Govindarajan Rajagopalan

Low incidence of spontaneous type 1 diabetes in non-obese diabetic mice raised on gluten-free diets is associated with changes in the intestinal microbiome.

Human and animal studies strongly suggest that dietary gluten could play a causal role in the etiopathogenesis of type 1 diabetes (T1D). However, the mechanisms have not been elucidated. Recent reports indicate that the intestinal microbiome has a major influence on the incidence of T1D. Since diet is known to shape the composition of the intestinal microbiome, we investigated using non-obese diabetic (NOD) mice whether changes in the intestinal microbiome could be attributed to the pro- and anti-diabetogenic effects of gluten-containing and gluten-free diets, respectively. NOD mice were raised on gluten-containing chows (GCC) or gluten-free chows (GFC). The incidence of diabetes was determined by monitoring blood glucose levels biweekly using a glucometer. Intestinal microbiome composition was analyzed by sequencing 16S rRNA amplicons derived from fecal samples. First of all, GCC-fed NOD mice had the expected high incidence of hyperglycemia whereas NOD mice fed with a GFC had significantly reduced incidence of hyperglycemia. Secondly, when the fecal microbiomes were compared, Bifidobacterium, Tannerella, and Barnesiella species were increased (p = 0.03, 0.02, and 0.02, respectively) in the microbiome of GCC mice, where as Akkermansia species was increased (p = 0.02) in the intestinal microbiomes of NOD mice fed GFC. Thirdly, both of the gluten-free chows that were evaluated, either egg white based (EW-GFC) or casein based (C-GFC), significantly reduced the incidence of hyperglycemia. Interestingly, the gut microbiome from EW-GFC mice was similar to C-GFC mice. Finally, adding back gluten to the gluten-free diet reversed its anti-diabetogenic effect, reduced Akkermansia species and increased Bifidobacterium, Tannerella, and Barnesiella suggesting that the presence of gluten is directly responsible for the pro-diabetogenic effects of diets and it determines the gut microflora. Our novel study thus suggests that dietary gluten could modulate the incidence of T1D by changing the gut microbiome.

HLA Class II Transgenic Mice Mimic Human Inflammatory Diseases

Population studies have shown that among all the genetic factors linked with autoimmune disease development, MHC class II genes on chromosome 6 accounts for majority of familial clustering in the common autoimmune diseases. Despite the highly polymorphic nature of HLA class II genes, majority of autoimmune diseases are linked to a limited set of class II-DR or -DQ alleles. Thus a more detailed study of these HLA-DR and -DQ alleles were needed to understand their role in genetic predisposition and pathogenesis of autoimmune diseases. Although in vitro studies using class-II restricted CD4 T cells and purified class II molecules have helped us in understanding some aspects of HLA class-II association with disease, it is difficult to study the role of class II genes in vivo because of heterogeneity of human population, complexity of MHC, and strong linkage disequilibrium among different class II genes. To overcome this problem, we pioneered the generation of HLA-class II transgenic mice to study role of these molecule in inflammatory disease. These HLA class II transgenic mice were used to develop novel in vivo disease model for common autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, insulin-dependent diabetes mellitus, myasthenia gravis, celiac disease, autoimmune relapsing polychondritis, autoimmune myocarditis, thyroiditis, uveitis, as well as other inflammatory disease such as allergy, tuberculosis and toxic shock syndrome. As the T-cell repertoire in these humanized HLA transgenic mice are shaped by human class II molecules, they show the same HLA restriction as humans, implicate potential triggering mechanism and autoantigens, and identify similar antigenic epitopes seen in human. This review describes the value of these humanized transgenic mice in deciphering role of HLA class II molecules in immunopathogenesis of inflammatory diseases.

Intranasal exposure to staphylococcal enterotoxin B elicits an acute systemic inflammatory response.

ABSTRACT Staphylococcus aureus produces a variety of superantigen exotoxins, including staphylococcal enterotoxin B (SEB). Little is known regarding the pathogenesis of SEB entering through the intranasal route. Intranasal exposure to SEB might occur because of nasal packing following surgical procedure, biologic warfare, or even S. aureus colonization. We evaluated the local and systemic effects of intranasally delivered SEB using a series of human leukocyte antigen (HLA) class II transgenic mice as conventional mice expressing endogenous class II molecules mount a poor immune response to SEB. Gene expression profiling using microarrays showed robust up-regulation of genes involved in several proinflammatory pathways as early as 3 h post-intranasal challenge with SEB in HLA class II transgenic mice. This was accompanied by a several hundred-fold increase in serum levels of pro-inflammatory cytokines such as IL-12, IL-6, TNF-&agr;, IFN-&ggr;, as well as MCP-1 in HLA class II transgenic mice but not in C57BL/6 mice; CD4+ or CD8+ T-cells independently contributed to the systemic cytokine response. Defective IL-12 or IL-4 receptor signaling significantly decreased or increased serum IFN-&ggr;, respectively. Intranasal exposure to SEB resulted in neutrophil influx into bronchoalveolar lavage fluid and caused expansion of both CD4+ and CD8+ T-cells expressing TCR V&bgr;8 in the spleen. This was accompanied by mononuclear cell infiltration in the liver reminiscent of the systemic inflammatory response syndrome. Thus, we have shown, for the first time, that intranasal administration of SEB can cause systemic immune activation.ABBREVIATIONS-BAL, bronchoalveolar lavage, SEB, staphylococcal enterotoxin B, STAT, signal transducer and activator of transcription

Intranasal Exposure to Bacterial Superantigens Induces Airway Inflammation in HLA Class II Transgenic Mice

ABSTRACT Staphylococcus aureus is widely prevalent in the nasopharynges of healthy individuals (carriers) but can also cause serious infections. S. aureus can elaborate a variety of superantigen exotoxins in “carrier” or “pathogenic” states. Streptococcus pyogenes can also colonize the nasopharynx and elaborate superantigens. Unlike the acute effects of superantigen exotoxins absorbed through the gut or vaginal mucosa, little is known regarding the pathogenesis of superantigens entering through the intranasal route. In the current study, we evaluated the local and systemic effects of staphylococcal enterotoxin B (SEB) and streptococcal pyrogenic exotoxin A (SPEA) delivered through the intranasal route. Superantigens were administered intranasally on multiple occasions, and experimental animals were sacrificed on day 8 for experimental analyses. SEB-induced airway inflammation was more pronounced for HLA-DR3 transgenic mice than for BALB/c mice, consistent with bacterial superantigens binding more efficiently to human than murine major histocompatibility complex class II. The nature of the airway inflammation in HLA-DR3 mice was determined by the concentration of SEB applied intranasally. Low concentrations (20 ng) induced eosinophilic airway inflammation as well as eosinophil degranulation, whereas intranasal exposure to higher concentrations (2,000 ng) resulted in neutrophilic airway inflammation, permanent airway destruction, toxic shock, and mortality. SEB-induced eosinophilic inflammatory response was enhanced in signal transducer and activator of transcription (STAT)-4-deficient HLA-DQ8 transgenic mice with defective interleukin-12 signaling. Intranasal administration of SPEA induced airway inflammation and systemic immune activation in HLA-DQ8 transgenic mice. In conclusion, repeated chronic intranasal exposure to bacterial superantigens causes airway inflammation and systemic immune activation.

In vitro and in vivo evaluation of staphylococcal superantigen peptide antagonists.

ABSTRACT Superantigen peptide antagonists failed to block T-cell activation and cytokine production as well as toxic shock induced by staphylococcal enterotoxin B (SEB) in HLA class II transgenic mice. They also failed to inhibit the binding of SEB to HLA class II molecules as well as activation of human T lymphocytes in vitro.

Potent Neutralization of Staphylococcal Enterotoxin B by Synergistic Action of Chimeric Antibodies

ABSTRACT Staphylococcal enterotoxin B (SEB), a shock-inducing exotoxin synthesized by Staphylococcus aureus, is an important cause of food poisoning and is a class B bioterrorism agent. SEB mediates antigen-independent activation of a major subset of the T-cell population by cross-linking T-cell receptors (TCRs) with class II major histocompatibility complex (MHC-II) molecules of antigen-presenting cells, resulting in the induction of antigen independent proliferation and cytokine secretion by a significant fraction of the T-cell population. Neutralizing antibodies inhibit SEB-mediated T-cell activation by blocking the toxins interaction with the TCR or MHC-II and provide protection against the debilitating effects of this superantigen. We derived and searched a set of monoclonal mouse anti-SEB antibodies to identify neutralizing anti-SEB antibodies that bind to different sites on the toxin. A pair of non-cross-reactive, neutralizing anti-SEB monoclonal antibodies (MAbs) was found, and a combination of these antibodies inhibited SEB-induced T-cell proliferation in a synergistic rather than merely additive manner. In order to engineer antibodies more suitable than mouse MAbs for use in humans, the genes encoding the VL and VH gene segments of a synergistically acting pair of mouse MAbs were grafted, respectively, onto genes encoding the constant regions of human Igκ and human IgG1, transfected into mammalian cells, and used to generate chimeric versions of these antibodies that had affinity and neutralization profiles essentially identical to their mouse counterparts. When tested in cultures of human peripheral blood mononuclear cells or splenocytes derived from HLA-DR3 transgenic mice, the chimeric human-mouse antibodies synergistically neutralized SEB-induced T-cell activation and cytokine production.

Interferon Gamma-Dependent Intestinal Pathology Contributes to the Lethality in Bacterial Superantigen-Induced Toxic Shock Syndrome

Toxic shock syndrome (TSS) caused by the superantigen exotoxins of Staphylococcus aureus and Streptococcus pyogenes is characterized by robust T cell activation, profound elevation in systemic levels of multiple cytokines, including interferon-γ (IFN-γ), followed by multiple organ dysfunction and often death. As IFN-γ possesses pro- as well as anti-inflammatory properties, we delineated its role in the pathogenesis of TSS. Antibody-mediated in vivo neutralization of IFN-γ or targeted disruption of IFN-γ gene conferred significant protection from lethal TSS in HLA-DR3 transgenic mice. Following systemic high dose SEB challenge, whereas the HLA-DR3.IFN-γ+/+ mice became sick and succumbed to TSS, HLA-DR3.IFN-γ−/− mice appeared healthy and were significantly protected from SEB-induced lethality. SEB-induced systemic cytokine storm was significantly blunted in HLA-DR3.IFN-γ−/− transgenic mice. Serum concentrations of several cytokines (IL-4, IL-10, IL-12p40 and IL-17) and chemokines (KC, rantes, eotaxin and MCP-1) were significantly lower in HLA-DR3.IFN-γ−/− transgenic mice. However, SEB-induced T cell expansion in the spleens was unaffected and expansion of SEB-reactive TCR Vβ8+ CD4+ and CD8+ T cells was even more pronounced in HLA-DR3.IFN-γ−/− transgenic mice when compared to HLA-DR3.IFN-γ+/+ mice. A systematic histopathological examination of several vital organs revealed that both HLA-DR3.IFN-γ+/+ and HLA-DR3.IFN-γ−/− transgenic mice displayed comparable severe inflammatory changes in lungs, and liver during TSS. Remarkably, whereas the small intestines from HLA-DR3.IFN-γ+/+ transgenic mice displayed significant pathological changes during TSS, the architecture of small intestines in HLA-DR3.IFN-γ−/− transgenic mice was preserved. In concordance with these histopathological changes, the gut permeability to macromolecules was dramatically increased in HLA-DR3.IFN-γ+/+ but not HLA-DR3.IFN-γ−/− mice during TSS. Overall, IFN-γ seemed to play a lethal role in the immunopathogenesis of TSS by inflicting fatal small bowel pathology. Our study thus identifies the important role for IFN-γ in TSS.

Expression and Function of Transgenic HLA-DQ Molecules and Lymphocyte Development in Mice Lacking Invariant Chain

Invariant chain (Ii) is a non-MHC-encoded molecule, which plays an accessory role in the proper assembly/expression of functional MHC class II molecules and there by plays an important role in Ag processing/presentation. The phenotype of mice lacking Ii depends on the allotype of the MHC class II molecule. In some mice strains, Ii deficiency results in reduction in expression of class II molecules accompanied by defective CD4+ T cell development. Responses to conventional Ags/superantigens are also compromised. In this study, we describe for the first time the functionality of human class II molecules, HLA-DQ6 and HLA-DQ8, in transgenic mice lacking Ii. HLA transgenic Ii−/− mice expressed very low levels of surface DQ6 and DQ8 accompanied by severe reduction in CD4+ T cells both in the thymus and periphery. In vitro proliferation and cytokine production to an exogenous superantigen, staphylococcal enterotoxin B (SEB) was diminished in HLA-transgenic Ii−/− mice. However, SEB-induced in vivo expansion of CD8+ T cells expressing TCR Vβ8 family in DQ8.Ii−/− mice was comparable with that of DQ8.Ii+/+ mice. Systemic IFN-γ production following in vivo challenge with SEB was reduced in DQ8.Ii−/− mice and were also protected from SEB-induced toxic shock. Although the T cell response to a known peptide Ag was diminished in DQ8.Ii−/− mice, DQ8.Ii−/− APCs were capable of presenting that peptide to primed T cells from wild-type DQ8 mice as well as to a specific T cell hybridoma. Differentiation of mature B cells was also affected to a certain extent in DQ8.Ii−/− mice.

Modulation of insulitis and type 1 diabetes by transgenic HLA-DR3 and DQ8 in NOD mice lacking endogenous MHC class II.

To evaluate the contributions of DR3 and DQ8 to the etiopathogenesis of type 1 diabetes in a diabetes-predisposing milieu, we developed human leukocyte antigen (HLA) transgenic mice on the nonobese diabetic (NOD) background in the absence of the endogenous class II molecule, I-A(g7) and studied the incidence of both spontaneous and experimental (induced) autoimmune diabetes. Transgenic expression of HLA-DR3 and -DQ8 (either alone or in combination) did not confer susceptibility to spontaneous or cyclophosphamide-induced type 1 diabetes. Expression of I-A(g7) was mandatory for development of spontaneous or cyclophosphamide-induced diabetes. However, multiple low doses of streptozotocin could induce diabetes in all groups of mice independent of the class II molecules expressed. In unmanipulated mice, only islets from I-A(g7+/+) mice revealed significant intra-islet infiltration. Although a characteristic peri-insulitis/peri-ductulitis was present in Abeta(0)/NOD mice, islets from DR3, DQ8 and DR3 x DQ8 double transgenic mice demonstrated significantly less infiltration. In conclusion, transgenic expression of HLA-DR3 and -DQ8 associated with predisposition to type 1 diabetes alone is not sufficient to induce spontaneous diabetes in NOD mice lacking endogenous class II molecules.

HLA-DR3 restricted T cell epitope mimicry in induction of autoimmune response to lupus-associated antigen SmD

Although systemic lupus erythematosus (SLE) is a multigenic autoimmune disorder, HLA-D is the most dominant genetic susceptibility locus. This study was undertaken to investigate the hypothesis that microbial peptides bind HLA-DR3 and activate T cells reactive with lupus autoantigens. Using HLA-DR3 transgenic mice and lupus-associated autoantigen SmD protein, SmD(79-93) was identified to contain a dominant HLA-DR3 restricted T cell epitope. This T cell epitope was characterized by using a T-T hybridoma, C1P2, generated from SmD immunized HLA-DR3 transgenic mouse. By pattern search analysis, 20 putative mimicry peptides (P2-P21) of SmD(79-93,) from microbial and human origin were identified. C1P2 cells responded to SmD, SmD(79-93) and a peptide (P20) from Vibro cholerae. Immunization of HLA-DR3 mice with P20 induced T cell responses and IgG antibodies to SmD that were not cross-reactive with the immunogen. A T-T hybridoma, P20P1, generated from P20 immunized mice, not only responded to P20 and SmD(79-93), but also to peptides from Streptococcus agalactiae (P17) and human-La related protein (P11). These three T cell mimics (P20, P11 and P17) induced diverse and different autoantibody response profiles. Our data demonstrates for the first time molecular mimicry at T cell epitope level between lupus-associated autoantigen SmD and microbial peptides. Considering that distinct autoreactive T cell clones were activated by different microbial peptides, molecular mimicry at T cell epitope level can be an important pathway for the activation of autoreactive T cells resulting in the production of autoantibodies. In addition, the novel findings reported herein may have significant implications in the pathogenesis of SLE.