Clayton E. Mathews

MerTK is required for apoptotic cell-induced T cell tolerance.

Self-antigens expressed by apoptotic cells (ACs) may become targets for autoimmunity. Tolerance to these antigens is partly established by an ill-defined capacity of ACs to inhibit antigen-presenting cells such as dendritic cells (DCs). We present evidence that the receptor tyrosine kinase Mer (MerTK) has a key role in mediating AC-induced inhibition of DC activation/maturation. Pretreatment of DCs prepared from nonobese diabetic (NOD) mice with AC blocked secretion of proinflammatory cytokines, up-regulation of costimulatory molecule expression, and T cell activation. The effect of ACs on DCs was dependent on Gas6, which is a MerTK ligand. NOD DCs lacking MerTK expression (NOD.MerTKKD/KD) were resistant to AC-induced inhibition. Notably, autoimmune diabetes was exacerbated in NOD.MerTKKD/KD versus NOD mice expressing the transgenic BDC T cell receptor. In addition, β cell–specific CD4+ T cells adoptively transferred into NOD.MerTKKD/KD mice in which β cell apoptosis was induced with streptozotocin exhibited increased expansion and differentiation into type 1 T cell effectors. In both models, the lack of MerTK expression was associated with an increased frequency of activated pancreatic CD11c+CD8α+ DCs, which exhibited an enhanced T cell stimulatory capacity. These findings demonstrate that MerTK plays a critical role in regulating self-tolerance mediated between ACs, DCs, and T cells.

Sepsis Induces Early Alterations in Innate Immunity That Impact Mortality to Secondary Infection

Sepsis, the systemic inflammatory response to microbial infection, induces changes in both innate and adaptive immunity that presumably lead to increased susceptibility to secondary infections, multiorgan failure, and death. Using a model of murine polymicrobial sepsis whose severity approximates human sepsis, we examined outcomes and defined requirements for survival after secondary Pseudomonas aeruginosa pneumonia or disseminated Listeria monocytogenes infection. We demonstrate that early after sepsis neutrophil numbers and function are decreased, whereas monocyte recruitment through the CCR2/MCP-1 pathway and function are enhanced. Consequently, lethality to Pseudomonas pneumonia is increased early but not late after induction of sepsis. In contrast, lethality to listeriosis, whose eradication is dependent upon monocyte/macrophage phagocytosis, is actually decreased both early and late after sepsis. Adaptive immunity plays little role in these secondary infectious responses. This study demonstrates that sepsis promotes selective early, impaired innate immune responses, primarily in neutrophils, that lead to a pathogen-specific, increased susceptibility to secondary infections.

HLA-A*0201-Restricted T Cells from Humanized NOD Mice Recognize Autoantigens of Potential Clinical Relevance to Type 1 Diabetes

In both humans and NOD mice, particular MHC genes are primary contributors to development of the autoreactive CD4+ and CD8+ T cell responses against pancreatic β cells that cause type 1 diabetes (T1D). Association studies have suggested, but not proved, that the HLA-A*0201 MHC class I variant is an important contributor to T1D in humans. In this study, we show that transgenic expression in NOD mice of HLA-A*0201, in the absence of murine class I MHC molecules, is sufficient to mediate autoreactive CD8+ T cell responses contributing to T1D development. CD8+ T cells from the transgenic mice are cytotoxic to murine and human HLA-A*0201-positive islet cells. Hence, the murine and human islets must present one or more peptides in common. Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is one of several important T1D autoantigens in standard NOD mice. Three IGRP-derived peptides were identified as targets of diabetogenic HLA-A*0201-restricted T cells in our NOD transgenic stock. Collectively, these results indicate the utility of humanized HLA-A*0201-expressing NOD mice in the identification of T cells and autoantigens of potential relevance to human T1D. In particular, the identified antigenic peptides represent promising tools to explore the potential importance of IGRP in the development of human T1D.

NADPH Oxidase Deficiency Regulates Th Lineage Commitment and Modulates Autoimmunity

Reactive oxygen species are used by the immune system to eliminate infections; however, they may also serve as signaling intermediates to coordinate the efforts of the innate and adaptive immune systems. In this study, we show that by eliminating macrophage and T cell superoxide production through the NADPH oxidase (NOX), T cell polarization was altered. After stimulation with immobilized anti-CD3 and anti-CD28 or priming recall, T cells from NOX-deficient mice exhibited a skewed Th17 phenotype, whereas NOX-intact cells produced cytokines indicative of a Th1 response. These findings were corroborated in vivo by studying two different autoimmune diseases mediated by Th17 or Th1 pathogenic T cell responses. NOX-deficient NOD mice were Th17 prone with a concomitant susceptibility to experimental allergic encephalomyelitis and significant protection against type 1 diabetes. These data validate the role of superoxide in shaping Th responses and as a signaling intermediate to modulate Th17 and Th1 T cell responses.

Neutrophil Mobilization from the Bone Marrow during Polymicrobial Sepsis Is Dependent on CXCL12 Signaling

Neutrophils are essential for successful host eradication of bacterial pathogens and for survival to polymicrobial sepsis. During inflammation, the bone marrow provides a large reserve of neutrophils that are released into the peripheral circulation where they traverse to sites of infection. Although neutrophils are essential for survival, few studies have investigated the mechanisms responsible for neutrophil mobilization from the bone marrow during polymicrobial sepsis. Using a cecal ligation and puncture model of polymicrobial sepsis, we demonstrated that neutrophil mobilization from the bone marrow is not dependent on TLR4, MyD88, TRIF, IFNARα/β, or CXCR2 pathway signaling during sepsis. In contrast, we observed that bone marrow CXCL12 mRNA abundance and specific CXCL12 levels are sharply reduced, whereas splenic CXCR4 mRNA and cell surface expression are increased during sepsis. Blocking CXCL12 activity significantly reduced blood neutrophilia by inhibiting bone marrow release of granulocytes during sepsis. However, CXCL12 inhibition had no impact on the expansion of bone marrow neutrophil precursors and hematopoietic progenitors. Bone marrow neutrophil retention by CXCL12 blockade prevented blood neutrophilia, inhibited peritoneal neutrophil accumulation, allowed significant peritoneal bacterial invasion, and increased polymicrobial sepsis mortality. We concluded that changes in the pattern of CXCL12 signaling during sepsis are essential for neutrophil bone marrow mobilization and host survival but have little impact on bone marrow granulopoiesis.

Anti-thymocyte globulin/G-CSF treatment preserves β cell function in patients with established type 1 diabetes

BACKGROUND Previous efforts to preserve β cell function in individuals with type 1 diabetes (T1D) have focused largely on the use of single immunomodulatory agents administered within 100 days of diagnosis. Based on human and preclinical studies, we hypothesized that a combination of low-dose anti-thymocyte globulin (ATG) and pegylated granulocyte CSF (G-CSF) would preserve β cell function in patients with established T1D (duration of T1D >4 months and <2 years). METHODS A randomized, single-blinded, placebo-controlled trial was performed on 25 subjects: 17 subjects received ATG (2.5 mg/kg intravenously) followed by pegylated G-CSF (6 mg subcutaneously every 2 weeks for 6 doses) and 8 subjects received placebo. The primary outcome was the 1-year change in AUC C-peptide following a 2-hour mixed-meal tolerance test (MMTT). At baseline, the age (mean ± SD) was 24.6 ± 10 years; mean BMI was 25.4 ± 5.2 kg/m²; mean A1c was 6.5% ± 1.1%; insulin use was 0.31 ± 0.22 units/kg/d; and length of diagnosis was 1 ± 0.5 years. RESULTS Combination ATG/G-CSF treatment tended to preserve β cell function in patients with established T1D. The mean difference in MMTT-stimulated AUC C-peptide between treated and placebo subjects was 0.28 nmol/l/min (95% CI 0.001-0.552, P = 0.050). A1c was lower in ATG/G-CSF-treated subjects at the 6-month study visit. ATG/G-CSF therapy was associated with relative preservation of Tregs. CONCLUSIONS Patients with established T1D may benefit from combination immunotherapy approaches to preserve β cell function. Further studies are needed to determine whether such approaches may prevent or delay the onset of the disease. TRIAL REGISTRATION Clinicaltrials.gov NCT01106157. FUNDING The Leona M. and Harry B. Helmsley Charitable Trust and Sanofi.

Constitutive differences in antioxidant defense status distinguish alloxan-resistant and alloxan-susceptible mice

Alloxan (AL), a potent generator of superoxide and hydroxyl radicals, selectively destroys rodent pancreatic beta-cells. Alloxan-susceptible (ALS/Lt) and AL-resistant (ALR/Lt) are inbred mouse strains derived in Japan by inbreeding CD-1 (ICR) mice with concomitant selection for high or low sensitivity to a relatively low AL dose. The present study was undertaken to examine whether resistance was mediated by differences in either systemic or beta-cell antioxidant defense status. Superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPX) activities were determined in tissues of AL-untreated ALR/Lt and ALS/Lt male mice at 7 weeks of age. Specific activities of pancreatic SOD1, GR, and GPX were significantly increased in ALR/Lt mice compared with ALS/Lt mice. ALR/Lt mice further exhibited higher levels of glutathione in plasma, blood, pancreas, and liver combined with lower constitutive lipid peroxides in serum, liver, and pancreas. These results support the hypothesis that the selection process leading to the development of an AL-resistant mouse strain entailed accumulation of a gene or genes contributing to upregulated antioxidant status.

Analysis of self-antigen specificity of islet-infiltrating T cells from human donors with type 1 diabetes

A major therapeutic goal for type 1 diabetes (T1D) is to induce autoantigen-specific tolerance of T cells. This could suppress autoimmunity in those at risk for the development of T1D, as well as in those with established disease who receive islet replacement or regeneration therapy. Because functional studies of human autoreactive T cell responses have been limited largely to peripheral blood–derived T cells, it is unclear how representative the peripheral T cell repertoire is of T cells infiltrating the islets. Our knowledge of the insulitic T cell repertoire is derived from histological and immunohistochemical analyses of insulitis, the identification of autoreactive CD8+ T cells in situ, in islets of human leukocyte antigen (HLA)-A2+ donors and isolation and identification of DQ8 and DQ2–DQ8 heterodimer–restricted, proinsulin-reactive CD4+ T cells grown from islets of a single donor with T1D. Here we present an analysis of 50 of a total of 236 CD4+ and CD8+ T cell lines grown from individual handpicked islets or clones directly sorted from handpicked, dispersed islets from nine donors with T1D. Seventeen of these T cell lines and clones reacted to a broad range of studied native islet antigens and to post-translationally modified peptides. These studies demonstrate the existence of a variety of islet-infiltrating, islet-autoantigen reactive T cells in individuals with T1D, and these data have implications for the design of successful immunotherapies.

New mouse model to study islet transplantation in insulin-dependent diabetes mellitus.

Background. Islet transplantation studies with diabetic rodents frequently use treatment with diabetogens such as alloxan or streptozotocin to render hosts hyperglycemic. These chemicals produce unwanted toxic side effects, which complicate interpretations of damage produced by hyperglycemia versus direct toxin-induced damage. A mouse that spontaneously developed insulin-sensitive diabetes without &bgr;-cell autoimmunity would provide an excellent vehicle for testing &bgr;-cell replacement protocols. The Ins2Akita mutation disrupts normal insulin processing and causes a failure in secretion of mature insulins, which results in the early development of hyperglycemia. This report examines the insulin sensitivity of mice that carry Ins2Akita and their responsiveness to engraftment with syngeneic pancreatic islets. Methods. Ten-week-old C57BL/6J-Ins2Akita/+ males were given 1 unit of insulin to determine insulin sensitivity. Also, 10-week-old, hyperglycemic B6-Ins2Akita/+ received either 400 islets isolated from syngeneic C57BL/6J males (n=7) or from allogeneic BALB/cJ males (n=5) under the renal capsule. These mice were followed for 8 weeks after engraftment or until remission of euglycemia. Nephrectomy of the graft-containing kidney was performed on mice that remained euglycemic. These mice were then followed for 2 weeks for return of hyperglycemia. Results. B6-Ins2Akita/+ mice are insulin responsive. Insulin treatment of hyperglycemic B6-Ins2Akita/+ males significantly lowered blood glucose values within 1 hr. In addition, B6-Ins2Akita/+ recipients of syngeneic islet grafts reversed their diabetic state in less than 72 hr. These islet-engrafted mice remained normoglycemic until removal of the graft-containing kidney. Removal of the graft resulted in a return to hyperglycemia. Mice that received allogeneic grafts efficiently rejected the graft. Conclusions. Our data support the hypothesis that B6-Ins2Akita/+ mice are insulin sensitive and provide an excellent model for islet transplantation studies. In addition, the reduced &bgr;-cell mass and the absence of &bgr;-cell autoimmunity, coupled to the fact that these mice also reject allografts, suggest that these mice may be useful for a variety of other applications, including testing functionality of human islets prepared for transplantation and perhaps also for exploring &bgr;-cell restorative therapy using pancreatic islet stem cells.

Immune Depletion With Cellular Mobilization Imparts Immunoregulation and Reverses Autoimmune Diabetes in Nonobese Diabetic Mice

OBJECTIVE The autoimmune destruction of β-cells in type 1 diabetes results in a loss of insulin production and glucose homeostasis. As such, an immense interest exists for the development of therapies capable of attenuating this destructive process through restoration of proper immune recognition. Therefore, we investigated the ability of the immune-depleting agent antithymocyte globulin (ATG), as well as the mobilization agent granulocyte colony–stimulating factor (GCSF), to reverse overt hyperglycemia in the nonobese diabetic (NOD) mouse model of type 1 diabetes. RESEARCH DESIGN AND METHODS Effects of each therapy were tested in pre-diabetic and diabetic female NOD mice using measurements of glycemia, regulatory T-cell (CD4+CD25+Foxp3+) frequency, insulitis, and/or β-cell area. RESULTS Here, we show that combination therapy of murine ATG and GCSF was remarkably effective at reversing new-onset diabetes in NOD mice and more efficacious than either agent alone. This combination also afforded durable reversal from disease (>180 days postonset) in animals having pronounced hyperglycemia (i.e., up to 500 mg/dl). Additionally, glucose control improved over time in mice subject to remission from type 1 diabetes. Mechanistically, this combination therapy resulted in both immunological (increases in CD4-to-CD8 ratios and splenic regulatory T-cell frequencies) and physiological (increase in the pancreatic β-cell area, attenuation of pancreatic inflammation) benefits. CONCLUSIONS In addition to lending further credence to the notion that combination therapies can enhance efficacy in addressing autoimmune disease, these studies also support the concept for utilizing agents designed for other clinical applications as a means to expedite efforts involving therapeutic translation.