Allograft inflammatory factor-1 in myeloid cells drives autoimmunity in type 1 diabetes
Diana M. Elizondo, Nailah Z.D. Brandy, Ricardo L. da Silva, Tatiana R. de Moura, Michael W. Lipscomb
Diana M. Elizondo, Nailah Z.D. Brandy, Ricardo L. da Silva, Tatiana R. de Moura, Michael W. Lipscomb
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Research Article Immunology

Allograft inflammatory factor-1 in myeloid cells drives autoimmunity in type 1 diabetes

Abstract

Allograft inflammatory factor-1 (AIF1) is a calcium-responsive cytoplasmic scaffold protein that directs hematopoiesis and immune responses within dendritic cells (DC) and macrophages. Although the role of AIF1 in transplant rejection and rheumatoid arthritis has been explored, little is known about its role in type 1 diabetes. Here, we show that in vivo silencing of AIF1 in NOD mice restrained infiltration of immune cells into the pancreas and inhibited diabetes incidence. Analyses of FACS-sorted CD45neg nonleukocyte populations from resected pancreatic islets showed markedly higher expression of insulin in the AIF1-silenced groups. Evaluation of CD45+ leukocytes revealed diminished infiltration of effector T cells and DC in the absence of AIF1. Transcriptional profiling further revealed a marked decrease in cDC1 DC-associated genes CD103, BATF3, and IRF8, which are required for orchestrating polarized type 1 immunity. Reduced T cell numbers within the islets were observed, with concomitant lower levels of IFN-γ and T-bet in AIF1-silenced cohorts. In turn, there was a reciprocal increase in functionally suppressive pancreas-resident CD25+Foxp3+CD4+ Tregs. Taken together, results show that AIF1 expression in myeloid cells plays a pivotal role in promoting type 1 diabetes and that its suppression restrains insulitis by shifting the immune microenvironment toward tolerance.

Authors

Diana M. Elizondo, Nailah Z.D. Brandy, Ricardo L. da Silva, Tatiana R. de Moura, Michael W. Lipscomb

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Figure 2

In vivo silencing of AIF1 in NOD mice restrains insulitis.

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In vivo silencing of AIF1 in NOD mice restrains insulitis. Six-week-old ...
Six-week-old NOD mice were injected i.p. with siRNA targeting AIF1 (siAIF1) (n = 8) (squares) or scrambled control (siScramble) (n = 8) (circles) oligonucleotides weekly for a total of 3 weeks. NOD/SCID mice (triangles) were used as internal controls (n = 8). Treated or control NOD mice were then monitored for (A) diabetes onset (presented as percentage free of diabetes) through 60 weeks of age. Arrows denote time points of siRNA i.p. injection. (B) Blood glucose levels were monitored weekly through 30 weeks of age. Dashed lane at 250 mg/dL represents the determinant level for diabetes. Arrows denote time points of siRNA i.p. injection. Serum collected biweekly from ages 6 to 30 weeks of NOD/SCID, siScramble, and siAIF1 groups were assessed for (C) insulin and IFN-γ (D) expression. Data are presented as an aggregate in a violin plot, with the mean represented as a solid line through each plot. Data sets are representative of pooled values with six mice per each cohort. (E) At 15 weeks of age (or a total of 9 weeks after initial treatment with siAIF1 or siScramble), mice were sacrificed. Pancreas islets were then isolated before staining for flow cytometric analyses. Dot plots represent FSC vs. CD45, with subsequent plots looking at TCRβ+ CD4+ vs. CD8+ T cell subsets gated from the CD45+ leukocyte populations. All gates established using isotype controls. Flow cytometric dot plots data sets are representative of three independent experiments (with 2–3 mice per group). For gene expression analyses, (F) CD45neg or (G) CD45+ subsets from the pancreata of NOD mice were FACS-sorted before performing qPCR analyses. Data are shown as mean ± SEM of three mice per control or treated group and are representative of three independent experiments. (H) Insulitis scoring was determined by histological analyses of the pancreas using a graded scale of 0 (no insulitis), 1 (peri-insulitis), 2 (moderate insulitis), or 3 (severe insulitis). Graph shows percentage of each score relative to the total. A total of 40 islets were counted per group. For all graphs, statistical significance was determined by the 2-tailed Student’s unpaired t test. *P < 0.05; **P < 0.01. AIF1, allograft inflammatory factor-1; ns, not significant; ND, not determined.