Immune Cell Infiltration into the Eye Is Controlled by IL-10 in Recoverin-Induced Autoimmune Retinopathy.

Autoimmune retinopathy (AIR) is a treatable condition that manifests in acute and progressive vision loss in patients. It has recently been determined that AIR is associated with an imbalance of TH1 versus regulatory T cell immunity toward the retinal protein, recoverin. This study describes a new murine model to understand the immunopathology of AIR and its association with T cell responses toward recoverin. Immunization of C57BL/6 mice with recoverin resulted in ocular inflammation including infiltration of CD4+ and CD8+ T lymphocytes, B cells, and CD11b+Ly6C+ inflammatory monocytes in the eyes. Production of IFN-γ and IL-17 from T cells was exacerbated in IL-10 knockout (KO) mice and kinetics of disease development was accelerated. Infiltration of T cells and inflammatory monocytes into the eyes dramatically increased in recoverin-immunized IL-10 KO mice. An immunodominant peptide of recoverin, AG-16, was capable of inducing disease in IL-10 KO mice and resulted in expansion of AG-16 tetramer-specific CD4+ T cells in lymphoid organs and eyes. Adoptive transfer of recoverin-stimulated cells into naive mice was sufficient to induce AIR, and immunization of B cell-deficient mice led to a milder form of the disease. This model supports the hypothesis that recoverin-specific T cell responses are major drivers of AIR pathogenesis and that IL-10 is an important factor in protection.

T Helper 1 Cellular Immunity Toward Recoverin Is Enhanced in Patients With Active Autoimmune Retinopathy.

Autoimmune retinopathy (AIR) causes rapidly progressive vision loss that is treatable but often is confused with other forms of retinal degeneration including retinitis pigmentosa (RP). Measurement of anti-retinal antibodies (ARA) by Western blot is a commonly used laboratory assay that supports the diagnosis yet does not reflect current disease activity. To search for better diagnostic indicators, this study was designed to compare immune biomarkers and responses toward the retinal protein, recoverin, between newly diagnosed AIR patients, slow progressing RP patients and healthy controls. All individuals had measurable anti-recoverin IgG and IgM antibodies by ELISA regardless of disease status or Western blot results. Many AIR patients had elevated anti-recoverin IgG1 levels and a strong cellular response toward recoverin dominated by IFNγ. RP patients and controls responded to recoverin with a lower IFNγ response that was balanced by IL-10 production. Both AIR and RP patients displayed lower levels of total peripheral blood mononuclear cells that were due to reductions of CD4+ TH cells. A comparison of messenger RNA (mRNA) for immune-related genes in whole blood of AIR patients versus RP patients or controls indicated lower expression of ATG5 and PTPN22 and higher expression of several genes involved in TH cell signaling/transcription and adhesion. These data indicate that an immune response toward recoverin is normal in humans, but that in AIR patients the balance shifts dramatically toward higher IFNγ production and cellular activation.

IL-22, cell regeneration and autoimmunity.

IL-22 as a cytokine is described with opposing pro-inflammatory and anti-inflammatory functions. Cell regeneration, tissue remodelling and balance between commensal bacteria in the gut and host immune system are considered as anti-inflammatory features of IL-22, whereas production of IL-22 from Th17 cells links this cytokine to pro-inflammatory pathways. Th17 cells and group 3 innate lymphoid cells (ILC3) are two major producers of IL-22 and both cell types express ROR-γt and Aryl hydrocarbon receptor (AhR) transcription factors. Typically, the immune system cells are the main producers of IL-22. However, targets of this cytokine are mostly non-hematopoietic cells such as hepatocytes, keratinocytes, and epithelial cells of lung and intestine. Association of IL-22 with other cytokines or transcription factors in different cell types might explain its contrasting role in health and disease. In this review we discuss the regulation of IL-22 production by AhR- and IL-23-driven pathways. A clear understanding of the biology of IL-22 will provide new opportunities for its application to improve human health involving many debilitating conditions.

Reciprocity in microbiome and immune system interactions and its implications in disease and health.

Adaptation of the whole microbial normal flora residing in a host to its natural habitat over an evolutionary peroid has resulted in peaceful coexistence with mutual benefits for both microbiota and host in steady state. This symbiotic relationship between host and microbiota has a significant impact on shaping the immune response in the host to achieve an immune tolerance to microbiota but retaining the ability to respond to invading pathogens. Perturbation of this balance by manipulation of microbial communities in the host can lead to immune dysregulation and susceptibility to diseases. By studying the host in the absence of microbiota or with alteration of microbiota the complexity of microbial impact on the immune system can be resolved. Conversely, the study of microbiota in the absence of immune system factors can show how the immune system contributes to preservation of the host-microbiota balance. The absence of molecules involved in innate or adaptive immunity in knockout models can perturb the balance between host and microbiota further adding to more immune dysregulation. A better understanding of Microbiome-immune system interaction provides a new opportunity to identify biomarkers and drug targets. This will allow the development of new therapeutic agents for modulating the immune system to improve health with little or no toxicity. The study of interplay between host and microbiota has a promising role in the design of therapeutic interventions for immunopathological diseases arising from imbalanced host and microbiota interactions.

Vasostatin-1 antigenic epitope mapping for induction of cellular and humoral immune responses to chromogranin A autoantigen in NOD mice.

The chromogranin A (ChgA) 29-42 sequence is the antigenic epitope for the BDC2.5 CD4(+) T-cell receptor in NOD mice (H-2(g7) ). We have now characterized the binding register of the ChgA 29-42 peptide for the I-A(g7) molecule. Truncation of the peptide demonstrated that the KCVLEVISD sequence 34-42 is the binding register and extension of this sequence by flanking residues increased its binding affinity and antigenic capacity. We employed anti-ChgA peptide antibodies generated against different fragments of ChgA for immunostaining of pancreatic islet sections from NOD mice. A strong immuno-staining pattern was observed for the ChgA 17-38 peptide antibodies that overlap with the ChgA 29-42 sequence. Moreover, sera from diabetic NOD mice showed elevated titers of autoantibodies to the ChgA 29-42 peptide. These findings indicate that peptides from the N-terminal region of ChgA are able to induce cellular and humoral immune responses in NOD mice.

Modulation of autoimmune diseases by interleukin (IL)-17 producing regulatory T helper (Th17) cells.

Following the discovery of interleukin (IL)-17 producing T helper (Th17) cells as a distinct lineage of CD4+ T helper cells it became clear that these cells play an important role in the host defense against extracellular fungal and bacterial pathogens and participate in the pathogenesis of multiple inflammatory and autoimmune disorders. Depending on the microenvironment, Th17 cells can alter their differentiation programme ultimately giving rise to either protective or pro-inflammatory pathogenic cells. We found that besides the conventional in vitro protocol for Th17 differentiation by transforming growth factor-beta (TGF-ß) plus IL-6 cytokines, a combination of IL-23 plus IL-6 can also induce Th17 cells. The Th17 cells induced by IL-23 plus IL-6 (termed as effector Th17, Teff17 cells) are pathogenic upon adoptive transfer into non-obese diabetic (NOD) mice contributing to the development of type 1 diabetes (T1D) while cells induced by TGF-ß plus IL-6 (termed as regulatory T cells, Treg17 cells) are non pathogenic and regulatory, and suppressed the pathogenic T cells in T1D. These cells differentially expressed a number of cytokines where Teff17 cells exhibited an increase in granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-22 whereas Treg17 cells demonstrated increased expression of IL-21 and immunosuppressive cytokine IL-10. Differentiation of Th17 cells is controlled by a transcription factor, RORγT although these cells also express variable levels of T-bet and FoxP3 transcription factors. This points to a dual functional role of Th17 subsets in autoimmune diseases particularly T1D. We suggest that similar to conventional regulatory T cells (Treg), induction of regulatory Treg17 cells could play an important role in modulating and preventing certain autoimmune diseases.

The involvement of interleukin-22 in the expression of pancreatic beta cell regenerative Reg genes.

BACKGROUND: In Type 1 diabetes, the insulin-producing ß-cells within the pancreatic islets of Langerhans are destroyed. We showed previously that immunotherapy with Bacillus Calmette-Guerin (BCG) or complete Freund's adjuvant (CFA) of non-obese diabetic (NOD) mice can prevent disease process and pancreatic ß-cell loss. This was associated with increased islet Regenerating (Reg) genes expression, and elevated IL-22-producing Th17 T-cells in the pancreas. RESULTS: We hypothesized that IL-22 was responsible for the increased Reg gene expression in the pancreas. We therefore quantified the Reg1, Reg2, and Reg3δ (INGAP) mRNA expression in isolated pre-diabetic NOD islets treated with IL-22. We measured IL-22, and IL-22 receptor(R)-α mRNA expression in the pancreas and spleen of pre-diabetic and diabetic NOD mice. Our results showed: 1) Reg1 and Reg2 mRNA abundance to be significantly increased in IL-22-treated islets in vitro; 2) IL-22 mRNA expression in the pre-diabetic mouse pancreas increased with time following CFA treatment; 3) a reduced expression of IL-22Rα following CFA treatment; 4) a down-regulation in Reg1 and Reg2 mRNA expression in the pancreas of pre-diabetic mice injected with an IL-22 neutralizing antibody; and 5) an increased islet ß-cell DNA synthesis in vitro in the presence of IL-22. CONCLUSIONS: We conclude that IL-22 may contribute to the regeneration of ß-cells by up-regulating Regenerating Reg1 and Reg2 genes in the islets.

Association of interleukin-2, interleukin-4 and transforming growth factor-beta gene polymorphisms with Behcet's disease.

Behçet's disease (BD) is a chronic immune-mediated disease, characterised by oral and genital lesions and ocular inflammation. As cytokines seem to have important roles in the pathogenesis of BD and production of cytokines could be affected by genetic polymorphisms, this study was performed to investigate gene polymorphisms of a number of cytokines in the patients with BD in comparison with control subjects. One hundred and fifty patients with BD were enrolled in this study. Interleukin (IL)-2 (-330, +166), IL-4 (-1098, -590, -33), IL-10 (-1082, -819, -592), IL-12 (-1188), IFN-γ (5644), transforming growth factor (TGF)-ß (codon 10, 25), and IL-4RA (+1902) typing were performed by polymerase chain reaction with sequence-specific primers. In the patients with BD, there were significantly increased frequency of IL-2 (-330) GG genotype (p<0.001), IL-4 (-33) CC genotype (p<0.001), and TGF-ß (codon 10) CC genotype (p=0.004). Meanwhile a significant decrease in the frequency of IL-4 (-33) TC genotype (p<0.001) was detected in the patient group in comparison with normal controls. The genotype CC of TGF-ß at codon 10 was also significantly overrepresented in the patient group (p=0.004). Haplotype frequencies of IL-4 (-1098, -590, -33) showed that the frequency of TTC haplotype was significantly increased in the patients (p<0.001), whereas TTT haplotype was significantly decreased in this group of patients (p<0.001). There was not any significant difference in allele and genotype frequencies of IL-10, IL-12, IFN-γ, and IL-4RA between patient and control groups. Cytokine single nucleotide polymorphisms could play a role in the pathophysiology of BD. The results of this study could suggest a tendency towards higher production of IL-2 and lower production of IL-4 in the patients with BD.

Immunomodulation and regeneration of islet Beta cells by cytokines in autoimmune type 1 diabetes.

Juvenile or type 1 diabetes (T1D) involves autoimmune-mediated destruction of insulin-producing ß cells in the islets of Langerhans in the pancreas. Lack of insulin prevents the absorption and metabolism of glucose throughout the body by interfering with cell signaling. Cytokines have been shown to play a key role in ß cell destruction and regulation of autoimmunity in T1D. The multiple roles of cytokines in T1D pathogenesis, regulation, and regeneration of ß cells presents both promise and challenge for their use in immunotherapy. We found that mycobacterial adjuvants induce various regulatory T cells in the non-obese diabetic (NOD) mouse model of T1D. Cytokines produced by these cells not only regulate innate and adaptive immunity but also prevent the development of diabetes and partially restored normoglycemia in diabetic NOD mice. We discovered that adjuvant immunotherapy upregulated Regenerating (Reg) genes in the islets and induced interleukin 22 (IL-22)-producing Th17 cells. IL-22 is known to upregulate Reg gene expression in islets and could potentially induce regeneration of ß cells and prevent their apoptosis. Therefore, cytokines both induce and regulate T1D and have the potential to regenerate and preserve insulin-producing ß cells in the islets.

Cutting edge: vasostatin-1-derived peptide ChgA29-42 is an antigenic epitope of diabetogenic BDC2.5 T cells in nonobese diabetic mice.

Mechanistic and therapeutic insights in autoimmune diabetes would benefit from a more complete identification of relevant autoantigens. BDC2.5 TCR transgenic NOD mice express transgenes for TCR Vα1 and Vß4 chains from the highly diabetogenic BDC2.5 CD4(+) T cell clone, which recognizes pancreatic ß cell membrane Ags presented by NOD I-A(g7) MHC class II molecules. The antigenic epitope of BDC2.5 TCR is absent in ß cells that do not express chromogranin A (ChgA) protein. However, characterization of the BDC2.5 epitope in ChgA has given inconclusive results. We have now identified a ChgA29-42 peptide within vasostatin-1, an N-terminal natural derivative of ChgA as the BDC2.5 TCR epitope. Having the necessary motif for binding to I-A(g7), it activates BDC2.5 T cells and induces an IFN-γ response. More importantly, adoptive transfer of naive BDC2.5 splenocytes activated with ChgA29-42 peptide transferred diabetes into NOD/SCID mice.

Proinflammatory cytokine gene polymorphisms in Behçet's disease.

Behçet's disease (BD) is a chronic, systemic disease, characterized by oral and genital lesions, and ocular inflammation. There is evidence indicating altered levels of proinflammatory cytokines, such as interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α) in patients with BD. This study involved 150 patients with BD and 140 healthy controls, and investigated the role of proinflammatory cytokine gene polymorphisms in the disease. The frequency of the TNF-α (-238) G/G genotype was significantly higher in the patient group, compared to the controls (p < 0.001), whilst the G/A genotype was significantly lower in the patients with BD (p < 0.001). Patients with BD showed a significant increase in the TNF-α (- 308, - 238) GG haplotype (p < 0.001), whilst there was a significant decrease in the GA haplotype (p < 0.001). The heterozygous, IL-6 (- 174) C/G genotype (p = 0.005), and the IL-6 (- 174, nt565) haplotype CG (p < 0.001), were significantly decreased in the patient group. The increased production of proinflammatory cytokines in BD could be a consequence of specific, cytokine gene polymorphisms. Particular genotypes and haplotypes in TNF-α were over-represented in BD, which may, in turn, predispose individuals to this disease.

Adjuvant immunotherapy increases beta cell regenerative factor Reg2 in the pancreas of diabetic mice.

Insulin-producing ß cells can partially regenerate in adult pancreatic tissues, both in human and animal models of type 1 diabetes (T1D). Previous studies have shown that treatment with mycobacterial adjuvants such as CFA and bacillus Calmette-Guérin prevents induction and recurrence of T1D in NOD mice with partial recovery of ß cell mass. In this study, we investigated factors involved in the regeneration of ß cells in the pancreas of NOD mice during diabetes development and after treatment with adjuvants. The Regeneration (Reg) gene family is known to be involved in regeneration of various tissues including ß cells. Reg2 expression was found to be upregulated in pancreatic islets both during diabetes development and as a result of adjuvant treatment in diabetic NOD mice and in C57BL/6 mice made diabetic by streptozotocin treatment. The upregulation of Reg2 by adjuvant treatment was independent of signaling through MyD88 and IL-6 because it was not altered in MyD88 or IL-6 knockout mice. We also observed upregulation of Reg2 in the pancreas of diabetic mice undergoing ß cell regenerative therapy with exendin-4 or with islet neogenesis-associated protein. Reg2 expression following adjuvant treatment correlated with a reduction in insulitis, an increase in insulin secretion, and an increase in the number of small islets in the pancreas of diabetic NOD mice and with improved glucose tolerance tests in streptozotocin-treated diabetic C57BL/6 mice. In conclusion, adjuvant immunotherapy regulates T1D in diabetic mice and induces Reg2-mediated regeneration of ß cells.

Th17 polarized cells from nonobese diabetic mice following mycobacterial adjuvant immunotherapy delay type 1 diabetes.

IL-17-producing T cells are regarded as potential pathogenic T cells in the induction of autoimmune diseases. Previously, we have shown that injection of adjuvants containing Mycobacterium, such as CFA or bacillus Calmette-Guérin, can prevent type 1 diabetes in NOD mice. We injected NOD mice with mycobacterial products s.c. and analyzed the IL-17-producing cells from the draining lymph nodes and spleen by restimulating whole-cell populations or CD4(+) T cells in vitro with or without IL-17-polarizing cytokines. Mice receiving CFA had a concomitant rise in the level of IL-17, IL-22, IL-10, and IFN-gamma in the draining lymph node and spleen. Adoptive transfer of splenocytes from CFA-injected NOD mice polarized with TGF-beta plus IL-6 or IL-23 delayed the development of diabetes in recipient mice. IL-17-producing cells induced by CFA maintained their IL-17-producing ability in the recipient mice. Injection of CFA also changed the cytokine profile of cells in pancreatic tissue by increasing IL-17, IL-10, and IFN-gamma cytokine gene expression. We suggest that the rise in the level of IL-17 after adjuvant therapy in NOD mice has a protective effect on type 1 diabetes development.

Dendritic cell differentiation induced by a self-peptide derived from apolipoprotein E.

Dendritic cells (DCs) are professional APCs and potent stimulators of naive T cells. Since DCs have the ability to immunize or tolerize T cells they are unique candidates for use in immunotherapy. Our laboratory has discovered that a naturally processed self-peptide from apolipoprotein E, Ep1.B, induces DC-like morphology and surface marker expression in a murine monocytic cell line (PU5-1.8), human monocytic cell line (U937), murine splenocytes, and human peripheral blood monocytes. Microscopy and flow cytometric analysis revealed that Ep1.B-treated cells display decreased adherence to plastic and increased aggregation, dendritic processes, and expression of DC surface markers, including DEC-205, CD11c, B7.1, and B7.2. These effects were observed in both PU5-1.8 cells and splenocytes from various mouse strains including BALB/c, C57BL/6, NOD/Lt, and C3H/HeJ. Coadministration of Ep1.B with OVA antigenic peptide functions in dampening specific immune response to OVA. Ep1.B down-regulates proliferation of T cells and IFN-gamma production and stimulates IL-10 secretion in immunized mice. Ep1.B-induced differentiation resulted in the activation of PI3K and MAPK signaling pathways, including ERK1/2, p38, and JNK. We also found that NF-kappaB, a transcription factor essential for DC differentiation, is critical in mediating the effects of Ep1.B. Ep1.B-induced differentiation is independent of MyD88-dependent pathway of TLR signaling. Cumulatively, these findings suggest that Ep1.B acts by initiating a signal transduction cascade in monocytes leading to their differentiation into DCs.

Therapeutic benefits of regulating inflammation in autoimmunity.

Autoimmunity results from the dysregulation of the immune system leading to tissue damage. Th1 and Th17 cells are known to be cellular mediators of inflammation in autoimmune diseases. The specific cytokine milieu within the site of inflammation or within secondary lymphatic tissues is important during the priming and effector phases of T cell response. In this review, we will address the nature of the inflammatory response in the context of autoimmune disease, specifically we will discuss the role of dendritic cells following stimulation of their innate pathogen recognition receptors in directing the development of T cell responses. We will focus on how dendritic cell subsets change the balance between major players in autoimmunity, namely Th1, Th17 and regulatory T cells. Th17 cells, once thought to only act as pathogenic effectors through production of IL-17, have been shown to have regulatory properties as well with co-production of the anti-inflammatory cytokine IL-10 by a subset now referred to as regulatory Th17 cells. IL-17 is important in the induction of autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) and inflammatory bowel disease (IBD). Study of the inflammatory process following encounter with agents that stimulate the innate immune responses such as adjuvants opens a new horizon for the discovery of therapeutic agents including those derived from microorganisms. Microbial products such as adjuvants that function as TLR ligands may stimulate the immune system by interacting with Toll-like receptors (TLR) on antigen-presenting cells. Microbial agents such as Bacille Calmette-Guérin (BCG) or Freund's adjuvant (CFA) that induce a Th17 response are protective in models of autoimmune diseases particularly EAE and type 1 diabetes (T1D). The induction of innate immunity by these microbial products alters the balance in the cytokine microenvironment and may be responsible for modulation of the inflammation and protection from autoimmunity.