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. Author manuscript; available in PMC: 2020 Dec 15.
Published in final edited form as: J Immunol. 2019 Nov 20;203(12):3256–3267. doi: 10.4049/jimmunol.1900695

MiR183 −96-182 cluster is involved in iNKT cells development, maturation and effector function

Jie Wang *,†,§, Guihua Li *,†,§, Xiaojun Wu *,†, Queping Liu *,†, Congcong Yin *,†, Stephen L Brown , Shunbin Xu ‖, Qing-Sheng Mi *,†,**, Li Zhou *,†,**
PMCID: PMC6986331  NIHMSID: NIHMS1541205  PMID: 31748350

Abstract

The development, differentiation and function of invariant natural killer T (iNKT) cells require a well-defined set of transcription factors, but how these factors are integrated to each other and the detailed signaling networks remain poorly understood. Using Dicer-deletion mouse model, our previous studies have demonstrated the critical involvement of miRNAs in iNKT cell development and function, but the role of individual miRNAs play in it is still not clear. Here we show the dynamic changes of miR183 cluster (miR183C) expression during iNKT cell development. Mouse with miR183C deletion showed a defective iNKT cell development, sub lineage differentiation and cytokine secretion function. miRNA target identification assays indicate the involvement of multiple target molecules. Our study not only confirmed the role of miR183C in iNKT cell development and function, but also demonstrated that miR183C achieved the regulation on iNKT cells through integrated targeting of multiple signaling molecules and pathways.

Introduction

Invariant natural killer T (iNKT) cells represent a rare and distinct subset of T lymphocytes that co-express T cell receptor (TCR) and various natural killer (NK) cell-related surface markers. Upon activation, iNKT cells rapidly secrete large quantities of cytokines that regulate immune responses associated with variety of diseases ranging from transplantation and tumors, to various forms of autoimmunity and infections. Unlike conventional T lymphocytes which express a diverse repertoire of TCRα and TCRβ, iNKT cells combine the Vα14i TCRα chain (Vα24-Jα18 in human) with a restricted repertoire of TCRβ chain that contain either Vβ8, Vβ7 or Vβ2 segments (Vβ11 in humans) in mice. After positive selection by CD4+CD8+ (DP) thymocytes (stage 0, CD24+CD44lo NK1.1−), iNKT cells downregulate CD24, proliferate and proceed through a three-stage maturation process from immature CD24−CD44loNK1.1− (Stage 1) to semi-mature CD24−CD44hiNK1.1− (Stage 2), and then to mature CD44hiNK1.1+ (Stage 3) iNKT cells. Recent studies have led to a new iNKT subsets model emphasizing their functional heterogeneity based on T-helper categories: NKT1, NKT2 and NKT17 (1, 2). These iNKT cells functional subsets can be recognized by expression patterns of specific transcription factors. The development and function of iNKT cells is under the control of a complex network including both transcriptional and post-transcriptional mechanisms. Recent studies have revealed the critical involvement of miRNAs, as a post-transcriptional mechanism, in the development and function of iNKT cells (3, 4). Nevertheless, the role of individual miRNAs in iNKT regulation and their position in the molecular network of iNKT regulation remain not completely understood.

The microRNA183 cluster (miR183C), comprised of miRs183, 96 and 182, is a miRNA family, of which the sequence homology and genomic organization is highly evolutionarily conserved (5). It has been shown that miR183C is highly expressed in sensory organs and are required for the maturation of these organs (6–8). Meanwhile, miR183C is frequently highly expressed in a variety of non-sensory organ diseases, including cancer, and auto-immune disorders (5). In macrophages and neutrophils, the miR183C modulate their phagocytosis and intracellular bacterial killing capacity (9), and production of pro-inflammatory cytokines (10). In T cells, the miR183C members regulate several pro-inflammatory cytokine pathways which is vital to the immune cell function (11, 12). However, the role of miR183C in iNKT cell development and function is completely unknown.

In the current study, we found that miR183C was highly expressed and dynamically regulated during iNKT cell development and maturation. Deletion of miR183C results in the defective iNKT cell development, sub-lineage differentiation and effector function. Multiple target genes potentially work in concert to mediate miR183C’s iNKT cell regulation. Collectively, our results demonstrate the role of miR183C in iNKT cell developmental and functional regulation.

Materials and Methods

Mice

The miR183C knock out (miR183C KO) mouse strain was derived from a gene-trap (GT) embryonic stem cell clone and described previously (8). Mice carrying a conditional floxed allele of FOXO1 (FOXO1fl/fl, from Jackson Laboratory) were mated to mice carrying the Csf1r-Cre allele in order to generate Csf1r-Cre+FOXO1fl/fl (FOXO1 KO) mice. The littermate Csf1r Cre−FOXO1fl/fl mice served as WT control. Csf1r-Cre mediated gene mutation occurs in most innate and adaptive immune cells including iNKT cells (13). Experiments were conducted on 6-8 weeks of age- and gender- matched KO and WT littermate controls. Mice were housed in a specific pathogen-free barrier unit. Handling of mice and experimental procedures were in accordance with requirements of the Institutional Animal Care and Use Committee.

Genotyping

MiR183C KO mice were genotyped using the following PCR primer pairs as describe previously (8): 5F intron l: 5’ GAA CGT GCT TGT GCT GTG CAC 3’. 3R intron l: CTA CAT CCT CTG CCA GGT CTC 3’. 353: 5’CAG GGT TTT CCC AGT CAC GAC 3’. The 5 F intron l/353 amplifies the KO (miR183C KO) allele with a 1.2kb RCR product, whereas the 5 Fintron l/3Rintron 1 detect a 523-bp product of the WT allele.

Flow cytometry analyses

Single-cell suspensions were washed twice with staining buffer (PBS, 2% FBS) and incubated with Fc block (clone 2.4G2). Cells were stained with CD1d-tetramers and/or the following conjugated monoclonal antibodies were used: TCRβ (H57-597), CD24 (30-F1), CD44 (IM7), CD122 (5H4), CD69 (H1.2F3), NK1.1 (PK136), Ki-67 (SolA15), Bcl-2 (10C4), PLZF (9E12), RORγt (B2D), T-bet (eBio17B7), IL-4 (11B11), IFN-γ (XMG1.2), IL-17 (eBio17B7), TNF-α (MP6-XP22), CD45.1 (A20), CD45.2 (104). All mAbs were purchased from eBioscience or Tonbo Bioscience. Data were analyzed using FlowJo 10.0 software. Apoptosis assays were carried out by staining with AnnexinV (eBiosciences), according to manufacturer’s instructions.

iNKT cell enrichment and sorting

Thymic iNKT cells were enriched from total thymocytes by depletion of CD8+ T cells using Magni Sort™ (eBioscience) with biotin-conjugated anti- mouse CD8 antibody and anti-biotin magnetic beads (eBioscience). Negatively selected CD8− cells were then stained with anti-mouse TCRβ, CD1d-tetramer, anti-mouse NK1.1 and anti-mouse CD44 Abs. Thymic iNKT cells of whole population or of different developmental stages were then sorted by BD FACS AriaII.

In vitro PMA and Ionomycin activation assay

Cells from WT and miR183C KO mice were cultured in T cell culture medium (RPMI 1640 with 10% FBS, HEPES, penicillin and streptomycin, pyruvate, nonessential amino acids, L-glutamine, and 2-ME) in the presence of PMA (50ng/ml) and Ionomycin (1μM) for total 4 hours, and Brefeldin A was added at last 2.5 hours before harvesting at final concentration of 1μM. The harvested cells were intracellularly stained with anti-mouse-IFN-γ, -IL-4, -IL-17 and -TNFα before flow cytometry analysis.

Mixed bone marrow transfer experiments

To generate bone marrow chimeras, 7- to 8 – weeks old C57BL/6. SJL (B6.SJL) recipient mice were lethally irradiated initially with 9.5 Gy with a dose rate of 2.5 Gy per minute. Quality assurance of the radiation exposure was performed using multiple dosimetry endpoints including an electrometer, micro-TLDs and Gafchromic film. Donor bone marrows (BM) were harvested from age- and sex-matched SJL (CD45.1+) and miR183C KO or WT control mice (CD45.2+). After erythrocyte lysis, mature T cells (CD3+) were depleted by biotin-conjugated anti-mouse CD3 (BD Biosciences) mAbs and anti-biotin magnetic beads (BD Biosciences) from bone marrows of each donor, using Magni Sort™. Over 90% of mature T cell depletion was confirmed by flow cytometry. CD45.1+ SJL and CD45.2+ miR183C KO or WT littermate control bone marrows were mixed at a 1:1 ratio, and 1 X107 cells per mouse (in a volume of 100 μl) were then injected into the irradiated recipients by tail vein. The chimeras were analyzed 8 weeks after reconstitution.

Quantitative RT-PCR analysis

Total RNA was isolated by Trizol reagent (Sigma) and was quantified by NanoDrop ND-1000 spectrophotometer. The A260/A280 ratio was >1.9 for all the samples. First-strand cDNA was prepared by using cDNA synthesis kit (Sigma) following manufacturer’s instructions. The PCR amplification was carried out on the Applied Biosystem 7900 Real-time RCR system; relative quantification using the ΔCT values in the cells from miR183C KO versus WT control mice was carried out; and fold changes were calculated.

Statistical analysis

Data were analyzed using GaphPad Prism 8, and two-tailed student t test was used. P<0.05 was set as the threshold to determine statistical significance.

Results

MiR183C expression is dynamically regulated during iNKT cell development and maturation

To investigate the possible regulatory role of miR183C in iNKT cell development and differentiation, we first assessed the expression of individual members of the miR183C in the developing iNKT cells. Wild Type (WT) thymus CD4+CD8+ double positive (DP) T cells and iNKT cells at different developmental stages were first sorted out based on CD44 and NK1.1 expression as shown previously (14). The expression of miR183C members were then evaluated and compared in DPT and iNKT cells at different developmental stages. As shown in Fig. 1A, the overall expression levels of miR183C were relative higher in iNKT cells than DP T cells. In different iNKT populations, miR182 and miR183 were relatively more abundant than that of miR96. Furthermore, the expression of miR183 and miR182 were both gradually down regulated during the development and maturation of iNKT cells and reached the lowest level at the final maturation stage 3 (CD44hi NK1.1+). Nevertheless, miR96 kept at a similar low expression in all stages of iNKT development (Fig.1A). These results showed the predominant expression of miR183C in iNKT cells, especially miR183/miR182, compared to T cell progenitors, and the dynamic regulation of miR182/miR183 during the development and maturation of iNKT cells. These results strongly suggest the potential regulatory role of miR183C in the development and differentiation of iNKT cells.

FIGURE 1.

FIGURE 1.

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MiR183C deficiency interferes with iNKT cell development. (A) Individual expression of miR183 cluster in wild-type (WT) and miR183C KO mice. Quantitative RT-PCR analysis of individual miR183 cluster in CD4+CD8+ double positive thymocytes (DP) and different developmental stages of purified iNKT cell populations in the thymus, presented relative to results obtained for the small nuclear RNA U6 (endogenic control). Data are from one experiment representative of triplicates. (B) Defective iNKT cell development in miR183C KO mice. Representative flow cytometric plots showing the percentages of iNKT cells (TCRβ+CD1d-tetramer+) in the thymus, spleen, lymph nodes, liver and lung of WT and miR183C KO mice. Numbers adjacent to outlined areas indicate percent of indicated populations. (C) The frequencies (left) and the absolute numbers (right) of TCRβ+CD1d-tetramer+ iNKT cells in the indicated organs of miR183C KO mice. *, P<0.05, **, P<0.01 and *** P<0.001, compared with WT controls. Data are from two to four independent experiments.

MiR183C deficiency interferes with overall iNKT cell development

To determine the role of miR183C in iNKT cells development, iNKT cells from thymus and different peripheral immune organs were evaluated and compared between miR183C KO and WT littermate controls. Based on the surface expression of TCRβ+ and CD1d-Tetramer, we detected a 2-fold reduction in thymus iNKT cell frequencies from miR183C KO mice, compared to WT controls (Fig. 1B, C), and the absolute number of thymus iNKT cells were reduced to a similar extent (Fig.1C). In peripheral immune organs, splenic iNKT cells from miR183C KO showed a 2-fold reduction in frequency and number, however, iNKT cell frequencies and numbers form lymph nodes (LN), livers and lungs remained comparable between miR183C KO and WT controls (Fig. 1B, C). To further evaluate the potential role of miR183C in conventional αβ T cells development, the frequencies of thymic DP T, CD4+CD8− or CD4−CD8+ single positive T cells (SPT) and CD4−CD8− double negative T cells (DN) T were compared between miR183C KO and WT controls. CD4 and CD8 SPT, and DN T frequencies were comparable, but a mild decrease of DPT frequencies was identified in miR183C KO compared to WT controls (Supplemental Fig. 1). Furthermore, no significant difference on CD4+ and CD8+ T cell frequencies in peripheral immune organs, including spleen, lymph nodes and liver, were identified comparing miR183C KO with WT controls (Supplemental Fig. 1). No difference was observed in thymus γδT cell and regulatory T cell frequencies between miR183C KO and WT controls (Supplemental Fig. 1).

Taken together, these data indicated that deletion of miR183C interrupted overall iNKT cell development, without substantial impact on the development of other T cell types.

MiR183C deletion interferes with iNKT cell maturation and lineage differentiation

iNKT progenitor cells originate from DP thymocytes and then undergo four developmental stages based on cell surface expression of CD24, CD44 and NK1.1(15). Here, we found that frequencies and numbers of Stage 0 (CD24+ CD44lo NK1.1−) and Stage 1 (CD24− CD44lo NK1.1−) thymic iNKT cells were comparable between miR183C KO and WT controls. However, increased Stage 2 (CD44hi NK1.1−) but decreased Stage 3 (CD44hi NK1.1+) thymic iNKT frequencies were observed in miR183C KO mice, as compared with WT controls. Consistent with the frequency changes, the absolute number of Stage 3 thymic iNKT cells reduced dramatically, while Stage 2 iNKT cells remained comparable due to the decreased total thymic iNKT cell number (Fig. 1A, B). These results indicated the requirement of miR183C in iNKT cell terminal maturation. In addition to NK1.1 and CD44, CD122 and CD69 are important markers reflecting the final maturation of iNKT cells. Interestingly, there are no alterations in the expressions of CD69 and CD122, even though NK1.1 expression was down regulated dramatically in thymic iNKT cells from miR183C KO mice (Fig. 2C, D). Further analysis showed the equivalent expression of CD69 and CD122 in the subsets of thymic iNKT cells based on NK1.1 expression in miR183C KO mice compared to WT controls (Fig.2C, D). In addition, similar downregulated NK1.1 and comparable CD69 and CD122 expression were identified in splenic iNKT cells (Fig. 2E). These results indicated that miR183C plays a role in iNKT cells final maturation and NK1.1 expression but is independent to other maturation marker CD69 and CD122 expression, suggesting the discordant role of miR183C in different iNKT cell maturation marker expressions.

FIGURE 2.

FIGURE 2.

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Deletion of miR183 cluster (miR183C) interferes with iNKT cell maturation. (A) The developmental stages of thymic iNKT cells was assessed by examining the surface levels of CD24, CD44 and NK1.1 expression on gated iNKT cells. Identified Stage 0 (ST0) as TCRβ+CD1d-Tetramer+CD24+, ST1 as TCRβ+CD1d-Tetramer+CD24−CD44−NK1.1−, ST2 as TCRβ+CD1d-Tetramer+CD24−CD44+ NK1.1− and ST3 as TCRβ+CD1d-Tetramer+CD24− CD44+NK1.1+. (B) The summary of thymic iNKT cell frequencies (left) and absolute numbers (right) in the indicated developmental stages of miR183C KO mice. *, P<0.05, **, P<0.01 and *** P<0.001, compared with WT controls. (C) The maturation status of thymus iNKT cells was assessed by examining the surface expression of NK1.1, CD69 and CD122 on gated iNKT cells. CD69 and CD122 were further analyzed in the subsets of iNKT cells based on NK1.1 expression. Numbers adjacent to outlined areas indicate the percentage of indicated populations. (D) The summary of thymic iNKT cell frequency of NK1.1, CD69 and CD122 expression from miR183C KO mice, *, P<0.05, **, P<0.01 and *** P<0.001, compared with WT controls. (E) The maturation status of spleen iNKT cells was assessed by examining the surface expression of NK1.1, CD69 and CD122 on gated iNKT cells. Numbers adjacent to outlined areas indicate percent of indicated populations. (F) The summary of splenic iNKT cell frequencies of NK1.1, CD69 and CD122 expression from miR183C KO mice, *, P<0.05, **, P<0.01 and *** P<0.001, compared with WT controls. Data are from four independent experiments.

Recently a new iNKT classification system categorizes iNKT cells into NKT1, NKT2 and NKT17, based on their transcription factors and cytokine expression profiles (1, 2). The new classification of iNKT cells alternative to the shared developmental stages favors functional heterogeneity and clear lineage separation. NKT1 cells are phenotypically PLZFlo T-bethi (mainly in stage 3), and mainly produce IFN-γ; NKT2 cells are phenotypically PLZFhi T-betlo RORγt−(mainly in stage 2), and mainly produce IL-4; NKT17 cells are defined as PLZFint RORγt+ (mainly in stage 2), and mainly produce IL-17. To evaluate the potential role of miR183C in iNKT functional lineage differentiation, iNKT cells from thymus, spleen, lymph nodes and lung were analyzed for related transcription factor expression and compared between miR183C KO and WT controls. In accordance with the defective maturation, thymic iNKT cells showed reduced NKT1, companied with upregulated NKT2 but unchanged NKT17 in miR183C KO compared to WT controls (Fig. 3A). Nevertheless, the thymic bias toward NKT2 differentiation in miR183C KO mice was absent in peripheral spleen (Fig. 3B), lung (Fig. 3C), and lymph node iNKT cells (Fig.3D, Supplementary Fig. 1D), presumably resulting from the differential migration and expansion of individual iNKT effector lineages after emigration from the thymus. Thus, these results indicate the role of miR183C in iNKT cell final maturation and functional sub-lineage differentiation especially in thymus, proposing the role of other mechanisms in peripheral iNKT cells.

FIGURE 3.

FIGURE 3.

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The role of miR183 cluster (miR183C) in iNKT cell lineage differentiation and homeostasis. Expression of transcription factors in thymus (A), spleen (B), lung (C) and lymph node (D) iNKT cells from WT and miR183C KO mice, assessed by intracellular staining for PLZF versus RORγt (left) or PLZF versus T-bet (right), identified NKT1 (N1) as PLZFloT-bet+, NKT2 (N2) as PLZFhiT-bet−RORγt− and NKT17 (N17) as T-bet− PLZFint RORγt+. Numbers adjacent to outlined areas indicate percentage of indicated populations. Left panels show the summary of frequencies of thymus (A), spleen (B), lung (C) and lymph node (D) iNKT sub lineages. (E and F) Representative flow cytometric plots showing the frequencies of iNKT cells stained for Annexin V, Bcl-2 and Ki-67 in thymus (E) and spleen (F) from WT and miR183C KO mice. Frequency of Annexin V, Bcl-2 and Ki-67 expression in indicated populations in thymus and spleen iNKT cells were summarized in the lower panel. (G and H) Representative flow cytometric plots showing the percentages of iNKT cell Ki-67 expression in the subpopulations of iNKT cells (NK1.1− and NK1.1+) in thymus (G) and spleen (H) from WT and miR183C KO mice. Frequency of Ki-67 expression in indicated sub populations in thymus and spleen iNKT cells were summarized in the left panel. *, P<0.05, **, P<0.01 and *** P<0.001, compared with WT controls. Data are from four independent experiments.

The role of miR183C in iNKT cell homeostasis

To assess whether the impairment of iNKT cell development in miR183C KO mice is related to iNKT cell homeostasis, the apoptosis and proliferation capacity of iNKT cells were evaluated. As shown in Fig.3E, a comparable thymic iNKT cells AnnexinV binding frequency was detected between miR183C KO and control mice. Consistent with unchanged cell apoptosis, similar expression of anti-apoptotic protein Bcl-2 was observed in thymic iNKT cells in miR183C KO mice compared to WT controls (Fig. 3E). Meanwhile, the similar unchanged apoptosis and Bcl-2 expression was also observed in the splenic iNKT cells of miR183C KO mice (Fig. 3F). To detect the proliferation capacity of iNKT cells, Ki-67 expression was evaluated by flow cytometry. As shown in Fig. 3E, increased Ki-67 expressing cells were observed in thymic iNKT cells in miR183C KO mice compared to WT controls. Meanwhile, trend of increased Ki-67 was also observed in splenic iNKT cells in miR183C KO mice, albeit the statistically significance was not reached (Fig. 3F). To dissect whether increased overall thymic iNKT cell Ki-67 expression in miR183C KO mice is related to their defective maturation phenotype, we further analyzed the Ki-67 expression in separated NK1.1+ and NK1.1− iNKT subsets. As shown in Fig.3G and H, we found that Ki-67 expression is comparable between miR183C KO and WT controls within the same iNKT subsets in both thymus and spleen. As immature thymic iNKT cells (NK1.1−) have relative higher proliferation capacity, the increased overall thymic iNKT cell proliferation observed from miR183C KO mice should be related to their immature phenotype compared to WT controls. In addition, we observed comparable Annexin V, Bcl-2 and Ki-67 expression in both thymic and splenic conventional T cells in miR183C KO mice compared to WT controls (Supplementary Fig. 2). Taken together, we concluded that miR183C has no significant impact on conventional T cells and iNKT cell homeostasis.

miR183C regulates NKT17 effector function

As miR183C plays a role in iNKT cell maturation and lineage differentiation (Fig. 2 and 3), here, we further assess whether miR183C is involved in iNKT cell effector function. To investigate the role of miR183C in the function of iNKT cells, we in vitro stimulated both thymocytes and splenocytes with PMA and Ionomycin (P/I) for four hours, and iNKT cell activation capacity and cytokine secretion function were evaluated via flow cytometry. After stimulation, thymic and splenic iNKT cells from miR183C KO mice showed similar magnitude of CD69 upregulation compared to that from WT controls (Fig. 4A, B), suggesting the comparable iNKT cell activation capacity in miR183C KO mice. Even though thymic iNKT cell lineage differentiation deflected from NKT1 toward NKT2 in miR183C KO mice (Fig. 2A), comparable frequencies of IFN-γ- and TNF-α-producing thymic iNKT cells were detected in miR183C KO mice compared to WT controls (Fig. 4A). However, thymic iNKT cells from miR183C KO showed trend of increased IL-4 secretion even though did not reach the statistical significance, supporting the upregulation of thymic NKT2 in miR183C KO. Meanwhile, comparable frequencies of splenic iNKT cells producing either IL-4 or IFN-γ and TNF-α after P/I stimulation were identified in miR183C KO versus WT control mice (Fig. 4B), which is consistent with the unchanged functional lineage in splenic iNKT cells in miR183C KO and WT controls. Interestingly, IL-17 production of thymic, splenic, and similarly stimulated lymph nodes and lung iNKT cells from miR183C KO mice concurrently decreased (Fig. 4A, B, C and D), even though no significant down regulation of NKT17 differentiation was observed in miR183C KO compared to WT controls (Fig. 3A, B, C and D). Similar functional phenotype changes were also found in CD4 T cells (Supplemental Fig. 3), indicating that miR183C promote the effector function of NKT17 and Th17 cells in the similar pattern.

FIGURE 4.

FIGURE 4.

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The role of miR183 cluster (miR183C) in iNKT cell cytokine production. (A and B) Flow cytometric plots showing the expression of CD69 and IL-4, IFN-γ, IL-17 and TNF-α production of iNKT cells from thymus (A) or spleen (B) of miR183C KO and WT controls post in vitro PMA/Ionomycin (P/I) stimulation. Lower panels show the summary of frequencies of iNKT cells expressing CD69 or producing IL-4, IFN-γ, IL-17 and TNF-α from miR183C KO versus WT controls. (C, D) showing IL-17 production of iNKT cells from lymph node (C) and lung (D) of miR183C KO and WT controls post in vitro P/I stimulation. *, P<0.05, **, P<0.01 and *** P<0.001, compared with WT controls. Data are from three independent experiments.

MiR183C regulation on iNKT cell development and maturation is cell-autonomous

Because the miR183C deletion model used in our study is conventional instead of tissue-specific, it is difficult to decipher whether the defect of iNKT cells identified in the mice is a direct effect of miR183C on iNKT cells and their precursors or the consequence of changed bone marrow (BM) or thymus environments involved in iNKT cell development and differentiation. To answer this question, we performed mixed BM chimera experiment in which BM cells from miR183C KO or WT littermates (CD45.2+) were mixed with BM cells from SJL mice (CD45.1+) at 1:1 ratio and transferred into lethally irradiated B6.SJL recipient mice (Fig.5A). Analysis of chimera mice 8 weeks post BM transfer revealed that less frequencies of thymic and splenic iNKT cells were derived from miR183C KO BM, compared with those derived from WT controls, albeit comparable LN and liver iNKT cell frequencies were observed (Fig. 5B). In addition, KO BM-derived thymus iNKT cells showed defected maturation based on NK1.1 expression, but comparable CD69 and CD122 expression (Fig. 5C). Consistent with thymus iNKT phenotype, spleen and liver iNKT cells derived from miR183C KO BM showed defective NK1.1, but comparable other maturation marker, CD69 and CD122, compared to that from WT BM (Fig. 5D and 5E). To further identify the cause of reduced iNKT cell number derived from KO BM, we evaluated the AnnexinV and Ki-67 expression in thymus and spleen iNKT cells. As shown in Fig. 5F and G, both thymus and spleen iNKT cells from KO BM showed comparable proliferation capacity compared to iNKT cells from WT BM, which is consistent with the iNKT phenotype from original miR183C KO mice. Nevertheless, thymus iNKT cells from KO BM showed clearly elevated Annexin V binding, while spleen iNKT cells from the KO BM showed the similar trend of change, albeit not statistically significant. This result does not recapitulate the phenotype observed in the original miR183C KO mice, indicating that the elevated apoptosis may be one of the major factors causing the defective thymus iNKT cell development. Overall, data from BM chimeras indicated that vast majority of the defective iNKT cell development and maturation observed in miR183C KO mice are cell intrinsic, while cell extrinsic factors may mask the cell autonomous defect in homeostasis in the iNKT cells with miR183C deletion.

FIGURE 5.

FIGURE 5.

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MiR183 cluster (miR183C) regulation on iNKT cell development and differentiation is cell autonomous. (A) Donor bone marrows (BM) harvested from age- and gender-matched SJL (CD45.1+) mice and miR183C KO (CD45.2+) or WT control (CD45.2+) mice with CD3 deletion were co-transferred at 1:1 ratio to 8 weeks-old B6.SJL recipient mice with lethally irradiated. (B) Representative flow cytometric plots showing the percentages of iNKT cells in thymus (Thy), spleen (Spl), lymph nodes (LN) and liver from CD45.2+ WT and CD45.2+ miR183C KO BM derived cells. The frequencies of iNKT cells in CD45.2+ population from indicated organs were shown in right panels. (C-E) Flow cytometric plots showing the NK1.1, CD69 and CD122 expression in thymic iNKT cell (C), spleen iNKT cell (D) and liver iNKT cells (E) from CD45.2+ WT and CD45.2+ miR183C KO mice. Bar graph showing the summary of frequencies of NK1.1, CD69 and CD122 expression in thymus (C) spleen (D) and liver (E) iNKT cells from CD45.2+ WT and CD45.2+ miR183C KO BM derived cells. (F and G) Flow cytometric plots showing the Annexin V binding (F) and Ki-67(G) staining in thymus and spleen iNKT cells from CD45.2+ WT and CD45.2+ miR183C KO BM derived cells. The summary frequencies of Annexin V and Ki-67 expression in indicated iNKT cells were shown in the right panel. *, P<0.05, **, P<0.01 and *** P<0.001, compared with WT controls. Data are from three independent experiments

miR183C regulates iNKT cell development, lineage differentiation and function through targeting multiple signaling molecules

To further investigate the potential molecular mechanisms of miR183C-mediated iNKT regulation, we sorted thymus iNKT cells from miR183C KO and WT littermate controls. Since iNKT cells from miR183C KO showed defective NK1.1 and related lineage development and distinct gene-expression program was displayed by different developmental stages (16), we sorted thymus iNKT cells of both miR183C KO and WT controls based on NK1.1 expression to get a more reliable target analysis (Fig. 6A). Total RNAs from sorted NK1.1− and NK1.1+ iNKT cells from both miR183C KO and WT littermate controls were purified and qRT-PCR was used to evaluate related potential miR183C target molecule expression levels. Recent studies indicated that miR183C could target Foxo1, Foxo3, Egr1 and Egr2, which are potentially related to iNKT development, differentiation and homeostasis (5, 12, 17). All four genes expressed at a relative higher level in NK1.1+ iNKT cells compared to their NK1.1− counterpart from WT mice (Fig. 6A), which is in reciprocal pattern of the miR183C expression (Fig 1A). This result suggest that these molecules may be the targets and under the tonic active regulation of miR183C during the iNKT cells differentiation. More interestingly, both NK1.1+ and NK1.1− iNKT cells with miR183C deletion showed upregulated expression of all four genes, except for Egr1 in NK1.1+, compared to their counterparts from WT controls. This further supports that Foxo1, Foxo3, Egr1 and Egr2 are the potential targets of miR183C in iNKT cells during their development and differentiation. These results suggest that miR183C are active regulators in iNKT development, differentiation and function through targeting multiple molecular pathways.

FIGURE 6.

FIGURE 6.

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MiR183 cluster (miR183C) regulates iNKT cell development, lineage differentiation and function through targeting multiple signaling molecules. (A) Quantitative RT-PCR analysis of Egr1, Egr2, Foxo1 and Foxo3 expression in purified thymic NK1.1− and NK1.1+ iNKT cell populations from miR183C KO mice and WT controls. Related gene expression are presented as relative to expression level of Gapdh. Data presented are the representative of two independent experiments. (B) Representative flow cytometric plots showing the percentages of iNKT cells in the thymus and spleen of Foxo1 KO mice and WT littermate controls. Numbers adjacent to outlined areas indicate percentage of indicated populations. (C) Frequencies of iNKT cells in the indicated organs of Foxo1 KO versus WT control mice. (D) Flow cytometric plots showing the percentages of thymus and spleen iNKT cells producing IL-17. (E) Summary of Frequencies of IL-17 producing iNKT cells from thymus or spleen of Foxo1 KO and WT control mice upon in vitro PMA/Ionomycin stimulation for 4 hours. *, P<0.05, **, P<0.01 and *** P<0.001, compared with WT controls. Data are from two independent experiments. (F) The speculated schematic model of miR183C regulation of iNKT cell development, maturation, homeostasis and function through targeting multiple signaling molecules and pathways.

iNKT effector function requires Foxo1

IL-17 producing NKT17 cells are key players in autoimmune diseases. Here, we found that deletion of miR183C resulted in an impairment in NKT17 effector function (Fig.4A). Previous study demonstrated that the miR183C regulate T helper 17 (Th17) cells by negatively regulating transcriptional factor Foxo1 expression (12). Given the upregulated Foxo1 and defective IL-17 production in iNKT cells with miR183C deletion, we speculated that Foxo1 deletion would result in elevated NKT17 effector function. Consistent with our expectation, both thymic and splenic iNKT cells from Foxo1 deletion mice produced excessive IL-17 upon PMA/Ionomycin stimulation compared to WT controls, although no gross alteration in iNKT cell frequencies in thymus and spleen was identified in these mice (Fig. 6B, C). These results further indicate that miR183C control NKT17 effector function through targeting Foxo1.

Discussion

MiRNAs have been identified as key regulators of immune cell development and function, as well as disease pathogenesis. Several individual miRNAs, such as miR-150, miR-155, miR-181ab, Let-7, and miR-17-92 family clusters have been shown to play a role in iNKT cell development (14, 18–21). Nevertheless, the overall and specific roles of miRNAs in the molecular networks that regulate iNKT cell positive selection, lineage specification, acquisition of functional activity and homeostasis remain poorly understood. In the current study, our data demonstrated that miR183C regulates iNKT cell development, homeostasis and effector function through targeting multiple transcription factors, which reflect previous observations that miRNAs target different molecules in different cell context.

iNKT cells with miR-183C deletion showed perturbed IL-17a production even though the differentiation of NKT17, based on RORγt expression, remains unchanged compared to WT controls. A recent study from Chen’s lab demonstrated that, miR-183C enhanced the IL-17 production and pathogenic function in Th17 cells through targeting Foxo1, which inhibited RORγt-induced IL-1R1 expression and subsequent IL-17 production without affecting RORγt expression (12). In concordance with this study, we observed an upregulation of Foxo1 in iNKT cells with miR183C deletion. In addition, we found that iNKT cells with Foxo1 deletion had dramatically increased IL-17 production capacity, which further indicates that miR-183C is involved in maintaining NKT17 effector function through targeting Foxo1.

As Foxo3 can bind NF-kB RelA in the cytosol and prevent RelA nuclear translocation(22), upregulation of Foxo3 could result in interrupted NK-κB signaling pathway. NF-κB signaling pathway is known to be critical for iNKT cell differentiation. NF-κB deficient mice, including NF-κB1, c-Rel and RelA KO mice, showed perturbed iNKT maturation, especially in RelA KO mice (23), which closely resembled the iNKT phenotypes in miR-183C KO mice, particularly with regard to the interrupted stage 3 maturation. As Foxo3 is one of the potential targets of miR183C in iNKT cells (Fig. 6A), these data thus strongly suggest that the defect of iNKT cell maturation in miR-183C KO mice are largely, if not uniquely, associated with the upregulation of Foxo3. In addition, miR-182 directly suppressed cylindromatosis (CYLD), an NF-κB negative regulator, to promote NF-κB activation in glioma (24). Furthermore, MiR-182 is upregulated after RET induced NF-κB translocation into the nucleus via binding of NF-κB to the miR-182 promoter (25). Therefore, these evidences support the notion that miR-183C regulate iNKT cell differentiation and maturation through targeting multiple NF-κB signaling pathway-related molecules, which cooperate additively in controlling NK-κB activation, and the reciprocal regulation between miR-182 and NF-κB may form a positive feedback loop in promoting iNKT cell development and differentiation (Fig. 6F).

Egr1 and Egr2 are among the earliest transcription factors induced by TCR signaling and play critical role in activating the survival program associated with the positive selection of T cells (26). Furthermore, they are also involved in iNKT cell development and differentiation through positive regulation of the iNKT lineage-specific transcription factor PLZF as well as IL2rβ (CD122) (27). As potential targets of miR183C, Egr1 and Egr2 were both upregulated in iNKT cells of miR-183C KO mice (Fig. 6A) Given the positive regulatory role of Egr2 on PLZF, the upregulation of PLZF and augmented NKT2 differentiation may be a result of the dysregulated Egr1 and Egr2, which may also explain the dissociation of normal CD122 expression and defective maturation in iNKT cells with miR183C deletion (Fig. 6F).

Even though iNKT cells going through apoptosis are comparable in miR183C KO mice and WT controls, KO BM-derived iNKT cells in the BM chimeric manifested dramatically elevated apoptosis, indicating the cell intrinsic effect of miR183C in the homeostasis of iNKT cells. As a potential target of miR183C, Foxo1 could be either pro-apoptotic or anti-apoptotic through its anti-oxidative stress capability (28, 29). The ultimate outcome may be determined by the basal levels of Foxo1 as well as the overall context of transcriptional landscapes of related cells (30, 31). Therefore, miR-183C regulates the homeostasis of iNKT cells at least partially through targeting Foxo1.

Both miR183 and miR182 were found to be transcriptionally up-regulated by TGF-β in separate studies (24, 32), while, TGF-β orchestrates lineage expansion and maturation of iNKT cells through concerted action of different pathways of TGF-β signaling (33, 34). Considering the role of miR183C in iNKT developmental and functional regulation, thus, miR183C may be involved in TGF-β -mediated iNKT cell regulation.

Overall, our data demonstrates the subtle but indispensable role of miR-183C in iNKT cell development, differentiation, homeostasis and effector function regulation through potentially targeting multiple molecules important for related signaling pathways (Fig. 6F).

Supplementary Material

1

Key Points.

miR183C expression dynamically changes during iNKT cell development.

The deletion of miR183C interferes with iNKT cell development and function.

Multiple target molecules are involved in miR183C-mediated iNKT regulation.

Acknowledgments

We thank NIH Tetramer Core Facility for supplying CD1d tetramers for mouse iNKT cell flow cytometry analysis and cell sorting.

This work was partially supported by the Henry Ford Immunology Program grants (T71017, L. Z.; T71016, Q.S. M.) and National Institutes of Health grants (1R01 AI119041-01A1, Q.S.M.; 1R56AI119041-01, Q.S.M.)

Abbreviations

iNKT

invariant natural killer T cells

miR183C

microRNA183 cluster

KO

knock out

WT

wild type

BM

bone marrow

Footnotes

Disclosures

The authors have no financial conflicts of interest.

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