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. 2017 Nov 28;114(48):E10418-E10427.
doi: 10.1073/pnas.1713543114. Epub 2017 Nov 13.

Resident Macrophages of Pancreatic Islets Have a Seminal Role in the Initiation of Autoimmune Diabetes of NOD Mice

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Free PMC article

Resident Macrophages of Pancreatic Islets Have a Seminal Role in the Initiation of Autoimmune Diabetes of NOD Mice

Javier A Carrero et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Treatment of C57BL/6 or NOD mice with a monoclonal antibody to the CSF-1 receptor resulted in depletion of the resident macrophages of pancreatic islets of Langerhans that lasted for several weeks. Depletion of macrophages in C57BL/6 mice did not affect multiple parameters of islet function, including glucose response, insulin content, and transcriptional profile. In NOD mice depleted of islet-resident macrophages starting at 3 wk of age, several changes occurred: (i) the early entrance of CD4 T cells and dendritic cells into pancreatic islets was reduced, (ii) presentation of insulin epitopes by dispersed islet cells to T cells was impaired, and (iii) the development of autoimmune diabetes was significantly reduced. Treatment of NOD mice starting at 10 wk of age, when the autoimmune process has progressed, also significantly reduced the incidence of diabetes. Despite the absence of diabetes, NOD mice treated with anti-CSF-1 receptor starting at 3 or 10 wk of age still contained variably elevated leukocytic infiltrates in their islets when examined at 20-40 wk of age. Diabetes occurred in the anti-CSF-1 receptor protected mice after treatment with a blocking antibody directed against PD-1. We conclude that treatment of NOD mice with an antibody against CSF-1 receptor reduced diabetes incidence and led to the development of a regulatory pathway that controlled autoimmune progression.

Keywords: islets of Langerhans; macrophage; nonobese diabetic mouse; regulation; type 1 diabetes.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Treatment of C57BL/6 mice with AFS98 antibody depleted their macrophages. (A) Female B6 mice aged 6–8 wk were administered 0.25, 0.50, or 2.0 mg of AFS98 antibody i.p. Islets were examined 7 and 14 d after injection for the presence of macrophages. Box indicates CD45+CD11c+MHCII+F4/80+CD11b+ cells as a percent of total islet cellularity. (B) Female B6 mice aged 6–8 wk were treated with 2.0 mg of AFS98, and their islets were examined at the time points indicated for the presence of macrophages. Top show the CD45+ cells, and Bottom show the CD45+CD11c+MHCII+ F4/80+CD11b+ cells as a percent of total islet cellularity. (C) Graph of the CD45+ cells found in mice 1–2 wk after treatment with 2.0 mg of AFS98. (D) Graph of CD45+F4/80+CD64+ islet stromal macrophage populations as a percent of CD45+ cells in control and AFS98-treated mice 2 wk after treatment. (E) Mice were treated for 1–2 wk with AFS98 antibody, and the percent of total macrophages in lung, liver, spleen, and pancreatic lymph nodes were determined. (F) Macrophages were examined as in E except plots show the percent of the indicated subpopulation of macrophages. For all graphs, controls were untreated age-matched mice. Flow cytometry plots in A and B are representative of individual islet, lung, liver, spleen, and lymph nodes, from two to three experiments with two to four mice per treatment group. Scatter plots in E and F were calculated from three independent experiments with two to three mice per group. P values were calculated using Mann–Whitney U test with the following style: not significant (n.s., >0.1234), *P = 0.0332, **P = 0.0021, ***P = 0.0002, ****P < 0.0001.
Fig. 2.
Fig. 2.
Islet function after depletion of macrophages by AFS98. (A) B6 mice were given 2.0 mg of AFS98 i.p. at 6–8 wk of age. Glucose tolerance assays were then performed on AFS98-treated and untreated mice. After the indicated number of days, the mice were fasted for 12 h and then injected with 2.0 g/kg glucose i.p. Blood glucose (mg/dL) was measured at the indicated time points. Results are pooled from two independent experiments (n = 2–3 mice per group). (B) Nine-week-old B6 females were left untreated or administered 2.0 mg of AFS98 i.p. Four days later, the mice were placed on a 20% sucrose diet for an additional 7 d, then returned to a normal diet for 2 d. The mice were then killed, and the insulin content of their islets was measured (n = 5 mice per group). (C) Three-week-old C57BL/6 mice were left untreated or administered 2.0 mg of AFS98 i.p. At 6 wk of age, their islets were isolated, total RNA was extracted, and transcripts were analyzed by microarray. Scatter plot shows the log2 mean expression values for four control and experimental mice. The dots highlighted in blue represent genes differentially expressed between treated and control mice at 99% confidence using moderated t test with Benjamini–Hochberg false discovery rate analysis. The selected genes are plotted in the heat map using Euclidean distance and normalized global expression as indicated.
Fig. 3.
Fig. 3.
Effect of AFS98 treatment on NOD mice. (A) Male NOD mice at 4–5 wk of age were administered 0.25, 0.50, or 2.0 mg of AFS98 i.p., and their islets were examined by flow cytometry. Plots show the CD45+CD11c+MHCII+ gate at either 7 or 14 d after treatment. Results are representative of two experiments performed in duplicate. Values in the box represent the percent of cells as a function of total islet cellularity. (B) Two-week-old male NOD mice were left untreated (Control) or injected i.p. with 0.5 mg of AFS98 at 2 wk of age and 2.0 mg of AFS98 at 4 wk of age (AFS98). At 6 wk of age, the islets were harvested, dispersed, and tested for their MHCII-peptide presentation to two T cell hybridomas that recognize insulin. High (25.0 mM) or low (5.0 mM) glucose and two different insulin peptides (Ins B:12–20 and Ins B:13–21) were evaluated. Bars represent the mean ± SD of 3H incorporation by the IL-2–dependent cell line CTLL-2.
Fig. 4.
Fig. 4.
AFS98 treatment does not affect T cell division in lymph nodes but prevents T cell entry into islets of Langerhans. NOD mice were injected with AFS98 antibody at a dose of 0.5 mg at 2 wk of age and 2.0 mg at 4 wk of age. Two TCR transgenic T cells, the CD4+ BDC2.5 and the CD8+ NY8.3, were isolated from lymph nodes and spleens of their respective mice. T cells were then labeled with CFSE and transferred into 6-wk-old NOD mice that had either been left untreated or treated with AFS98. (A and B) Seven days after T cell transfer, the pancreatic and inguinal lymph nodes were isolated and analyzed by flow cytometry. (A) Dilution of CFSE in either inguinal (Upper) or pancreatic (Lower) lymph nodes for an individual mouse per treatment is shown. Cells were gated on forward and side-scatter, CD45, CD3, and either CD4 (BDC2.5) or CD8 (NY.8.3). (B) Summary of division index and proliferation index for individual mice examined as in A. Results show three or four individual mice per group. (C) Ten days after TCR transgenic T cell transfer, islets of Langerhans were isolated and examined for entry of either the BDC2.5 or NY8.3 T cells by flow cytometry. The Left two images are gated on forward and side-scatter, CD45, and CD3. The Right images are gated on forward and side-scatter. BDC T cells were identified using a clonotypic antibody to its T cell receptor. NY8.3 T cells were identified by a CD45.2 congenic label. Numbers indicate the percent of cells in each selection as a function of CD45+ cells. T cell islet entry results are representative of two to three independent experiments with two to four individual mice per group.
Fig. 5.
Fig. 5.
Lymph node priming is not reduced by AFS98 treatment. B6.g7 or NOD mice were treated with 2 mg of AFS98 antibody for 1 wk and then injected with 10 nmols INS:9–23, HEL, or IGRP peptides in complete Freund’s adjuvant. The draining popliteal lymph nodes were isolated and tested by ELISPOT for IL-2 (A) and IFN-γ (B) production. Recall antigens for the ELISPOT are shown in the figure and include the following: insulin protein (INS) and INS:9–23, HEL protein and peptides that elicit CD4 (11–25) and CD8 (20–35) responses, and the IGRP peptides that elicit CD4 (128–142) and CD8 (206–214) responses. Results are taken from two individual mice tested in duplicate or triplicate.
Fig. 6.
Fig. 6.
Treatment with AFS98 protects against autoimmune diabetes. (A) Female NOD mice were left untreated, or injected with Rat IgG2a or AFS98 at the ages and doses indicated in C and followed for diabetes incidence. Control mice represent the pooled results of the three experiments. (B) Splenocytes were isolated from nondiabetic 44- to 50-wk-old mice taken from A, 107 were transferred into NOD.Rag1−/− mice, and recipients were followed for diabetes incidence. (C) Summary of primary and transfer diabetes incidence for three AFS98 treatment protocols as well as the effect of PD-1 treatment on the AFS98 protection. Weeks indicate ages of mice at treatment, and dose is milligrams per mouse for either control or AFS98 antibodies.
Fig. 7.
Fig. 7.
Early treatment with AFS98 reduces infiltrating leukocytes in NOD mice up to 12 wk of age. (A and B) Female NOD mice were left untreated or injected with 0.5 mg of AFS98 i.p. at 2 wk of age and 2.0 mg of AFS98 i.p. at 4, 7, and 10 wk of age. The islets of mice at 6 (B), 8 (A and B), and 12 (A) wk of age were isolated and analyzed by flow cytometry. (A) Shows the flow cytometry plots of myeloid and T cell compartments. Gating is indicated over the plots. Flow cytometry plots are representative of individual islet preparation from three control or four AFS98-treated mice. (B) Summarizes the flow cytometry data for all time course experiments (three to four mice per group). Bars represent the mean ± SD for two independent experiments with two to three replicates per group. (C) Flow cytometry plots of immune cell populations in islets isolated from nondiabetic control or AFS98-treated mice taken from Fig. 6 A and C early treatment examined at 40–44 wk of age. Plots were generated from individual mice. Gates are indicated on the Top of each column of plots. Flow cytometry plots are representative of individual islets preparations isolated from two control and eight AFS98-treated mice. (D) Summary of the data shown in C. (E and F) Hematoxylin/eosin staining of pancreatic sections isolated from nondiabetic AFS98-treated mice taken from Fig. 6A: The mice were treated early, and their islets were examined at 40–44 wk of age. (Scale bars, 400 μm.)
Fig. 8.
Fig. 8.
Leukocyte infiltrates are reduced following macrophage depletion at 10 wk of age. NOD mice were either treated with AFS98 or control Rat IgG2a starting at 10 wk of age. At 22 or 40 wk of age, the islet cells of control or AFS-treated mice were isolated and analyzed by flow cytometry as indicated. Values in the CD45 by SSC plot represent the percent of leukocytes in islets. Values in the CD3e by I-Ag7 plot represent the percent CD3e+ or I-Ag7+ cells as a percent of CD45+ cells. Values in the T cell (CD3e+, I-Ag7−) and APC (CD3e, I-Ag7+) plots represent the percent of cells in each quadrant as a function of total islet cellularity. Gating is indicated over the top for each column of plots. Results are representative of two mice per group.

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