MicroRNA-155 promotes autoimmune inflammation by enhancing inflammatory T cell development - PubMed (original) (raw)

MicroRNA-155 promotes autoimmune inflammation by enhancing inflammatory T cell development

Ryan M O'Connell et al. Immunity. 2010.

Abstract

Mammalian noncoding microRNAs (miRNAs) are a class of gene regulators that have been linked to immune system function. Here, we have investigated the role of miR-155 during an autoimmune inflammatory disease. Consistent with a positive role for miR-155 in mediating inflammatory responses, Mir155(-/-) mice were highly resistant to experimental autoimmune encephalomyelitis (EAE). miR-155 functions in the hematopoietic compartment to promote the development of inflammatory T cells including the T helper 17 (Th17) cell and Th1 cell subsets. Furthermore, the major contribution of miR-155 to EAE was CD4(+) T cell intrinsic, whereas miR-155 was also required for optimum dendritic cell production of cytokines that promoted Th17 cell formation. Our study shows that one aspect of miR-155 function is the promotion of T cell-dependent tissue inflammation, suggesting that miR-155 might be a promising therapeutic target for the treatment of autoimmune disorders.

Copyright © 2010 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. _Mir155_−/− mice are resistant to EAE induced by MOG35–55

A. EAE was induced in Mir155+/+ and _Mir155_−/− mice by immunizing both groups with 100 µg of the MOG35–55 peptide followed by administration of pertussis toxin. Their disease severity was scored regularly based upon clinical symptoms (n=10). Data represent three independent experiments. B. Disease incidence was assessed for each group (n=10). C. Representative H&E stained brain sections from Mir155+/+ or _Mir155_−/− mice harvested on day 25 post-immunization. D. Average histology score for each group (n=4). E. Number of live LN cells (left) and their lineage composition was assessed by flow cytometry (right) using LNs from Mir155+/+ and _Mir155_−/− mice 25 days after immunization (n=4). F. Number of splenocytes (left) and their lineage composition as determined by flow cytometry (right) using spleens from both groups 25 days after immunization (n=4). G. WT mice were lethally irradiated and reconstituted with Mir155+/+ or _Mir155_−/− BM. 4 months later, expression of miR-155 in LPS activated splenic B cells was assessed. H. MOG35–55-induced EAE was induced in mice with WT or _Mir155_−/− hematopoietic cells and disease was scored over a time course (n=5–7). I. 12 days following induction of EAE in WT mice with MOG35–55, splenocytes were harvested and cultured in 20 µg/ml MOG35–55 and 20ng/ml IL-12 p70 for 48 hours. Cells were then washed and 25×106 cells were injected intravenously into Mir155+/+ and _Mir155_−/− mice followed by administration of pertussis toxin. Mice were monitored regularly and disease severity was scored (n=5). Data represent two independent experiments. Error bars represent +/−SEM and * denotes statistical significance with a p value of <0.05 according to a student’s two-tailed t-test. See also Figure S1

Figure 2

Figure 2. _Mir155_−/− mice exhibit defective inflammatory T cell development during EAE

Mir155+/+ and _Mir155_−/− mice were harvested 25 days following immunization with MOG35–55. A. Intracellular staining was conducted to identify total lymph node cells (top) and CD4+ lymphocytes (bottom) producing IL-17A and/or IFN-γ (n=4). B. Splenocytes were analyzed as in (A) (n=4). C. Mir155+/+ or _miR155_−/− splenocytes harvested from mice 25 days after EAE induction were labeled with CFSE. CFSE loss by CD4+ proliferating cells from both groups was assayed by flow cytometry following restimulation with MOG35–55 (20 µg/ml) for 72 hours (n=4). D. 3[H] thymidine incorporation was also assays using replicate cultures (n=4). 2 naïve WT mice were also included as controls. E. Production of IL-17A and IFN-γ by cells from (C.) was determined by ELISA (n=4). Data represent two independent experiments. Error bars represent +/−SEM and * denotes statistical significance with a p value of <0.05 according to a student’s two-tailed t-test. +/+ = Mir155+/+; −/− = _Mir155_−/−. See also Figure S2.

Figure 3

Figure 3. miR-155 is required for inflammatory T cell development during the induction phase of EAE

Mir155+/+ and _Mir155_−/− mice were harvested 13 days following immunization with MOG35–55. A. Mononuclear cells were purified from Mir155+/+ and _Mir155_−/− brains and intracellular staining was conducted to identify CD4+ lymphocytes producing IL-17A and/or IFN-γ (n=5). B. Total numbers of Th17 and Th1 cells in the brain, in addition to the percentage of Th17 and Th1 cells among total CD4+ T cells is shown on the right (n=5). C. WT and _Mir155_−/− splenocytes were analyzed for expression of BIC, IL-17A and IL-23 p19 mRNA by qPCR (n=5) and D. intracellular staining was used to determine the number of Th17 and Th1 cells (n=5). E. Splenocytes were restimulated with MOG35–55 and E. proliferation was assayed by 3[H] thymidine incorporation (n=5) and F. the production of IL-17A, IFN-γ, IL-6 and GM-CSF measured by ELISA (n=5). G. Expression of BIC and IL-17A mRNA in the LNs was assayed by qPCR (n=5) and H. the number of Th17 and Th1 cells was also quantified by flow cytometry (n=5). Error bars represent +/−SEM and * denotes statistical significance with a p value of <0.05 according to a student’s two-tailed t-test. +/+ = Mir155+/+; −/− = _Mir155_−/−. See also Figure S3.

Figure 4

Figure 4. _Mir155_−/− mice have reduced foot pad inflammation during DTH

Mir155+/+ and _Mir155_−/− mice were immunized with 100 µg of KLH in CFA and 8 days later injected with 50 µg of KLH in one footpad and PBS in the other. A. Increases in footpad inflammation were measured for both groups (n=5). B. Total numbers of splenocytes and LN cells was assessed (n=5). C. Proliferation of splenocytes and LN cells following in vitro restimulation with KLH was determined by assaying 3[H] thymidine incorporation (n=5). D. Production of IL-17A, IFN-γ and IL-6 from the cells in (C.) was determined by ELISA (n=5). Error bars represent +/−SEM and * denotes statistical significance with a p value of <0.05 according to a student’s t-test. +/+ = Mir155+/+; −/−= _Mir155_−/−.

Figure 5

Figure 5. miR-155 expression by CD4+ T cells is necessary for proper Th17 cell development in vitro

A. CD4+ T cells were isolated from Mir155+/+ or _Mir155_−/− spleens and cultured in the presence of plate bound CD3 and soluble CD28 antibodies, with (Th17 cell) and without (Th0 cell) IL-6 (50 ng/ml) and TGF-β (2 ng/ml). After 96 hours, expression of IL-17A and IFN-γ was assayed by intracellular staining following by flow cytometry. B. Results from a representative experiment are represented graphically (n=2). Data represent three independent experiments. C. Expression of Mir155 was measured by qPCR before and after activation with CD3 and CD28 antibodies (n=3). D. Expression of BIC, Mir155, and IL-17A in WT and _Mir155_−/− CD4+ T cells was assayed by qPCR following 96 hours of culture with CD3 and CD28 antibodies alone, or in Th17 cell skewing conditions (n=2). Data represent two independent experiments. Error bars represent +/−SEM.

Figure 6

Figure 6. Expression of miR-155 by CD4+ T cells is required for proper development of inflammatory T cells during EAE

A. 5×106 WT or _Mir155_−/− CD4+ T cells from naïve mice were injected i.v. into _Rag1_−/− recipients, EAE was induced with MOG35–55 24 hours later, and disease was scored over a time course (n=5–6). Data represent two independent experiments. B. Mice were harvested and engraftment of CD3+CD4+ T cells was assayed by flow cytometry using splenocytes (top). Expression of IL-17A and IFN-γ by CD4+ cells in the spleens and LNs was assayed by intracellular staining followed by flow cytometry. A representative plot from the LNs is shown (bottom). C. The averages of 5–6 mice per group are shown graphically. D. Mir155+/+ and _Mir155_−/− mice were injected with 1×107 WT CD45.1+CD4+ naïve T cells, and EAE was induced 24 hours later (n=5). Disease symptoms were scored over a time course. Data represent two independent experiments. E. Mice were harvested and CD4+ T cells in the brains were analyzed by flow cytometry to detect cells expressing CD45.1, IL-17A and IFN-γ. F. The average of 5 mice per group from (E.). Error bars represent +/−SEM and * denotes statistical significance with a p value of <0.05 according to a student’s two-tailed t-test. +/+ = Mir155+/+; −/− = _Mir155_−/−. See also Figure S4.

Figure 7

Figure 7. miR-155 expression by LPS-activated, GM-CSF-derived myeloid dendritic cells is necessary for proper production of Th17 cell relevant inflammatory cytokines

A. CD11c+ DCs were derived using GM-CSF at 20 ng/ml. B. Expression of BIC (top) and mature miR-155 (bottom) before and after 20 hours of LPS stimulation (100 ng/ml) was assayed using qPCR. C. Total RNA was next used for a microarray analysis to determine mRNA expression differences between Mir155+/+ and _Mir155_−/− LPS treated DCs. Several selected targets of miR-155 were expressed in higher amounts in _Mir155_−/− DCs, while a subset of selected proinflammatory cytokines were expressed at lower amounts. Red = higher expression and Green = lower expression in the _Mir155_−/− vs. Mir155+/+ DCs. D. Expression of SHIP1 and SOCS1 mRNAs was assessed by qPCR and by Western blotting (n=3). E. qPCR was also used to assay expression of IL-23 p19, IL-6, IL-12p40 and TNF-α mRNA amounts (n=3). Data represent two independent experiments. F. Concentrations of the cytokines from (E) in the culture supernatants were determined by ELISAs (n=3). Data represent two independent experiments. G. GM-CSF-derived DCs overexpressing miR-155, a miR-155 “seed” mutant or a control vector were stimulated with LPS for 20 hours and expression of IL-23 p19, IL-6, IL- 12p40 and TNF-α mRNA was assayed by qPCR. Data represent two independent experiments. H. Proliferation of 2D2 or OT2 CD4+ T cells in response to their respective antigens presented by WT or _Mir155_−/− DCs was assessed by assaying 3[H] thymidine incorporation (n=3). Data represent two independent experiments. Error bars represent +/−SEM and * denotes statistical significance with a p value of <0.05 according to a student’s two-tailed t-test.

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