DQ8 Mice Research Articles

Concurrent Brain Subregion Microgliosis in an HLA-II Mouse Model of Group A Streptococcal Skin Infection.

The broad range of clinical manifestations and life-threatening infections caused by the Gram-positive bacterium, Streptococcus pyogenes or Group A Streptococcus (GAS), remains a significant concern to public health, with a subset of individuals developing neurological complications. Here, we examined the concurrent neuroimmune effects of subcutaneous GAS infections in an HLA-Class II (HLA) transgenic mouse model of subcutaneous GAS infection. To investigate changes in the skin-brain axis, HLA-DQ8 (DQA1*0301/DQB1*0302) mice (DQ8) were randomly divided into three groups: uninfected controls (No Inf), GAS infected and untreated (No Tx), and GAS infected with a resolution by clindamycin (CLN) treatment (CLN Tx) (10 mg/kg/5 days) and were monitored for 16 days post-infection. While the skin GAS burden was significantly reduced by CLN, the cortical and hippocampal GAS burden in the male DQ8 mice was not significantly reduced with CLN. Immunoreactivity to anti-GAS antibody revealed the presence of GAS bacteria in the vicinity of the neuronal nucleus in the neocortex of both No Tx and CLN Tx male DQ8 mice. GAS infection-mediated cortical cytokine changes were modest; however, compared to No Inf or No Tx groups, a significant increase in IL-2, IL-13, IL-22, and IL-10 levels was observed in CLN Tx females despite the lack of GAS burden. Western blot analysis of cortical and hippocampal homogenates showed significantly higher ionized calcium-binding adaptor-1 (Iba-1, microglia marker) protein levels in No Tx females and males and CLN Tx males compared to the No Inf group. Immunohistochemical analysis showed that Iba-1 immunoreactivity in the hippocampal CA3 and CA1 subregions was significantly higher in the CLN Tx males compared to the No Tx group. Our data support the possibility that the subcutaneous GAS infection communicates to the brain and is characterized by intraneuronal GAS sequestration, brain cytokine changes, Iba-1 protein levels, and concurrent CA3 and CA1 subregion-specific microgliosis, even without bacteremia.

HLA Class II Polymorphisms Influence Gut Microbiota Composition and Modulate Disease in Transgenic Mice Model of Multiple Sclerosis

Abstract Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system with unknown etiology. The interaction of both genetic and environmental factors plays an important role in MS pathogenesis. Recently, gut microbiota has emerged as a potential environmental factor in MS pathology due to its ability to modulate host immune response. Yet, it is unknown whether genetic factors, such as HLA class II gene(s), can influence gut microbiota which then can modulate MS through regulation of the host immune response. In the present study, we investigated the involvement of gut microbiota in HLA class II genes mediated susceptibility and resistance to MS/experimental autoimmune encephalomyelitis-EAE (an animal model of MS). Previously, we showed that HLA-DR3 transgenic mice lacking endogenous mouse class II genes (AE-KO) were susceptible to proteolipid protein-PLP91–110 induced EAE, whereas AE-KO.HLA-DQ8 transgenic mice were resistant to PLP91–110. Surprisingly, HLA-DR3.DQ8 double transgenic mice showed increased EAE severity compared with HLA-DR3 mice. Distinct microbiota in HLA-DR3, HLA-DQ8, and HLA-DR3.DQ8 transgenic mice compared to AE-KO mice suggested an important role of HLA class-II gene in shaping the gut microbiota composition. We also observed that gut microbiota of HLA-DQ8 mice was more similar to HLA-DR3.DQ8 than HLA-DR3. As the presence of HLA-DQ8 on an HLA-DR3 background increases disease severity, our data suggest that HLA-DQ8 restricted microbiota may contribute to disease severity in HLA-DR3.DQ8 mice. Altogether, our study provides evidence that the HLA-DR and -DQ genes linked to specific gut microbiota may contribute to EAE susceptibility or resistance in a transgenic animal model of MS. The authors acknowledge funding from the National Multiple Sclerosis Society (RG 5138A1/1T), NIAID/NIH (1R01AI137075-01), a Carver Trust Medical Research Initiative Grant, and the University of Iowa Environmental Health Sciences Research Center, NIEHS/NIH (P30 ES005605). SA was supported by the Emory Warner Fellowship, which provides medical students at the Carver College of Medicine the opportunity to take a full year out of their medical school curriculum to work in a laboratory in the University of Iowa Department of Pathology.

HLA-II Alleles Influence Physical and Behavioral Responses to a Whey Allergen in a Transgenic Mouse Model of Cow's Milk Allergy.

The symptoms of food allergies vary significantly between individuals, likely due to genetic determinants. In humans, allergy development is initiated by antigen-presenting cells via class II human leukocyte antigen (HLA-II). The HLA-II gene is highly polymorphic, and its allelic variance is thought to influence the susceptibility of individuals to a particular allergen. However, whether antigen presentation by different HLA-II variants contributes to symptom variation is not clear. We hypothesized that HLA-II allelic variance affects symptom phenotypes, including immediate physical reactions and delayed behavioral changes, in individuals with food hypersensitivity. To test our hypothesis, male and female mice of three transgenic strains expressing an HLA-II variant, DR3, DR15, or DQ8, were used to establish a cow's milk allergy model. Mice were sensitized to a bovine whey allergen, β-lactoglobulin (BLG; Bos d 5), weekly for 5 weeks, followed by an acute oral allergen challenge. At 30 min post-challenge, BLG-sensitized DR3 mice showed moderate to severe anaphylaxis resulting in perioral redness, swelling, and death. In contrast, DQ8 and DR15 mice were generally asymptomatic. The production of allergen-specific immunoglobulins was also HLA- and sex-dependent. Both male and female DR3 and female DR15 mice significantly increased BLG-specific IgE production, while robust elevation in BLG-specific IgG1 was observed in sensitized DQ8 mice of both sexes and, to a lesser extent, in DR15 males. Furthermore, BLG-sensitized DR15 mice showed sex-specific behavior changes, with males exhibiting mobility changes and anxiety-like behavior and females showing spatial memory impairment. When splenocytes from transgenic mice were stimulated in vitro with BLG, phenotypes of immune cells were HLA- and sex-specific, further underscoring the influence of HLA-II on immune responses. Our results support that HLA-II alleles influence behavioral responses in addition to immune and physical reactions of food allergy, suggesting that certain HLA-II variants may predispose individuals to food-allergy-associated behavioral changes.

Transamidated wheat gliadin induces differential antigen recognition in the small intestine of HLA/DQ8 transgenic mice.

A lifelong gluten-free diet (GFD) is currently the only available therapy for coeliac disease (CD). However, GFD compliance is difficult and alternative strategies are envisaged in the near future. We previously found that wheat gliadin following transamidation by microbial transglutaminase (mTG) does not induce IFN-γ secretion by intestinal T cells from CD patients. Fully transamidated gliadin with lysine ethyl ester can be recovered in a soluble protein fraction (spf) generated by the enzymatic treatment of wheat flour. Herein, we analysed the performance of transamidation by mTG on a pilot-scale (1L) by evaluating the reaction kinetics and its biological effect on the intestinal immune response in HLA/DQ8 transgenic mice, a model of gluten sensitivity. At 1 h, all gliadin fractions showed a faster electrophoretic mobility by acid-polyacrylamide gel electrophoresis (A-PAGE) following transamidation in comparison with their native counterparts. In parallel, the yield of residual native gliadin dropped (30% at 180 min), confirming our previous findings on a lab scale. Mucosal sensitisation of mice with gliadin via the intranasal route induced a Th1 phenotype in mesenteric lymph nodes (MLNs). Importantly, IFN-γ secretion was significantly reduced when gliadin-specific MLN cells were challenged in vitro with spf (P < 0.001). Multiplex analysis revealed that the adaptive immune response evoked by spf involved a distinct cell population characterised by secretion of IL-2, IL-3 and IL-5. Notably, spf stimulated in vitro a reduced or null secretion of all of the examined pro-inflammatory markers mainly associated to innate immunity. In conclusion, our data revealed the ability of transamidated gliadin to modulate both innate and adaptive mechanisms involved in the inflammatory response induced by wheat gliadin in the small intestine of DQ8 mice.

HLA Class II Polymorphisms Modulate Gut Microbiota and Experimental Autoimmune Encephalomyelitis Phenotype.

Multiple sclerosis (MS) is an autoimmune disease of the CNS in which the interaction between genetic and environmental factors plays an important role in disease pathogenesis. Although environmental factors account for 70% of disease risk, the exact environmental factors associated with MS are unknown. Recently, gut microbiota has emerged as a potential missing environmental factor linked with the pathobiology of MS. Yet, how genetic factors, such as HLA class II gene(s), interact with gut microbiota and influence MS is unclear. In the current study, we investigated whether HLA class II genes that regulate experimental autoimmune encephalomyelitis (EAE) and MS susceptibility also influence gut microbiota. Previously, we have shown that HLA-DR3 transgenic mice lacking endogenous mouse class II genes (AE-KO) were susceptible to myelin proteolipid protein (91-110)-induced EAE, an animal model of MS, whereas AE-KO.HLA-DQ8 transgenic mice were resistant. Surprisingly, HLA-DR3.DQ8 double transgenic mice showed higher disease prevalence and severity compared with HLA-DR3 mice. Gut microbiota analysis showed that HLA-DR3, HLA-DQ8, and HLA-DR3.DQ8 double transgenic mice microbiota are compositionally different from AE-KO mice. Within HLA class II transgenic mice, the microbiota of HLA-DQ8 mice were more similar to HLA-DR3.DQ8 than HLA-DR3. As the presence of DQ8 on an HLA-DR3 background increases disease severity, our data suggests that HLA-DQ8-specific microbiota may contribute to disease severity in HLA-DR3.DQ8 mice. Altogether, our study provides evidence that the HLA-DR and -DQ genes linked to specific gut microbiota contribute to EAE susceptibility or resistance in a transgenic animal model of MS.

First morphological-level insights into the efficiency of green tea catechins and grape seed procyanidins on a transgenic mouse model of celiac disease enteropathy.

Alternative or complementary treatments to a gluten-free diet are urgently needed for Celiac Disease. By exploiting the health-promoting properties of polyphenols on a transgenic mouse model of Celiac Disease enteropathy, this study provides the first in vivo evidence regarding the ability of 1 mg day-1 doses of green tea catechins and grape seed procyanidins to ameliorate some of the most characteristic histological changes of gliadin-treated DQ8 mice, including villus flattening, crypt hyperplasia, and infiltration of intraepithelial lymphocytes. Mechanistically, polyphenols were found to increase the intestinal nucleophilic tone of DQ8 mice by orchestrating an adaptive antioxidant response characterized by enhanced GSR enzyme activity and GSH content. Taken together, this work constitutes a highly relevant breakthrough as it provides the fundamental basis concerning the significance of natural polyphenols to be used in, for instance, the development of innovative functional foods aimed at CD individuals.

Retro-inverso D-peptides as a novel targeted immunotherapy for Type 1 diabetes

Over the past four decades, the number of people with Type 1 Diabetes (T1D) has increased by 4% per year, making it an important public health challenge. Currently, no curative therapy exists for T1D and the only available treatment is insulin replacement. HLA-DQ8 has been shown to present antigenic islet peptides driving the activation of CD4+ T-cells in T1D patients. Specifically, the insulin peptide InsB:9-23 activates self-reactive CD4+ T-cells, causing pancreatic beta cell destruction. The aim of the current study was to identify retro-inverso-d-amino acid based peptides (RI-D-peptides) that can suppress T-cell activation by blocking the presentation of InsB:9-23 peptide within HLA-DQ8 pocket. We identified a RI-D-peptide (RI-EXT) that inhibited InsB:9-23 binding to recombinant HLA-DQ8 molecule, as well as its binding to DQ8 expressed on human B-cells. RI-EXT prevented T-cell activation in a cellular antigen presentation assay containing human DQ8 cells loaded with InsB:9-23 peptide and murine T-cells expressing a human T-cell receptor specific for the InsB:9-23–DQ8 complex. Moreover, RI-EXT blocked T-cell activation by InsB:9-23 in a humanized DQ8 mice both ex vivo and in vivo, as shown by decreased production of IL-2 and IFN-γ and reduced lymphocyte proliferation. Interestingly, RI-EXT also blocked lymphocyte activation and proliferation by InsB:9-23 in PBMCs isolated from recent onset DQ8-T1D patients. In summary, we discovered a RI-D-peptide that blocks InsB:9-23 binding to HLA-DQ8 and its presentation to T-cells in T1D. These findings set the stage for using our approach as a novel therapy for patients with T1D and potentially other autoimmune diseases.

Prevotella histicola treatment reduces arthritic pain and partially normalizes gut microbiota and metabolites

Abstract We have previously shown that treatment with a novel human commensal, Prevotella histicola, isolated from a duodenal biopsy, can protect HLA-DQ8 mice from collagen induced arthritis (CIA), a model for RA, by modulating the systemic immune response. The present study analyzed the functional and mechanistic impact of P. histicola by comparing pain, motor function, gut microbiota and metabolites in treated and non-treated arthritic DQ8 mice. P. histicola treatment reduced the arthritis-associated pain by improving the motor function when compared to control mice. Analysis of synovium showed that P. histicola treatment increased T regulatory cells and reduced the levels of IL6 in the synovium of arthritic mice which were associated with reduced pain. Treated mice showed P. histicola colonization in the duodenum, a niche from where it was isolated. Further, P. histicola treatment showed varied effects based on circadian rhythm. A longitudinal follow-up of the gut microbial profile in different gut sections suggested that dysbiosis caused due to arthritis in DQ8 is partly normalized to naïve profile after treatment with P. histicola. Treated mice showed an expansion of genus Allobaculum, a butyrate-producing Firmicutes in the guts of DQ8 arthritic mice as compared to pre-treatment. The post-treatment restoration was also reflected in short-chain fatty acids and glucose assimilation rates in T cells. The present study proposes that P. histicola treatment of arthritis in DQ8 mice leads to gut homeostasis. Normalized microbial diversity and metabolites lead to immune regulation resulting in reduced inflammation with improved physical function. The observations suggest P. histicola as a strong candidate for monoclonal bio-therapeutics.

Effector T cell stimulation using Staphylococcal enterotoxin G and I loaded HLA transgenic dendritic cells

Abstract Staphylococcal enterotoxins (SE) induce powerful T cell stimulation with specificity for the variable beta (Vβ) region of the T cell receptor (TCR). Additionally, MHC in mice (H2) and humans (human leukocyte antigen; HLA) differ in binding superantigens and, in turn, provide different T cell activating potentials. We have previously demonstrated that SEG and SEI, administered together, elicited progression free survival of B16–F10 challenged HLA-DQ8 (DQ8; DQA1*0301 and DQB1*0302) transgenic mice. C57BL/6 (B6) mice did not benefit from SEG/SEI given post B16–F10 challenge, as expected, given a markedly reduced T cell proliferation in the B6 mice compared to the DQ8 mice. We provide evidence here of the potential of dendritic cells (DCs) to be the vector for a SEG and SEI immunotherapy. Primary antigen presenting cells, DCs and monocytes, were isolated from the bone marrow of DQ8 or HLA-DR3 (DRB1*0301) transgenic mice. SEG/SEI loaded DQ8 and DR3 DCs drove a strong B6 CD8 T cell response at low T cell:DC ratios; whereas both DQ8 and DR3 DCs at a high T cell:DC ratios activated CD4 T cells. Collectively, these data support incorporating SEG and SEI into the current DC based immunotherapeutic strategies to induce a stronger and direct anti-cancer T cell response.

Autoimmunity-Associated Gut Commensals Modulate Gut Permeability and Immunity in Humanized Mice.

Although the etiology of rheumatoid arthritis (RA) is unknown, recent studies have led to the concept that gut dysbiosis may be involved in onset. In this study, we aimed to determine if human gut commensals modulate the immune response and gut epithelial integrity in DQ8 mice. DQ8 mice were orally gavaged with RA-associated (Eggerthella lenta or Collinsella aerofaciens) and non-associated (Prevotella histicola or Bifidobacterium sp.) on alternate days for 1 week in naïve mice. Some mice were immunized with type II collagen and oral gavage continued for 6 weeks and followed for arthritis. Epithelial integrity was done by FITC-Dextran assay. In addition, cytokines were measured in sera by ELISA and various immune cells were quantified using flow cytometry. Gut permeability was increased by the RA-associated bacteria and was sex and age-dependent. In vivo and in vitro observations showed that the RA-non-associated bacteria outgrow the RA-associated bacteria when gavaged or cultured together. Mice gavaged with the RA-non-associated bacteria produced lower levels of pro-inflammatory MCP-1 and MCP-3 and had lower numbers of Inflammatory monocytes CD11c+Ly6c+, when compared to controls. E. lenta treated naïve mice produce Th17 cytokines. Our studies suggest that gut commensals influence immune response in and away from the gut by changing the gut permeability and immunity. Dysbiosis helps the growth of RA-associated bacteria and reduces the beneficial bacteria.

Mechanism of Th1/Th2 Cytokines in HLA-DQ8 Transgenic Mice Model of Ocular Experimental Autoimmune Myasthenia Gravis

Objective To analyze the levels of cytokines (IL-2,IFN-γ,IL-6,IL-10) associated with Th1 and Th2 cells in HLA-DQ8 transgenic mice model of ocular experimental autoimmune myasthenia gravis (oEAMG) induced by recombinant H-AChR γ subunit immunization.Methods DQ8 mice were immunized with 20 μg of AChR γ subunit,20 μg of crude E. coli extract (E. coli group),or complete Freund's adjuvant (CFA) only (CFA group). All mice were immunized on days 0,30,and 60. Mice were euthanized 28 days after the third immunization,and draining lymph node cells (LNC) and spleen lymphocytes were cultured in vitro. The supernatant was collected to observe the interleukin(IL)-2,interferon(IFN)-γ,IL-6,IL-10 production by ELISA.Results LNCs and spleen lymphocytes of H-AChR γ subunit-immunized mice exhibited significantly enhanced IFN-γ (F=76.332,P<0.001;F=34.865,P<0.001) and IL-2 (F=42.835,P<0.001;F=38.030,P<0.001),which associated with Th1 cells,as compared to E. coli group and CFA group. There were no significant differences in IL-6 (F=1.325,P=0.284;F=1.935,P=0.166) and IL-10 (F=0.908,P=0.417;F=1.189,P=0.322) levels,which secreted by Th2 cells,among these three groups.Conclusion Th1 cytokines play key roles in the pathogenesis of oEAMG,while the mechanism of Th2 cytokines for oEAMG remains unclear.

Major Histocompatibility Complex Class II Alleles Influence Induction of Pathogenic Antiphospholipid Antibodies in a Mouse Model of Thrombosis.

Both environmental and genetic factors are important in the development of antiphospholipid antibodies (aPL) in patients with antiphospholipid syndrome (APS). Currently, the only available data on predisposing genetic factors have been obtained from epidemiologic studies, without mechanistic evidence. Therefore, we studied the influence of major histocompatibility complex (MHC) class II alleles on the production of aPL in a mouse model of APS. Three groups of mice, MHC class II-deficient (MHCII-/- ) mice, MHCII-/- mice transgenic for human HLA-DQ6 (DQ6), DQ8, or DR4 alleles, and the corresponding wild-type (WT) mouse strains were immunized; half were immunized with human β2 -glycoprotein I (β2 GPI), and the other half were immunized with control ovalbumin (OVA) protein. Thrombus formation in vivo, tissue factor activity in carotid and peritoneal macrophages, and serum levels of tumor necrosis factor (TNF), IgG anticardiolipin (aCL), antibodies, and anti-OVA antibodies were determined. Immunization with β2 GPI induced significant production of aCL and anti-β2 GPI in WT mice compared with control mice immunized with OVA (P < 0.001) but diminished aCL (P < 0.001) and anti-β2 GPI (P = 0.016) production in MHCII-/- mice. Anti-β2 GPI production was fully restored in DQ6 and DQ8 mice, while levels of anti-β2 GPI in DR4 mice and aCL in all transgenic lines were only partially restored (P < 0.001 to P < 0.046). Thrombus size in WT mice was twice that in MHCII-/- mice (P < 0.001) but similar to that in all transgenic lines. Carotid and peritoneal macrophage tissue factor levels decreased by >50% in MHCII-/- mice compared with wild-type B6 mice and were restored in DQ8 mice but not DR4 mice or DQ6 mice. TNF levels decreased 4-fold in MHCII-/- mice (P < 0.001) and were not restored in transgenic mice. Our mechanistic study is the first to show that MHC class II alleles influence not only quantitative aPL production but also the pathogenic capacity of induced aPL.

Suppression of Inflammatory Arthritis by Human Gut-Derived Prevotella histicola in Humanized Mice.

The gut microbiome regulates host immune homeostasis. Rheumatoid arthritis (RA) is associated with intestinal dysbiosis. This study was undertaken to test the ability of a human gut-derived commensal to modulate immune response and treat arthritis in a humanized mouse model. We isolated a commensal bacterium, Prevotella histicola, that is native to the human gut and has systemic immune effects when administered enterally. Arthritis-susceptible HLA-DQ8 mice were immunized with type II collagen and treated with P histicola. Disease incidence, onset, and severity were monitored. Changes in gut epithelial proteins and immune response as well as systemic cellular and humoral immune responses were studied in treated mice. When treated with P histicola in prophylactic or therapeutic protocols, DQ8 mice exhibited significantly decreased incidence and severity of arthritis compared to controls. The microbial mucosal modulation of arthritis was dependent on regulation by CD103+ dendritic cells and myeloid suppressors (CD11b+Gr-1+ cells) and by generation of Treg cells (CD4+CD25+FoxP3+) in the gut, resulting in suppression of antigen-specific Th17 responses and increased transcription of interleukin-10. Treatment with P histicola led to reduced intestinal permeability by increasing expression of enzymes that produce antimicrobial peptides as well as tight junction proteins (zonula occludens 1 and occludin). However, the innate immune response via Toll-like receptor 4 (TLR-4) and TLR-9 was not affected in treated mice. Our results demonstrate that enteral exposure to P histicola suppresses arthritis via mucosal regulation. P histicola is a unique commensal that can be explored as a novel therapy for RA and may have few or no side effects.

Protective antibodies against a sphingomyelinase D from Loxosceles intermedia spider venom elicited in mice with different genetic background

In the present investigation we used a recombinant LiD1 toxin, named rLiD1his, from Loxosceles intermedia brown spider to elicit specific antibodies in mice carrying different Human Leukocyte Antigens class II (HLAII) {DRB1.0401 (DR4), DQB1.0601 (DQ6) and DQB1.0302 (DQ8)} as well as in BALB/C and C57BL/6 control mice. All mice strains produced high antibody titers against rLiD1his but DR4 mice antibodies (the lower responder mice) were not able to recognize L. intermedia crude venom. The anti-rLiD1his sera, except from DR4 mice, were able to neutralize dermonecrotic, hemorrhagic and edematogenic effects of rLiD1his in naïve rabbits. Overlapping peptides from the amino acid sequence of LiD1 toxin were prepared by SPOT method and differences in LiD1 epitope recognition were observed using different mice anti-rLiD1his sera. The region 160DKVGHDFSGNDDISDVGK177 was recognized by transgenic DQ8 and DQ6 mice sera. Other epitopes were recognized by at least two different animals’ sera including 10MGHMVNAIGQIDEFVNLG27, 37FDDNANPEYTYHGIP51, 70GLRSATTPGNSKYQEKLV87 and 259AAYKKKFRVATYDDN273. Among these epitopes, the epitopes 37–51 and 160–177 have already been shown in previously studies as good candidates to be used alone or combined with other peptides to induce protective immune response against Loxosceles venoms. The results presented here highlight the importance of HLAII in antibody response and recognition of specific B-cell epitopes of rLiD1his spider toxin according to HLAII type and impact in the epitopic vaccine development against this spider.

B cells influence sex specificity of arthritis via myeloid suppressors and chemokines in humanized mice

Rheumatoid arthritis (RA) occurs two times more often in women than men. B cell depletion has been shown to be efficacious in treating RA. Our previous studies suggested that antigen presentation via B cells results in a sex-specific immune response in DR4 and DR4/DQ8 mice. Here we evaluated the mechanism of efficacy of the B cell depletion in treating arthritis-susceptible DQ8 mice. The data show that arthritic DQ8 mice treated with anti-CD20 antibody in therapeutic protocols show milder disease severity in females as compared to males, which is associated with decreased antibodies to citrullinated proteins and reduced levels of IL-23 and CCL5. Treatment led to significantly increased numbers of T regulatory and monocyte-derived suppressor F4/80+Gr1hi cells in females as compared to male DQ8 mice. Our observations suggest that therapeutic strategies that target B cells may benefit females while functions of DCs might be relatively more important for men than women.

Mucosal immunity induced by gliadin-presenting spores of Bacillus subtilis in HLA-DQ8-transgenic mice

The induction of mucosal immunity requires efficient antigen delivery and adjuvant systems. Probiotic bacterial strains are considered to be very promising tools to address both of these needs. In particular, Bacillus subtilis spores are currently under investigation as a long-lived, protease-resistant adjuvant system for different antigens. Furthermore, a non-recombinant approach has been developed based on the stable adsorption of antigen on the spore surface. In the present study, we explored this strategy as a means of modulating the immune response to wheat gliadin, the triggering agent of celiac disease (CD), an enteropathy driven by inflammatory CD4+ T cells. Gliadin adsorption was tested on untreated or autoclaved wild-type (wt) and mutant (cotH or cotE) spores. We found that gliadin was stably and maximally adsorbed by autoclaved wt spores. We then tested the immune properties of the spore-adsorbed gliadin in HLA–DQ8-transgenic mice, which express one of the two HLA heterodimers associated with CD. In vitro, spore-adsorbed gliadin was efficiently taken up by mouse dendritic cells (DCs). Interestingly, gliadin-pulsed DCs efficiently stimulated splenic CD4+ T cells from mice immunised with spore-adsorbed gliadin. Nasal pre-dosing with spore-adsorbed gliadin failed to down-regulate the ongoing cellular response in gliadin-sensitised DQ8 mice. Notably, naïve mice inoculated intranasally with multiple doses of spore-adsorbed gliadin developed an intestinal antigen-specific CD4+ T cell-mediated response. In conclusion, our data highlight the ability of spore-adsorbed gliadin to elicit a T-cell response in the gut that could be exploitable for developing immune strategies in CD.

Sex-dependent response to treatment for arthritis in humanized mice (THER5P.903)

Abstract Rheumatoid arthritis (RA) occurs twice more often in women than men. B cell depletion has been shown to be efficacious in treating RA though it is unknown if efficacy is dependent on the sex of the patient. Our previous studies suggested that antigen presentation via B cells results in a sex-specific immune response and cytokines in humanized mice. Now we show that the efficacy of B cell depletion in arthritic DQ8 mice is sex-dependent. Arthritic female DQ8 mice treated with anti-CD20 antibody in therapeutic protocols show milder disease severity associated with decreased antibodies to citrullinated proteins along with increased numbers of T regulatory as compared to male DQ8 mice. The milder disease severity in CD20-depleted females was associated with reduced levels of IL-23 and CCL5 with increased IL-13 as compared to males. Treated male mice showed higher numbers of subsets of dendritic cells, known producers of pro-inflammatory cytokines, as compared to females. A window where males can benefit from B cell depletion needs to be defined. Our observations suggest that in therapeutic strategies that target B cells during established disease may benefit females while DCs might be relatively more important for men than women.

Identification of genes/pathways responsible for resistance vs susceptibility to EAE utilizing HLA class-II transgenic mice (BA8P.163)

Abstract MS is an autoimmune disease of the CNS and among all the genetic factors; MHC class-II molecules show the strongest association. To better understand the role of MHC class-II (HLA-DR/DQ) genes in susceptibility and resistance to MS, we utilized transgenic (Tg) mice expressing HLA class-II gene lacking endogenous mouse class II genes. HLA-DR3 Tg mice are susceptible to PLP91-110 induced experimental autoimmune encephalomyelitis (EAE), whereas DQ8 (DQB1*0302) transgenic mice are resistant. To identify the gene expression changes and pathways required for disease susceptibility vs resistance to EAE, we performed mRNA expression profiling of CD4+ T cells from HLA-DR3 (susceptible) and HLA-DQ8 (resistant) Tg mice. Whole genome expression analysis identified 2945 genes in HLA-DR3 Tg mice (PLP91-110 immunized vs. naïve) and 1053 genes in HLA-DQ8 Tg mice (PLP91-110 immunized vs. naïve). Comparison of modulated genes between DR3 and DQ8 identified 772 genes common to both strains, 2173 genes unique to DR3 whereas 281 genes were unique to DQ8 mice. Ingenuity pathway analysis showed up regulation of pathways involved in T cell signaling in inflammation, NF-kB signaling and those responsible for communication between innate and adoptive immunity. Majority of these pathways have IL1β as a major upstream regulator in disease susceptible HLA-DR3 Tg mice. Thus microarray data indicates an important role of IL1β induced encephalitogenic T helper cell differentiation in disease susceptibility.

Adaptive response activated by dietary cis9, trans11 conjugated linoleic acid prevents distinct signs of gliadin-induced enteropathy in mice.

The beneficial effects of conjugated linoleic acid (CLA) mixture (cis9, trans11, c9; trans10, cis12, t10) against gliadin-induced toxicity in HLA-DQ8-transgenic mice (DQ8) have been associated with improved duodenal cytoprotective mechanisms [nuclear factor-E2-related factor-2, Nrf2; acylpeptide hydrolase (APEH)/proteasome]. The present study was aimed at investigating the ability of individual CLA isomers to improve the efficacy of these defensive mechanisms and to protect against duodenal injury caused by the combined administration of gliadin and indomethacin (GI). Gluten-mediated enteropathy was induced in DQ8 mice by three intra-gastric administration of gliadin (20 mg kg(-1)/bw) and indomethacin (15 mg L(-1)) in drinking water for 10 days (GI). C9 or t10 CLA (520 mg kg(-1)/bw/day) were orally administered for 2 weeks. Pro-oxidant and toxic effects associated with GI treatment, anti-oxidant/detoxifying ability of c9 or t10-CLA and the protective effect induced by c9 pre-treatment (c9 + GI) were evaluated in DQ8 mice duodenum by combining enzymatic, immunoblotting, histological evaluation and quantitative real-time PCR assays. GI treatment produces the time-dependent decline of the considered detoxifying mechanisms thus leading to pro-apoptotic and pro-oxidant effects. APEH/proteasome pathway was not markedly affected by individual CLA isomers, but duodenal redox status and activity/mRNA levels of Nrf2-activated enzymes were significantly improved by c9 administration. c9 pre-treatment protects against GI-mediated accumulation of oxidative stress markers, and histological examination reveals the increase of goblet cells number in mouse duodenum but induces only a partial recovery of APEH/proteasome activity. The activation of and adaptive response by low doses of c9 supplementation prevents distinct signs of gliadin-induced enteropathy in DQ8 mice.

Absence of IFN-γ Increases Brain Pathology in Experimental Autoimmune Encephalomyelitis–Susceptible DRB1*0301.DQ8 HLA Transgenic Mice through Secretion of Proinflammatory Cytokine IL-17 and Induction of Pathogenic Monocytes/Microglia into the Central Nervous System

Abstract Multiple sclerosis is an inflammatory, demyelinating disease of the CNS of presumed autoimmune origin. Of all the genetic factors linked with multiple sclerosis, MHC class II molecules have the strongest association. Generation of HLA class II transgenic (Tg) mice has helped to elucidate the role of HLA class II genes in chronic inflammatory and demyelinating diseases. We have shown that the human HLA-DRB1*0301 gene predisposes to proteolipid protein (PLP)–induced experimental autoimmune encephalomyelitis (EAE), whereas HLA-DQβ1*0601 (DQ6) was resistant. We also showed that the DQ6 molecule protects from EAE in DRB1*0301.DQ6 double-Tg mice by producing anti-inflammatory IFN-γ. HLA-DQβ1*0302 (DQ8) Tg mice were also resistant to PLP91–110–induced EAE, but production of proinflammatory IL-17 exacerbated disease in DRB1*0301.DQ8 mice. To further confirm the role of IFN-γ in protection, we generated DRB1*0301.DQ8 mice lacking IFN-γ (DRB1*0301.DQ8.IFN-γ−/−). Immunization with PLP91–110 peptide caused atypical EAE in DRB1*0301.DQ8.IFN-γ−/− mice characterized by ataxia, spasticity, and dystonia, hallmarks of brain-specific disease. Severe brain-specific inflammation and demyelination in DRB1*0301.DQ8.IFN-γ−/− mice with minimal spinal cord pathology further confirmed brain-specific pathology. Atypical EAE in DRB1*0301.DQ8.IFN-γ−/− mice was associated with increased encephalitogenicity of CD4 T cells and their ability to produce greater levels of IL-17 and GM-CSF compared with DRB1*0301.DQ8 mice. Further, areas with demyelination showed increased presence of CD68+ inflammatory cells, suggesting an important role for monocytes/microglia in causing brain pathology. Thus, our study supports a protective role for IFN-γ in the demyelination of brain through downregulation of IL-17/GM-CSF and induction of neuroprotective factors in the brain by monocytes/microglial cells.