmune mice were subsequently restimulated in vitro with B10.A lymphocytes and/or B10.T(6R) lymphocytes, thus presenting them with the immunizing H-2K and H-2 I alloantigens independently. When stimulated with B10.A lymphocytes, alloimmune lymphocytes develop significant cytotoxicity against the immunizing H-2K target antigens. When stimulated with a similar number of B10.T(6R) spleen cells, alloimmune lymphocytes undergo a prominant proliferative response, but develop little, if any, cytotoxicity against the immunizing H-2 K target antigens. The most efficient restimulation of cytotoxicity occurs when the alloimmune spleen cells are simultaneously restimulated by B10.A and B10.T(6R) lymphocytes. Stimulation with the immunizing H-2 I alloantigens alone is not sufficient for regeneration of detectable cytotoxic responses from alloimmune spleen populations. Stimulation with the immunizing H-2K alloantigens alone appears to be both necessary and sufficient to stimulate alloimmune cytotoxic responses. Although the immunizing H-2 I alloantigens are apparently not required to generate alloimmune cytotoxic responses, they markedly potentiate the cytotoxic responses induced by the immunizing H-2K alloantigens.     

Prostaglandin E2 Induction during Mouse Adenovirus Type 1 Respiratory Infection Regulates Inflammatory Mediator Generation but Does Not Affect Viral Pathogenesis

Respiratory viruses cause substantial disease and are a significant healthcare burden. Virus-induced inflammation can be detrimental to the host, causing symptoms during acute infection and leading to damage that contributes to long-term residual lung disease. Prostaglandin E₂ (PGE₂) is a lipid mediator that is increased in response to many viral infections, and inhibition of PGE₂ production during respiratory viral infection often leads to a decreased inflammatory response. We tested the hypothesis that PGE₂ promotes inflammatory responses to mouse adenovirus type 1 (MAV-1) respiratory infection. Acute MAV-1 infection increased COX-2 expression and PGE₂ production in wild type mice. Deficiency of the E prostanoid 2 receptor had no apparent effect on MAV-1 pathogenesis. Virus-induced induction of PGE₂, IFN-γ, CXCL1, and CCL5 was reduced in mice deficient in microsomal PGE synthase-1 (mPGES-1^-/- mice). However, there were no differences between mPGES-1^+/+ and mPGES-1^-/- mice in viral replication, recruitment of leukocytes to airways or lung inflammation. Infection of both mPGES‑1^+/+ and mPGES-1^-/- mice led to protection against reinfection. Thus, while PGE₂ promotes the expression of a variety of cytokines in response to acute MAV-1 infection, PGE₂ synthesis does not appear to be essential for generating pulmonary immunity.     

Dissociation between lymphocyte activation for proliferation and for the capacity to adoptively transfer uveoretinitis

We have shown previously that immunization with bovine interphotoreceptor retinoid-binding protein (IRBP) induces in rats severe eye disease, experimental autoimmune uveoretinitis (EAU). This study examined the uveitogenic capacity of IRBP of another species, the monkey, and tested the cross-antigenicity between these two proteins by a battery of immunological assays. Monkey IRBP was found to be approximately 20 times less uveitogenic in Lewis rats than bovine IRBP. High levels of cross-reactivity between bovine and monkey IRBP were demonstrated by antibodies as measured by the enzyme-linked immunosorbent assay, and by the radiometric ear test of delayed-type hypersensitivity, by using rats immunized with either one of the IRBP. On the other hand, lymphocytes from these rats failed to detect the cross-reactivity between the two IRBP by the proliferation response in culture. Yet, such lymphocytes did recognize the nonimmunizing IRBP when activated in culture for acquiring the capacity to adoptively transfer EAU into naive recipients. The data are discussed with regard to the limited usefulness of the lymphocyte proliferation assay for detection of immunopathogenic processes and the role of cross-reacting antigens in initiation of autoimmune responses.     

Perforin deficiency attenuates collagen-induced arthritis

Collagen-induced arthritis (CIA), an approved animal model for rheumatoid arthritis, is thought to be a T cell-dependent disease. There is evidence that CD8⁺ T cells are a major subset controlling the pathogenesis of CIA. They probably contribute to certain features of disease, namely tissue destruction and synovial hyperplasia. In this study we examined the role of perforin (pfp), a key molecule of the cytotoxic death pathway that is expressed mainly in CD8⁺ T cells, for the pathogenesis of CIA. We generated DBA/1J mice suffering from mutations of the pfp molecule, DBA/1J-pfp^-/-, and studied their susceptibility to arthritis. As a result, pfp-deficient mice showed a reduced incidence (DBA/1J-pfp^+/+, 64%; DBA/1J-pfp^-/-, 54%), a slightly delayed onset (onset of disease: DBA/1J-pfp^+/+, 53 ± 3.6; DBA/1J-pfp^-/-, 59 ± 4.9 (mean ± SEM), and milder form of the disease (maximum disease score: DBA/1J-pfp^+/+, 7.3 ± 1.1; DBA/1J-pfp^-/-, 3.4 ± 1.4 (mean ± SEM); P < 0.05). Concomitantly, peripheral T cell proliferation in response to the specific antigen bovine collagen II was increased in pfp^-/- mice compared with pfp^+/+ mice, arguing for an impaired killing of autoreactive T cells caused by pfp deficiency. Thus, pfp-mediated cytotoxicity is involved in the initiation of tissue damage in arthritis, but pfp-independent cytotoxic death pathways might also contribute to CIA.     

Enhancement of Glucose Uptake in Mouse Skeletal Muscle Cells and Adipocytes by P2Y6 Receptor Agonists

Glucose uptake by peripheral tissues such as skeletal muscles and adipocytes is important in the maintenance of glucose homeostasis. We previously demonstrated that P2Y₆ receptor (P2Y₆R) agonists protect pancreatic islet cells from apoptosis and stimulate glucose-dependent insulin release. Here, we investigated the effects of P2Y₆R activation on glucose uptake in insulin target tissues. An agonist of the P2Y₆R, P¹-(5′-uridine)-P³-(5′-N⁴-methoxycytidine)-triphosphate (MRS2957), significantly increased the uptake of [³H]2-deoxyglucose in mouse C2C12 myotubes and 3T3-L1 adipocytes, and this stimulation was significantly decreased by a selective P2Y₆R antagonist N,N″-1,4-butanediyl-bis[N′-(3-isothiocyanatophenyl)thiourea] (MRS2578). Pre-incubation with Compound C (an inhibitor of 5′-AMP-activated protein kinase, AMPK), or AMPK siRNA abolished the stimulatory effect of MRS2957 on glucose uptake. Also, MRS2957 (60 min incubation) increased recruitment of the facilitated glucose transporter-4 (GLUT4) to the cell membrane, which was blocked by MRS2578. Treatment of C2C12 myotubes with MRS2957 induced significant phosphorylation of AMPK, which increase GLUT4 expression through histone deacetylase (HDAC)5 signaling. Glucose uptake in primary mouse adipocytes from wild-type mice was stimulated upon P2Y₆R activation by either MRS2957 or native agonist UDP, and the P2Y₆R effect was antagonized by MRS2578. However, in adipocytes from P2Y₆R-knockout mice P2Y₆R agonists had no effect on glucose uptake, and there was no change in the glucose uptake by insulin. Our results indicate that the P2Y₆R promotes glucose metabolism in peripheral tissues, which may be mediated through AMPK signaling.     

Paradoxical Effect of Pertussis Toxin on the Delayed Hypersensitivity Response to Autoantigens in Mice

Background

Pertussis toxin (PTX), an exotoxin of Bordetella pertussis, enhances the development of experimental autoimmune diseases such as experimental autoimmune uveitis (EAU) and experimental autoimmune encephalomyelitis (EAE) in rodent models. The mechanisms of the promotion of experimental autoimmune diseases by PTX may be based upon PTX-induced disruption of the blood eye/brain barriers facilitating the infiltration of inflammatory cells, the modulation of inflammatory cell migration and the enhancement of the activation of inflammatory cells. We hypothesized that the facilitation of experimental autoimmunity by PTX suggests that its influence on the in vivo immune response to auto-antigen may differ from its influence on non-self antigens.

Methodology/Principal Findings

We have evaluated the effect of PTX on the simultaneous generation of delayed type hypersensitivity (DTH) responses and autoimmune responses to uveitogenic interphotoreceptor retinoid binding protein peptide (IRBP_161–180), encephalitogenic myelin oligodendrocyte glycoprotein peptide (MOG_35–55) or ovalbumin (OVA). PTX injection of mice immunized to IRBP peptide_161–180 led to (i) the development of EAU as shown by histopathology of the retina, (ii) pro-inflammatory cytokine production by splenocytes in response to IRBP peptide _161–180, and (iii) symptomatic EAE in mice immunized with encephalitogenic MOG peptide_35–55. However, mice that received PTX had a reduced DTH response to IRBP_161–180 peptide or MOG peptide_35–55 when challenged distal to the site affected by autoreactive T cells. Moreover, footpad challenge with MOG_35–55 peptide reduced EAE in mice immunized with MOG peptide. In contrast, the use of PTX when immunizing with OVA protein or an OVA immunogenic peptide did not affect the DTH response to OVA.

Conclusions/Significance

The results suggest that that the reduced DTH response in mice receiving PTX may be specific for autoantigens and autoantigen-reactive T cells are diverted away from ectopic sites that received the autoantigen and towards the tissue site of the autoantigen.     

Sialylation and Muscle Performance: Sialic Acid Is a Marker of Muscle Ageing

Sialic acids (Sia) are widely expressed as terminal monosaccharides on eukaryotic glycoconjugates. They are involved in many cellular functions, such as cell–cell interaction and signal recognition. The key enzyme of sialic acid biosynthesis is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE), which catalyses the first two steps of Sia biosynthesis in the cytosol. In this study we analysed sialylation of muscles in wild type (C57Bl/6 GNE ^+/+) and heterozygous GNE-deficient (C57Bl/6 GNE ^+/−) mice. We measured a significantly lower performance in the initial weeks of a treadmill exercise in C57Bl/6 GNE ^+/− mice compared to wild type C57Bl/6 GNE ^+/+animals. Membrane bound Sia of C57Bl/6 GNE ^+/− mice were reduced by 33–53% at week 24 and by 12–15% at week 80 in comparison to C57Bl/6 GNE ^+/+mice. Interestingly, membrane bound Sia concentration increased with age of the mice by 16–46% in C57Bl/6 GNE ^+/+, but by 87–207% in C57Bl/6 GNE ^+/−. Furthermore we could identify specific morphological changes in aged muscles. Here we propose that increased Sia concentrations in muscles are a characteristic feature of ageing and could be used as a marker for age-related changes in muscle.     

Irgm 1参与动脉粥样硬化斑块的形成

目的:探讨免疫相关GTP酶1(Irgm 1)对小鼠血管动脉粥样硬化(AS)斑块形成的影响。方法:高脂饲料喂养野生型(WT)、ApoE~(-/-)Irgm 1~(+/+)和ApoE~(-/-)Irgm1~(+/-)小鼠3个月,建立AS模型;取小鼠主动脉弓,免疫荧光染色方法观察WT和ApoE~(-/-)Irgm 1~(+/+)小鼠血管AS斑块中Irgm 1的表达情况及部位;Western blot方法检测WT和ApoE~(-/-)Irgm 1~(+/+)小鼠血管AS斑块中Irgm 1蛋白表达情况;Q-PCR方法检测WT和ApoE~(-/-)Irgm 1~(+/+)小鼠血管AS斑块中Irgm 1 m RNA表达情况;油红O染色观察ApoE~(-/-)Irgm1~(+/+)和ApoE~(-/-)Irgm1~(+/-)小鼠血管AS斑块形成情况;结果:与WT组相比,ApoE~(-/-)Irgm 1~(+/+)组小鼠主动脉弓AS斑块中Irgm 1+细胞明显增多,Irgm 1+细胞主要位于血管AS斑块的表面;与WT组相比,ApoE~(-/-)Irgm 1~(+/+)组小鼠血管AS斑块中Irgm 1蛋白表达显著增多(P0.001),Irgm 1 m RNA表达显著增多(P0.01);与ApoE~(-/-)Irgm1~(+/-)组相比,ApoE~(-/-)Irgm1~(+/+)组小鼠主动脉弓AS斑块面积显著增大(P0.01);结论:Irgm 1能够促进血管AS斑块的形成。