(C) PLOS One
This story was originally published by PLOS One and is unaltered.
 . . . . . . . . . .



A microRNA atlas of CD4+ T cell subsets in experimental autoimmune encephalomyelitis identifies regulators of disease pathogenesis [1]

['Carolina Cunha', 'Gulbenkian Institute For Molecular Medicine', 'Lisbon', 'Daniel Inácio', 'Paula Vargas Romero', 'Ana Teresa Pais', 'Catarina Pelicano', 'Marina Costa', 'Sofia Mensurado', 'Natacha Gonçalves-Sousa']

Date: 2026-08 

MicroRNAs (miRNAs) are key regulators of CD4 + T cell differentiation, but how they contribute to the course of an autoimmune disease in vivo remains poorly studied. Given the known roles in autoimmunity of pro-inflammatory T helper 1 (Th)1 and Th17 cells, and anti-inflammatory Foxp3 + regulatory cells, we established a triple reporter mouse for Ifng, Il17 and Foxp3, and subjected it to experimental autoimmune encephalomyelitis (EAE) to characterize the miRNomes of the corresponding CD4 + T cell subsets. We identified 110 miRNAs differentially expressed between the pro-inflammatory (Th1 and Th17 cells) and the Treg cell subsets. Among these, we found novel functions for miR-122-5p and miR-1247 as regulators of Th17 cell proliferation and Th1 cell differentiation, thus impacting the course or severity of EAE, respectively. Importantly, their expression patterns suggest miR-122-5p and miR-1247 act as peripheral brakes to CD4 + T cell pathogenicity that are subverted in the inflamed central nervous system.

Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Daniel Neves is currently employed by SGS Global Biosciences Center. This employment began after completion of the research reported in this article. SGS Global Biosciences Center had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The other authors have declared that no competing interests exist.

Funding: This work was funded by the European Research Council ( https://erc.europa.eu/homepage ) (CoG_646701 to B.S.-S.), by “la Caixa” Foundation ( https://lacaixafoundation.org/en/ ) (LCF/PR/HR24/00929 to B.S.S.); and by Fundação para a Ciência e Tecnologia ( https://www.fct.pt ) (SFRH/BD/145352/2019 PhD grant to D. I.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: CD4+ T cell RNA-seq data are available from the Sequence Read Archive (SRA) under the accession code PRJNA1067547 ( https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1067547 ). The raw flow cytometry data (FCS files) have been deposited in Zenodo and are available at https://doi.org/10.5281/zenodo.21341947 . All other data that support the findings of this study are available in the article and Supplementary Information.

Building on these foundations, we set out to characterize the global miRNomes of Th1, Th17 and Treg cells in EAE, with the ultimate goal of identifying novel miRNA regulators of CD4 + T cell functions in vivo. In order to gain resolution compared to previous studies, where candidates were selected based on the analysis of total CD4 + T cells of EAE mice or MS patients (compared to controls), we established a triple reporter mouse for IFN-γ (YFP + ), IL-17A (GFP + ) and Foxp3 (hCD2 + ) to isolate the corresponding CD4 + T cell subsets upon EAE induction. This allowed us to dissect their distinct miRNomes, and to identify new miRNAs, miR-122-5p and miR-1247-5p, that respectively regulate Th17 and Th1 cells, and thus impact EAE disease onset or severity in vivo.

MicroRNAs (miRNAs) have emerged as key players in the fine-tuning of gene expression at the post-transcriptional level. They are highly conserved small (~22 nucleotide-long) non-coding RNAs that bind to the 3´UTR, and less frequently to the coding sequence (CDS), of target mRNAs to induce either degradation or translation repression [ 14 , 15 ]. miRNAs are known to regulate CD4 + T cell differentiation; most strikingly, the ablation of (all) miRNAs in T cells caused impaired T cell proliferation and survival, reduced Treg numbers, and increased production of IFN-γ and IL-17A by CD4 + T cells, leading to spontaneous lethal inflammatory disease [ 16 – 19 ]. However, only a few specific miRNAs have been identified as underlying such overt phenotypes. For example, miR-29 [ 20 ] and the miR-106-363 cluster [ 21 ] were shown to inhibit Th1 and Th17 differentiation, respectively, whereas miR-125a promoted Treg activity [ 22 ]. In the EAE/ MS setting, miR-326 associated with Th17 cells and disease severity, and in vivo miR-326 silencing reduced Th17 numbers and EAE pathogenesis [ 23 ]. Moreover, MS-enriched miR-92 limited Treg induction while supporting Th17 responses, and its inhibition ameliorated EAE [ 24 ].

Immunosuppressive Tregs also play a role in MS by reducing the function of effector cells, often prevailing in remitting phases of the disease [ 9 ]. In fact, when Treg cells were administered therapeutically in the EAE model, there was a reduction in disease severity, which coincided with limited immune cell infiltration, principally Th17 cells, into the CNS [ 13 ]. These results reinforce the concept that the balance between Teff and Treg subsets is a major determinant of the outcome of the local inflammatory/ autoimmune response [ 2 , 3 ]. As such, it is important to identify molecular mechanisms that control Teff or Treg cells across their transition from the periphery to the target CNS.

More specifically in MS, effector Th1 and Th17 CD4 + T cells contribute to disease pathogenesis after differentiation in the periphery and infiltration into the central nervous system (CNS), causing local inflammation and demyelination [ 5 , 6 ]. With regard to Th1 cells, genetic polymorphisms of IFN-γ have been associated with susceptibility to MS, and increased T cell-derived IFN-γ levels preceded disease exacerbation and correlated with active lesions in vivo, and killed oligodentrocytes in vitro (reviewed in [ 7 ]). Th17 cells, on the other hand, activate microglia and recruit neutrophils, macrophages, and lymphocytes [ 8 ] through synergy of IL-17A with other proinflammatory cytokines such as IL-6, IL-23, IL-1β and GM-CSF [ 9 , 10 ]. Critically, myelin oligodendrocyte glycoprotein (MOG)-specific Th1 and Th17 cells were shown to induce experimental autoimmune encephalomyelitis (EAE), the well-established mouse model of MS, upon adoptive transfer [ 6 , 11 , 12 ].

CD4 + T cells can differentiate into multiple functional subsets through networks of cytokines and transcription factors (TFs) that dictate lineage-specific transcriptional programs. Interferon-γ (IFN-γ)-producing T helper 1 (Th1) and interleukin (IL)-17A-producing Th17 cells are two of the key effector CD4 + T cell populations that orchestrate complementary immune responses to major pathogens, such as viruses and intracellular bacteria (Th1), or fungi and extracellular bacteria (Th17). Critically, these effector CD4 + T cells are also involved in the development of chronic inflammatory and autoimmune diseases such as type I diabetes, rheumatoid arthritis, psoriasis, colitis or multiple sclerosis (MS) [ 1 , 2 ]. On the other hand, CD4 + T cells can also differentiate, either in the thymus or in the periphery, into anti-inflammatory regulatory T cells (Treg) that are able to curtail the function of effector cells (Teff) [ 2 , 3 ], and whose relevance is highlighted by the very severe autoimmune manifestations of Treg-defective IPEX patients and Foxp3 mutant mice [ 4 ].

We next analyzed the in vivo effects of miR-1247 manipulation. Unlike the previous antagomiRs ( S6a Fig ), the miR-1247-5p antagomir paradoxically led to increased levels of miR-1247-5p in various tissues ( S9a Fig ), thus constituting an overexpression strategy. Consistent with increased miR-1247-5p activity, we observed a trend towards downregulation of Trapc9 and Synj2bp—miR-1247-5p target candidates identified by Ago2 CLIP-seq [ 36 ]—in matching organs ( S9b Fig ). While the progression of symptoms followed a similar course ( S10a Fig ), we observed decreased EAE severity, as well as reduced weight loss, in mice overexpressing miR-1247-5p compared with the control group ( Fig 6a ). Furthermore, this phenotype was associated with a selective decrease in the numbers of IFN-γ + CD4 + T cells ( Fig 6b and 6c ), without affecting the total CD4 + T cell frequency ( S10b Fig ), in the draining lymph nodes of miR-1247-5p-treated group. Of note, the frequency of other immune cells also did not change upon miR-1247-5p overexpression ( S10c Fig ). Thus, the upregulation of miR-1247 expression restricts IFN-γ production by Th1 cells and reduces EAE severity.

To assess the selectivity of these phenotypes, we studied the impact of an anti-miR-126a antagomiR. While this resulted in the downregulation of miR-126a ( S8a Fig ), it had no impact neither on clinical scores nor on weight loss, since the specific antagomir-treated mice followed a similar disease course as the control group ( S8b Fig ), and no differences were found in the frequency of IFN-γ + and IL-17A + nor Treg (Foxp3 + ) cell populations ( S8c Fig ). These data highlight our finding that miR-122-5p inhibition selectively increases the frequency of Th17 cells and anticipates EAE onset.

(a) Mice treated with control and miR-1247-5p antagomiRs were scored for EAE clinical signs and weighed daily for 28 days. Results are mean ± SD from 3 independent experiments (n = 10–11). *p < 0.05 and **p < 0.01 (two-way ANOVA followed by Sidak’s multiple comparisons test). (b) Representative flow cytometry plots of IFN-γ + and IL-17A + CD4 + T cells in the draining lymph nodes (inguinal, axillary and brachial) of triple Foxp3-hCD2.Ifng-YFP.Il17a-GFP reporter mice with EAE and treated with control and miR-1247-5p antagomiRs. (c) Frequency and absolute numbers of IFN-γ + CD4 + T cells in the draining lymph nodes (inguinal, axillary and brachial) of triple Foxp3-hCD2.Ifng-YFP.Il17a-GFP reporter mice with EAE and treated with control and miR-1247-5p antagomiRs. Results are mean + SD from 3 independent experiments (n = 10–11). Each dot represents an individual mouse. *p < 0.05 (non-parametric Mann–Whitney test). The data underlying this Figure can be found in S1 and https://doi.org/10.5281/zenodo.21341947 .

Analysis of cervical and lumbar LNs (cLNs) showed no differences in the total CD4 + T cell frequency S6c Fig ), but an increased percentage of IL-17A + CD4 + T cells in mice subjected to miR-122-5p antagomir treatment when compared with controls ( Fig 6c and 6d ), consistent with the early onset of the disease. We found no differences in the frequency and total cell numbers of Th1 (IFN-γ + ) and Treg (Foxp3 + ) cell populations in this case ( Figs 6d , S6 d, and S6 e). Moreover, no differences were observed in the frequency of other immune cells between mice treated with miR-122-5p or control antagomiRs ( S6f Fig ) based on the gating strategy highlighted in S7a Fig (T cell populations) and S7b Fig (other immune cell populations).

(a) Experimental setup: triple Foxp3-hCD2.Ifng-YFP.Il17a-GFP reporter mice were immunized s.c. in both flanks with 100 µg of myelin oligodendrocyte glicoprotein (MOG) 35-55 peptide emulsified in CFA solution (day 0). On the day of immunization and 2 days later, mice received a retro-orbital injection of 200 ng of pertussis toxin. At days 7, 9 and 11, mice received 5 µmol/kg of specific antagomiRs (retro-orbital injection). AntagomiR for cel-miR-67-3p was used as control. (b) Mice treated with control and miR-122-5p antagomiRs were scored for EAE clinical signs and weighed daily for 28 days. Results are mean ± SD from 3 independent experiments (n = 9–11). *p < 0.05 and **p < 0.01 (two-way ANOVA followed by Sidak’s multiple comparisons test). (c) Representative flow cytometry plots of IFN-γ + and IL-17A + CD4 + T cells in the cervical and lumbar lymph nodes of triple Foxp3-hCD2.Ifng-YFP.Il17a-GFP reporter mice with EAE and treated with control and miR-122-5p antagomiRs. (d) Frequency and absolute numbers of IL-17A + CD4 + T cells in the cervical and lumbar lymph nodes of triple Foxp3-hCD2.Ifng-YFP.Il17a-GFP reporter mice with EAE and treated with control and miR-122-5p antagomiRs. Results are mean + SD from 3 independent experiments (n = 9–11). Each dot represents an individual mouse. *p < 0.05 (non-parametric Mann–Whitney test). The data underlying this Figure can be found in S1 and https://doi.org/10.5281/zenodo.21341947 .

Next, to assess if the candidate miRNAs play a role in EAE pathophysiology, we treated mice challenged with EAE with synthetic antagomiRs and evaluated their impact on disease course and on the frequency of Teff and Treg subsets in the lymph nodes of antagomiR-treated mice at the end of the experiment. As depicted in Fig 5a , we injected the antagomiRs of the candidate miRNAs at days 7, 9 and 11 after immunization, which corresponds to a preclinical stage (onset of symptoms usually occurs around day 10). Focusing first on miR-122-5p antagomiR treatment—which efficiently reduced miR-122-5p expression levels ( S6a Fig ) —we observed an anticipation of EAE disease onset as mice started to develop symptoms and lose weight earlier than those treated with the control antagomiR ( Fig 5b ). Importantly, all mice that were subjected to miR-122-5p antagomiR injections were symptomatic already at day 12 post immunization (p.i.), whereas the control group needed an additional 5 days (day 17 p.i.) to present symptoms, thus entering the peak plateau stage later on ( S6b Fig ).

We employed the same experimental approach to identify miR-1247-5p targets in the context of Th1 cell differentiation. The differential Ago2 RIP-seq ( S4a Fig ) showed that the RNA-induced silencing complex (RISC) of miR-1247-overexpressing Th1 cells (as confirmed by RT-qPCR, S4b Fig ) was enriched (compared to mock-transduced Th1 cells) in 644 mRNAs, of which 423 (65.7%) had predicted binding sites for miR-1247-5p ( S4c Fig ). Upon examination of our mRNA-seq data for Th1 cells, we found 41 mRNAs upregulated (FC > 1.5 and p > 0.05) in the CNS ( Fig 4d ), of which 18 had relevant functions for Th1 cell biology ( S2 Table ). Based on literature curation, we selected 5 genes—Gng2, Arfip2, C2h2c3, Rasal3 and Wipf1—to further test in vitro, i.e., upon retroviral transduction of the native stem-loop of miR-1247 in Th1 cells ( Fig 4e ). Of these, Gng2 and Rasal3 were validated as miR-1247-5p targets both in vitro, as shown by their downregulation upon miR-1247-5p overexpression ( Fig 4e ), and in luciferase assays demonstrating direct binding of Gng2 and Rasal3 using the same strategy as described above for miR-122-5p target genes ( Fig 4f ). More specifically, the 3′UTR regions of both wild-type and mutant Gng2 and Rasal3 ( Fig 4f ) were introduced into a pmirGLO Dual-luciferase miRNA target expression vector and co-expressed with either miR-1247-5p or a control unrelated miRNA. These assays showed a decrease in luminescence in the presence of the miRNA that was abolished when the 3′UTR target regions were mutated ( Fig 4f ). Thus, our results indicate that miR-1247-5p targets Gng2 and Rasal3. Moreover, they provide the community with a comprehensive Ago2 RIP-seq data set for additional studies on miR-1247-5p in the context of effector T cell differentiation.

Together, these data suggest that miR-122-5p is able to repress Th17 cell proliferation in vitro by targeting Nudt4 and Sap130. Furthermore, it provides a much larger Ago2 RIP-seq data set that constitutes a resource for those interested in assessing miR-122-5p in future studies on T cell differentiation.

(a) Gene expression determined by RNA-seq of candidate miR-122-5p mRNA targets that are upregulated in Th17 cells isolated from the CNS of triple Foxp3-hCD2.Ifng-YFP.Il17a-GFP reporter mice at EAE peak plateau stage when compared to Th17 isolated from the periphery (lymph nodes and spleen) of the same mice (FC > 1.5; p < 0.05). Results are represented as mean of log fold change (logFC) between periphery- and CNS-derived Th17 cells. (b) RT-qPCR analysis of candidate miR-122-5p mRNA targets in retrovirally transduced Th17 cells expressing a control vector (RV-empty) or miR-122 (RV-122). Results are presented as fold vs. RV-empty (n = 4). Gray dashed lines link cells from the same mouse. Black dashed line represents the baseline expression level of controls. *p < 0.05 (paired Student t test). (c) Luciferase activity in HEK293T cells co-transfected with a pmirGLO Dual-Luciferase miRNA target expression vector containing either the WT or mutated miR-122-5p-binding sites of Sap130, Il18rap and Nudt4 mRNAs plus a vector containing miR-122-5p or an unrelated miRNA. An unrelated construct (without miR-122-5p binding sites) and a positive construct (Cpeb1) were included. Schematic representation of miR-122-5p putative binding sites of in the miR response elements of Sap130, Il18rap and Nudt4 mRNAs are included. Results are presented as fold vs. unrelated miRNA and represent mean + SD (n = 5). *p < 0.05 and **p < 0.01 (paired students t test). Representative histogram of proliferating CTV + cells (d) and replication index (e) of Th17 cells knocked-out (CRISPR-mediated) for target genes: Sap130, Il18rap and Nudt4. CD5 was used as a positive control for CRISPR-Cas9 gene edition. Data (n = 3) are normalized to control cells (Cas9). *p < 0.05 and **p < 0.01 (Paired t test). (f) Gene expression determined by RNA-seq of candidate miR-1247-5p mRNA targets that are upregulated in Th1 cells isolated from the CNS of triple Foxp3-hCD2.Ifng-YFP.Il17a-GFP reporter mice at EAE peak plateau stage when compared to Th1 isolated from the periphery (lymph nodes and spleen) of the same mice (FC > 1.5; p < 0.05). Results are represented as mean of log fold change (logFC) between periphery- and CNS-derived Th1 cells. (g) RT-qPCR analysis of candidate miR-1247-5p mRNA targets in retrovirally transduced Th1 cells expressing a control vector (RV-empty) or miR-1247 (RV-1247). Results are presented as fold vs. RV-empty (n = 3). Gray dashed lines link cells from the same mouse. Black dashed line represents the baseline expression level of controls. *p < 0.05 (paired Student t test). (h) Luciferase activity in HEK293T cells co-transfected with a pmirGLO Dual-Luciferase miRNA target expression vector containing either the WT or mutated miR-1247-5p-binding sites of Rasal3 and Gng2 mRNAs plus a vector containing miR-1247-5p or an unrelated miRNA. An unrelated construct (without miR-1247-5p binding sites) and a positive construct (Trappc9) were included. Schematic representation of miR-1247-5p putative binding sites of in the miR response elements of Rasal3 and Gng2 mRNAs are included. Results are presented as fold vs. unrelated miRNA and represent mean + SD (n = 5). *p < 0.05 and **p < 0.01 (paired Student t test). The data underlying this Figure can be found in S1 and https://doi.org/10.5281/zenodo.21341947 .

Starting with miR-122-5p, RT-qPCR analysis after the Ago-IP confirmed its enrichment in the Argonaute complex of retrovirally miR-122-transduced Th17 cells (RV-122) relative to the empty vector control (RV-empty), whereas other miRNAs, such as miR-126a-5p or miR15b-5p, showed similar levels between RV-122 and RV-empty-treated cells ( S4b Fig ). The results of the differential Ago2 RIP-seq in Th17 cells identified 587 mRNAs overexpressed in miR-122-5p-transduced Th17 cells, of which 339 (57.8%) had predicted miR-122-5p binding sites ( S4c Fig ). From these, as proof-of-concept, we inspected our mRNA-seq data for those upregulated in the CNS compared to peripheral Th17 cells, based on the previous observation that miR-122-5p had the opposite expression pattern ( Fig 3b ). This retrieved 27 mRNAs (FC > 1.5, p < 0.05) ( Fig 4a ), including 16 whose function was of relevance for Th17 cell biology and/or proliferation ( S2 Table ). Based on literature curation, we further analyzed the expression levels of 5 genes (Pttglip, Nudt4, Il18rap, Spred2 and Sap130) in retrovirally transduced Th17 cells with the native stem loop of miR-122 (RV-122), and observed that Nudt4, Il18rap and Sap130 were specifically downregulated in miR-122 overexpressing (versus control) cells, further reinforcing their potential to be functional targets of miR-122 in the context of Th17 cells in EAE ( Fig 4b ). Finally, to biochemically validate these 3 mRNAs as direct miR-122-5p targets, we performed luciferase assays. The three putative targets have predicted miR-122-5p binding sites in the 3’UTR of Nudt4 and Il18rap and the CDS of Sap130 ( Fig 4c ). To determine whether miR-122-5p was directly targeting these mRNAs, we designed reporter constructs in a pmirGLO Dual-luciferase miRNA target expression vector for the 3′UTRs of Nudt4 and Il18rap and the CDS of Sap130. We additionally included a negative control vector containing a 3′UTR without miR-122-5p binding sites and a positive control containing the 3′UTR of Cpeb1, a previously validated miR-122-5p target [ 34 ]. We transiently transfected these constructs into human embryonic kidney (HEK) 293 T cells together with an expression plasmid for either miR-122-5p or an unrelated miRNA. Co-transfection of miR-122-5p (but not an unrelated miRNA) showed a significant repression of luciferase activity compared with control for the three mRNA targets ( Fig 4c ). Mutations in the miR-122-5p binding sites of Nudt4, Il18rap and Sap130 led to significant recoveries of luciferase levels, attesting specificity ( Fig 4c ). To further confirm that these targets mediate the repressive effect of miR-122 in Th17 cell proliferation, we knocked-out Nudt4, Il18rap and Sap130 in vitro in naïve CD4 + T cells undergoing Th17 cell differentiation using CRIPSR-Cas9 gene editing, namely by electroporating them with a ribonucleoprotein (RNP) consisting of Cas9 combined with a single-guide RNA (sgRNA) against each target ( S3 Table ) or Cas9 alone as a control [ 35 ]. Additionally, we included an sgRNA against the surface marker Cd5 as a positive control for the efficiency of the CRISPR approach, and observed a decrease in the frequency of CD5 + cells from 99% to around 4% in Cd5 knocked-out cells comparing to controls ( S5 Fig ). Importantly, we observed a decreased proliferative capacity (as indicated by CTV dilution) of Th17 cells upon Nudt4 and Sap130 knock-out compared to the control condition ( Fig 4d and 4e ), thus recapitulating the in vitro phenotype of Th17 cells overexpressing miR-122.

Given that miRNAs exert their functions by suppressing mRNAs, and each miRNA can impact the expression of many mRNAs [ 33 ], we next aimed at identifying the repertoires of mRNAs directly regulated by miR-122-5p and miR-1247-5p. We devised a strategy based on differential Ago2-immunoprecipitation followed by RNA-seq (Ago2 RIP-seq) in lineage-specific cells overexpressing each candidate miRNA (upon retroviral transduction) versus control cells (mock-transduced) ( S4a Fig ). Briefly, naïve CD4 + T cells were sorted and activated under polarizing conditions (Th1 or Th17 as appropriate), transduced at day 1 with pre-miR RV particles (as used above for the overexpression experiments) and further incubated for 36 h, when GFP + T cells were sorted and subjected to UV-cross-linking and Ago2 immunoprecipitation with high-affinity antibody ( S4a Fig ).

We performed similar studies for miR-1247, the Th1-biased miRNA that downregulated IFN-γ production in vitro ( Fig 2g ). Although miR-1247-5p was upregulated on in vivo-generated Th1 cells ( Fig 1e and 1f ), its expression levels were low in Th1 cells differentiated in vitro in the presence of IL-12 and anti-IL-4 antibody ( Fig 3d ). We also tested IL-18, IL-15 and IFN-γ, since IL-18 and IL-15 can promote IFN-γ production in the presence of IL-12 [ 31 ]; and IFN-γ regulates its own production in an autocrine loop [ 32 ]. However, none of these cytokines was able to induce miR-1247-5p expression in Th1 cells ( Fig 3d ). We then reasoned that, as miR-1247-5p limits IFN-γ production by Th1 cells, its expression might be controlled by anti-inflammatory cytokines. Indeed, we found that TGF-β and IL-10 were the key inducers of miR-1247-5p expression in vitro, even in the presence of IL-12 ( Fig 3d ). Moreover, when we challenged our triple reporter mice with EAE, we observed that, similarly to miR-122-5p ( Fig 3b ), the expression levels of miR-1247-5p collapsed in Th1 cells isolated from the CNS when compared to peripheral Th1 cells ( Fig 3e ). Furthermore, upon mRNA-seq analysis of Th1 cells isolated from both sites (spleen/lymph nodes versus CNS), we established a CNS-specific pro-inflammatory signature, including the signature Th1 cytokine (Ifng) and TF (Tbx21), as well as chemokines (Ccl3 and Ccl4) and receptors (Il12rb2, Il18r1, Il18rap), which contrasted with a set of “self-regulatory genes”, including Il10, that were upregulated in Th1 cells from the secondary lymphoid organs ( Fig 3f ). These data suggest that miR-1247 is part of an anti-inflammatory (TGF-β/ IL-10-mediated) regulatory loop that is subverted once Th1 cells invade the CNS and shut down its expression.

(a) RT-qPCR analysis of miR-122 expression in CD4 + T cells cultured in vitro for 4 days in the presence of indicated cytokines and antibodies. Results are presented relative to miR-423-3p expression and represent mean ± SD (n = 6–7). *p < 0.05 and **p < 0.01 (non parametric Mann–Whitney test). (b) RT-qPCR analysis of miR-122 expression in Treg, Th1 and Th17 cells isolated and pooled from the periphery (lymph nodes and spleen) and the CNS of triple Foxp3-hCD2.Ifng-YFP.Il17a-GFP reporter mice at EAE peak plateau stage. Results are presented as fold vs. Treg in each specific tissue (ddct) and represent mean ± SD (n = 7). miR-423-3p was used as reference gene. *p < 0.05 (Wilcoxon test). (c) Gene expression determined by RNA-seq of pathogenic vs. non-pathogenic signature genes in Th17 cells isolated and pooled from the periphery (lymph nodes and spleen) and the CNS of triple Foxp3-hCD2.Ifng-YFP.Il17a-GFP reporter mice at EAE peak plateau stage. Results are represented as mean of log fold change (logFC) between periphery and CNS Th17 cells. (d) RT-qPCR analysis of miR-1247 expression in CD4 + T cells cultured in vitro for 4 days in the presence of indicated cytokines and antibodies. Results are presented relative to miR-423-3p expression and represent mean ± SD (n = 2–12). *p < 0.05 and p < 0.01 (non parametric Mann–Whitney test). (e) RT-qPCR analysis of miR-1247 expression in Treg, Th1 and Th17 cells isolated and pooled from the periphery (lymph nodes and spleen) and the CNS of triple Foxp3-hCD2.Ifng-YFP.Il17a-GFP reporter mice at EAE peak plateau stage. Results are presented as fold vs. Treg in each specific tissue (ddct) and represent mean ± SD (n = 7). miR-423-3p was used as reference gene. *p < 0.05 (Wilcoxon test). (f) Gene expression determined by RNA-seq of self-regulation vs. proinflammatory genes in Th1 cells isolated and pooled from the periphery (lymph nodes and spleen) and the CNS of triple Foxp3-hCD2.Ifng-YFP.Il17a-GFP reporter mice at EAE peak plateau stage. Results are represented as mean of log fold change (logFC) between periphery and CNS Th1 cells. The data underlying this Figure can be found in S1 Data .

We next aimed at dissecting the molecular cues that regulate miR-122-5p and miR-1247-5p expression. For this, we sorted naïve CD4 + T cells from the spleen and lymph nodes of WT mice and cultured them in the presence of different cytokines involved in Th17 or Th1 cell differentiation, respectively. Starting with the Th17-biased miR-122-5p, its expression reached maximal levels in the presence of IL-6 and TGF-β, but decreased when stimulated with IL-1β and especially with IL-23 ( Fig 3a ). IL-6 and TGF-β are the key cytokines involved in the polarization of naïve CD4 + T cells into the Th17 subset, while IL-1β and IL-23 are critical for lineage maintenance and the acquisition of a pathogenic phenotype [ 26 ]. Thus, our data suggests that miR-122 might limit the pathogenicity of Th17 cells but is downregulated in a strong (IL-23/IL-1β-rich) inflammatory environment. To build on this premise in vivo, we characterized miR-122-5p expression levels in CD4 + T cell subsets isolated from the CNS of triple Foxp3 hCD2 .Ifng YFP .Il17a GFP reporter mice challenged with EAE. Interestingly, our data showed that miR-122-5p was markedly downregulated in cells isolated from the CNS compared with peripheral Th17 cells ( Fig 3b ). To further provide biological context to these observations, we performed mRNA-seq in Th17 cells isolated from both sites, i.e., spleen/lymph nodes versus CNS. This showed that genes associated with a pathogenic phenotype—such as those encoding key cytokines as IFN-γ and GM-CSF (Ifng and Csf2), chemokines (Ccl4 and Cxcl3) and TFs (Tbx21 and Runx1) - were all increased in CNS-derived Th17 cells when compared to peripheral Th17 cells ( Fig 3c ) [ 26 ]. These results link miR-122 expression dynamics with the pathophysiology of EAE, with its downregulation in the CNS associating with enhanced pathogenicity of Th17 cells.

In what refers to miR-1247-5p, its overexpression with retroviral vectors in differentiating Th1 cells ( Figs 2g and S3d ) led to a decreased frequency of IFN-γ + CD4 + T cells ( Fig 2h and 2i ), but did not affect cell proliferation ( Fig 2j – 2l ), nor Treg or Th17 in vitro polarization ( S3c and S3e Fig ). These results indicate that miR-1247-5p selectively downregulates IFN-γ production in Th1 cells.

(a) Experimental setup: naive CD4 + T cells were sorted from WT mice and differentiated in vitro towards Th17 cells for 4 days. At day 1, cells were transduced with retroviral vectors encoding the precursor forms of miR-122 and differentiation was analyzed by flow cytometry. Empty vector was used as control. (b) Representative flow cytometry plots of IL-17A + cells in retrovirally transduced Th17 cells expressing a control vector (RV-empty) or miR-122 (RV-122) and frequency of IL-17A + cells in retrovirally transduced Th17 cells expressing a control vector (RV-empty) or miR-122 (RV-122) from 5 independent experiments (c) . Dotted lines link cells from the same mouse. (d) Representative histogram of proliferating CTV + cells in retrovirally transduced Th17 cells expressing a control vector (RV-empty) or miR-122 (RV-122). (e) Frequency of proliferating CTV + cells in retrovirally transduced Th17 cells expressing a control vector (RV-empty) or miR-122 (RV-122). Results are mean ± SD from 2 independent experiments (n = 4). *p < 0.05 and **p < 0.01 (two-way ANOVA followed by Sidak’s multiple comparisons test). (f) Replication index of retrovirally transduced Th17 cells expressing a control vector (RV-empty) or miR-122 (RV-122). Dotted lines link cells from the same mouse. *p < 0.05 (Paired t test). (g) Experimental setup: naive CD4 + T cells were sorted from WT mice and differentiated in vitro towards Th1 cells for 4 days. At day 1, cells were transduced with retroviral vectors encoding the precursor forms of miR-1247 and differentiation was analyzed by flow cytometry. Empty vector was used as control. (h) Representative flow cytometry plots of IFN-γ + cells in retrovirally transduced Th1 cells expressing a control vector (RV-empty) or miR-1247 (RV-1247) and frequency of IFN-γ + cells in retrovirally transduced Th1 cells expressing a control vector (RV-empty) or miR-122 (RV-122) from 5 independent experiments (i) . Dotted lines link cells from the same mouse. *p < 0.05 (Paired t test). (j) Representative histogram of proliferating CTV + cells in retrovirally transduced Th1 cells expressing a control vector (RV-empty) or miR-1247 (RV-1247). (k) Frequency of proliferating CTV + cells in retrovirally transduced Th1 cells expressing a control vector (RV-empty) or miR-1247 (RV-1247). Results are mean ± SD from 3 independent experiments (n = 5). (l) Replication index of retrovirally transduced Th1 cells expressing a control vector (RV-empty) or miR-1247 (RV-1247). Dotted lines link cells from the same mouse. The data underlying this Figure can be found in S1 and https://doi.org/10.5281/zenodo.21341947 .

We inquired whether these miRNAs might have a cell-intrinsic role in Th17 (miR-122-5p) or Th1 (miR-1247) cells by performing overexpression experiments in in vitro-differentiated cells (from isolated naïve CD4 + T cells— S2 Fig ). Upon transduction with retroviral GFP reporter vectors (RVs) encoding the native stem-loop of miR-122 (RV-122) ( Fig 2a ), we observed an upregulation of miR-122-5p levels in in vitro-differentiated Th17 cells ( S3a Fig ). While no differences were observed in the frequency of IL-17A + ( Fig 2b and 2c ), Th17 cells overexpressing miR-122 had a significantly lower proliferation rate when compared with control cells (RV-empty), as observed by the frequency of proliferating GFP + T cells upon CTV labeling as well as their replication index ( Fig 2d – 2f ). Notably, miR-122 overexpression did not impact Th1 or Treg cell differentiation in vitro ( S3b and S3c Fig ). These results suggest that miR-122-5p negatively regulates Th17 cell function by limiting their proliferative capacity.

As the response is triggered in peripheral lymphoid organs, we isolated these populations by FACS from the spleen and lymph nodes of EAE-induced triple Foxp3 hCD2 .Ifng YFP .Il17a GFP reporter mice at the peak-plateau phase of the disease, and extracted RNA to perform both small RNA- and messenger-RNA sequencing (small RNA-seq and mRNA-seq, respectively) ( Figs 1b and S1a ). Both datasets showed that the isolated YFP + , GFP + and hCD2 + CD4 + T cell populations segregated unambiguously on a principal component analysis ( S1b and S1c Fig , respectively), and importantly, differentially expressed the signature genes of each corresponding CD4 + T cell subset ( S1d Fig ), thus attesting the reliability and reproducibility of our experimental system. The small RNA-seq data revealed clearly distinct miRNomes, with 110 miRNAs differentially expressed between Teff and Treg cell populations in vivo ( Fig 1c and 1d ; S1 Table ). These data are a valuable resource for the community, from which we decided to zoom in on miRNAs specifically enriched or depleted in one of the CD4 + T cell subsets. Interestingly, within this candidate list of differentially expressed miRNAs, only nine were specifically up- or down-regulated in one subset (i.e., Th1, Th17 or Treg) compared with the other two subsets ( Fig 1e ): miR-1247-5p was up-regulated in Th1 cells; miR-126a-5p, miR-5108 and miR-122-5p were up-regulated in Th17 cells, miR-211-5p, miR-15b-5p, miR-151-3p and miR-467a-5p were up-regulated in Treg cells; and miR-125a-5p was down-regulated in Th17 cells. We performed validation studies by RT-qPCR analysis of independent Th1, Th17 and Treg samples isolated by FACS from the spleen/lymph nodes of triple reporter mice with EAE. These confirmed the expression profiles of five of the candidate miRNAs ( Fig 1f ), namely miR-1247-5p, miR-122-5p, miR-125a-5p, mir-211-5p and miR-15b 5p. Notably, two of the validated miRNAs, miR-15b-5p and miR-125a-5p, have already been thoroughly studied and, consistent with our expression data, shown to be involved in Treg or Th17 differentiation and/or function [ 22 , 28 – 30 ]. Thus, for subsequent “proof-of-concept” functional assays, and since the effector cell populations are the major drivers of EAE, we focused on the candidates miR-1247-5p and miR-122-5p, highly expressed inTh1 and Th17 cells, respectively ( Fig 1e and 1f ). Of note, analysis of these candidates at pre-symptomatic stages showed that Th17-associated miRNAs did not exhibit significant differences at this earlier phase, while the remaining candidates displayed a consistent pattern with that observed at the peak/plateau phase ( S1e Fig ).

(a) Schematic representation of the genomic alterations introduced in the triple Foxp3-hCD2.Ifng-YFP.Il17a-GFP reporter mouse strain with the respective CD4 + T cell subset. In the loci of each reporter gene, a sequence for an independently translated reporter protein was inserted downstream of the gene endogenous translational stop codon. (b) Experimental setup: triple Foxp3-hCD2.Ifng-YFP.Il17a-GFP reporter mice were subjected to EAE and at the peak plateau stage Treg, Th1 and Th17 cells were isolated from the spleen and lymph nodes by FACS, according with each reporter gene. After RNA isolation samples were subjected to both mRNA-seq and small RNA-seq. (c, d) Heatmaps depicting differentially expressed miRNAs between Treg and effector Th (up in Treg vs. Th1 or Treg vs. Th17 (c) ; vs. up in Th1 vs. Treg or Th17 vs. Treg (d) ). Colors indicate the direction and magnitude of calculated z-scores, with red representing higher expression of a miRNA in a given cell subset and blue, the opposite. (e) Volcano plots depicting differentially expressed miRNAs between Th1, Th17 and Treg subsets. miRNAs names are indicated when differentially expressed (log2FC > 1; FDR < 0.05) in two comparisons. (f) RT-qPCR analysis of the 9 candidate miRNAs’ expression in Treg, Th1 and Th17 cells isolated and pooled from the lymph nodes and spleen of triple Foxp3-hCD2.Ifng-YFP.Il17a-GFP reporter mice at EAE peak plateau. Box-and-whiskers indicate median, 25% and 75% percentiles, minimum and maximum expression levels of candidate miRNAs. Results are presented relative to miR-423-3p expression from 7 independent experiments. *p < 0.05 and **p < 0.01 (Kruskal–Wallis test). The data underlying this Figure can be found in https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1067547 (c, d) and in S1 Data (e, f).

In order to identify miRNA repertoires of CD4 + T cell subsets under inflammatory conditions (EAE) in vivo, we established a triple reporter mouse for IFN-γ (YFP + ), IL-17A (GFP + ) and Foxp3 (hCD2 + ) (triple Foxp3 hCD2 .Ifng YFP .Il17a GFP reporter mice Fig 1a ), by crossing previously described single reporter lines [ 25 – 27 ]. While Treg cells were isolated based on hCD2 expression, driven by the activity of the Foxp3 promoter, which defines this lineage independently of activation status, Th1 and Th17 cells were purified based on YFP and GFP expression, thus requiring cell activation for cytokine gene expression.

Discussion

This study aimed to provide novel insights on how miRNAs regulate CD4+ T cell subsets during an autoimmune response in vivo. Previous studies on the topic had focused on a limited set of miRNAs, such as miR-29 [20] and the miR-106-363 cluster [21] which were shown to inhibit Th1 and Th17 differentiation, respectively, by directly targeting either T-bet (and Eomes) or RORγt, TFs that directly regulate the production of IFN-γ or IL-17. Furthermore, several miRNAs had been shown to be either up-regulated—e.g., let-7e, miR-155, miR-223 and miR-326—or down-regulated—e.g., miR-20b, miR-26a and miR-30a—in CD4+ Th cell subsets of MS patients or EAE-challenged mice, with some being functionally implicated in MS or EAE—e.g., miR-326 upregulation stimulated the production of IL-17 in Th17 cells and enhanced pathogenesis [23] while miR-92 restricted Treg induction and supported Th17 responses, consistent with its elevated levels in CD4+ T cells of MS patients [24]. Notably, two of our validated miRNAs, miR-15b-5p and miR-125a-5p, have already been shown to control Treg/Th17 cell function in EAE. Thus, miR-15b-5p, which was downregulated in EAE and MS, suppressed Th17 cells while increasing peripheral Treg differentiation [28,29]; and miR-125a-5p stabilized the immunoregulatory capacity of Treg cells, thereby impacting EAE severity [22,30].

We now provide a global resource on miRNAs, as well as mRNAs, expressed on Th1, Th17 and Treg cell subsets from the peripheral lymphoid organs and the CNS (for mRNA-seq) of EAE-challenged mice, which can be exploited in further studies. The functional analyses of miR-122-5p and miR-1247 as “proof-of-concept” for our resource provided a key conceptual insight: they constitute molecular brakes to pro-inflammatory Th1 and Th17 cells that are subverted in the CNS. Thus, miR-122-5p and miR-1247 were strikingly downregulated in, respectively, Th17 and Th1 cells from the CNS compared with their lymphoid organ counterparts, and this was accompanied by an upregulation of pro-inflammatory/pathogenic signatures (like Ifng, Tbx21 or Csf2, the later encoding GM-CSF) at the mRNA level. We further identified the molecular cues that regulate the dynamic expression of these two miRNAs. For Th17-biased miR-122, we found it to be induced by IL-6 and TGF-β, but downregulated by IL-1β and especially IL-23. This is particularly interesting because while IL-6 and TGF-β are the key cytokines promoting Th17 cell polarization from naïve CD4+ T cells, IL-1β and IL-23 are critical for Th17 cell maintenance, expansion and, most importantly, the acquisition of a pathogenic phenotype in the target organ [26]. As for the Th1-biased miR-1247-5p, we identified TGF-β and IL-10 as the key inducers of its expression, even in the presence of IL-12. Given that TGF-β induces the expression of IL-10 in Th1 cells to reduce their encephalogenicity [37,38], we conceptualize miR-1247 as part of this self-regulatory mechanism to control excessive IFN-γ levels in Th1 cells.

With regard to the unexpected upregulation of miR-1247-5p expression upon antagomiR administration, one explanation could be an indirect derepression mechanism: if the miRNA targets a repressor of its own expression, silencing the miRNA may release and upregulate its expression via a feedback loop [39]. Another possibility could be a competing endogenous RNA (ceRNA) network disrupted by the antagomiR, thus shifting the miRNA-target equilibrium, potentially unleashing mRNAs acting as positive regulators of the miRNA transcription [40]. While the precise mechanism remains to be elucidated, the biological consequences of this paradoxical effect of the antagomiR were entirely consistent with miR-1247-5p gain-of-function, since it drove a reduction in IFN-γ⁺ Th1 cells and amelioration of EAE severity.

To gain molecular insight on miR-122-5p and miR-1247 functions, we aimed to characterize direct mRNA targets relevant to Th17 or Th1 cell biology, respectively. In fact, mRNA target identification arguably remains the greatest challenge in miRNA biology, as it is now clear that each miRNA can fine-tune the expression of hundreds of mRNAs in any given cell type [33]. To tackle this problem, and in line with the resource nature of our study, we used differential Ago2 RIP-seq on in vitro-polarized CD4+ T cells: by overexpressing the candidate miRNA, we promoted its binding to target mRNAs, thus enriching them in Ago2 complexes that are UV-cross-linked and immunoprecipitated, followed by RNA-seq. This resulted in large Ago2 RIP-seq datasets that can be used by the community for other studies on these miRNAs in effector CD4+ T cells. Here we zoomed in on candidate mRNAs targets using the following criteria: (1) having predicted binding sites for the candidate miRNA; (2) being upregulated in the CNS compared to secondary lymphoid organs, thus inversely correlating with the miRNA expression; (3) being downregulated upon retroviral overexpression of the miRNA in vitro; (4) directly binding to the mRNA sequence as indicated by luciferase assays.

Using this pipeline, we identified Nudt4, Il18rap and Sap130 as putative validated targets of miR-122-5p in Th17 cells. Furthermore, using CRISPR-mediated deletion of these target genes in Th17 cells, we have observed that both Nudt4 and Sap130 knock-outs significantly reduced Th17 cell proliferation in vitro, thus phenocopying the effect of miR-122 overexpression. Of note, Nudt4 is a hydrolase that catalyzes the m7G tRNA modification that promotes the translation of cell cycle mRNAs [41], consistent with the Th17 cell proliferation phenotypes upon miR-122-5p modulation. Although the function of Sap130 remains more enigmatic, it is known to associate with Sin3A [42], which is essential for IL-17 production [43]. Collectively, the identification of these direct targets provides new biological context to miR-122-5p-mediated regulation of Th17 cells.

With regard to miR-1247, our analysis pinpointed Gng2 and Rasal3 as direct targets in Th1 cells. Gng2, G-protein gamma subunit 2, acts as a tumor suppressor gene by inhibiting Akt and Erk activity [44]. In turn, Erk1-deficient mice exhibit increased Th1 cell differentiation and earlier EAE onset than wild-type controls [45]. As for Rasal3, Ras GTPase activating protein-like 3, it is a member of the GTPase-activating proteins family required for survival of naïve and activated T cells [46]. Importantly, both Rasal3 and its partner GTPase, Rac2 [47] have been shown to promote T-cell production of IFN-γ [48,49]. This previous knowledge on Gng2 and Rasal3 nicely fits our proposed model where miR-1247-mediated targeting impacts Th1 responses in EAE. Overall, by meeting the criteria stated above, these validated mRNA targets offer mechanistic insight into the functions of miR-122-5p and miR-1247 in regulating the pathogenicity of T helper cell subsets in EAE.

Notably, our functional analyses demonstrated the pathophysiological impact of miR-122 and miR-1247 on EAE onset and severity, which associated with intrinsic effects on Th17 cell proliferation and Th1 cell differentiation, respectively. As both miRNAs suppress these pro-inflammatory responses, our findings highlight the potential of modulating specific miRNAs to control the pathogenic immune populations implicated in autoimmune diseases like MS.

A limitation of our current study is that it does not address the role of miRNAs in CD4+ T cell plasticity during EAE [50]. Th17 cells, in particular, are known to transdifferentiate into Th1 cells under the IL-23-rich inflammatory environment of EAE [51], but we opted for a snapshot of the effector CD4+ T cell subsets at the peak of EAE using dynamic reporter mice, rather than fate mapping [50]. We also did not address the impact of our miRNA candidates on the production of GM-CSF, which has been shown by Becher and colleagues to initiate autoimmune neuroinflammation [52], particularly with its secretion by Th1 and Th17 cells being sufficient to induce EAE [5,53]. Another caveat of our study is that in vivo modulation of miRs was achieved through systemic administration of antagomiRs, which while effective at reducing miR levels in peripheral immune cells, does not exclude potential effects on other cell types where these miRs may be abundant, such as hepatocytes in the case of miR-122-5p. To overcome these limitations, future studies may address the encephalitogenic potential of the candidate miRs through targeted approaches such as (i) T cell-specific conditional miR deletion, e.g., through Cre-mediated knockout driven by the CD4 promoter; (ii) T cell-targeted lipid nanoparticle delivery systems [54] or aptamer-antimiR chimeras [55]; (iii) use of MOG-specific Th17 and Th1 cells with modulated miR-122-5p and miR-1247-5p expression in an adoptive transfer model of EAE [6].

Conversely, the key contributions of this study are the global resource on miRNAs expressed in Th1, Th17 and Treg cells at the peak of EAE disease severity; the identification of two new miRNA determinants of Th1/Th17 cell function, their repertoires of putative mRNA targets in those cells, the intricate regulation by a network of cytokines and their expression dynamics in vivo across lymphoid organs and the CNS, which open new avenues to explore in both mice and humans, with the ultimate goal of devising miRNA-based immunotherapies for MS.

[END]
---
[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3003271

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/