Deficiency of MicroRNA-10a in CD4⁺ T Cells Protects Against Intestinal Infection through Mitochondrial Oxidation-IL-22 Pathway

Abstract

Interleukin-22 (IL-22) produced by CD4⁺ T cells plays an important role in regulating intestinal immune responses during inflammation and infection, but the mechanisms controlling IL-22 expression in T cells remain incompletely understood. MicroRNA-10a (miR-10a) is known to regulate CD4⁺ T cell function, but its role in IL-22 production has not been defined. Here, using mouse CD4⁺ T cell-specific miR-10a knockout models, we examined how miR-10a regulates IL-22 expression and the underlying metabolic mechanisms. MiR-10a deficiency led to increased IL-22 production in CD4⁺ T cells both in vitro and in vivo, under steady and inflammatory conditions. CD4⁺ T cell-specific miR-10a knockout mice were resistant to Citrobacter rodentium infection, and the protection was abolished when blocking the IL-22 pathway in mice. Mechanistically, miR-10a-deficient CD4⁺ T cells exhibited increased mitochondrial oxidative metabolism and membrane potential. Pharmacologic inhibition of mitochondrial Complex III with antimycin A suppressed the enhanced IL-22 production in miR-10a-deficient T cells. We further identified Uqcrq, a subunit of mitochondrial Complex III, as a direct target of miR-10a, and loss of Uqcrq suppressed IL-22 production in CD4⁺ T cells. Together, these findings identify miR-10a as a T cell-intrinsic regulator of mitochondrial oxidative metabolism that constrains IL-22 production in the intestine.

Introduction

Intestinal epithelial cells (IECs) are the front line of defense against enteric pathogens while simultaneously maintaining a balanced relationship with commensal microbes.[1] Interleukin-22 (IL-22), mainly produced by CD4⁺ T cells and innate lymphoid cells (ILCs), plays an essential role in maintaining intestinal homeostasis by promoting tissue repair and upregulating antimicrobial peptides and mucus production.[2, 3] Therefore, IL-22 is considered a potential therapeutic target for inflammatory bowel disease (IBD).[4] In addition, CD4⁺ T cell production of IL-22 is indispensable in protecting the host against enteric pathogens such as Citrobacter rodentium.[2]

MicroRNAs (miRNAs) are key post-transcriptional regulators that affect a variety of cells, including T cells. Among them, miR-10a has been reported to be dysregulated in the inflamed intestine of IBD patients.[5] Our previous study demonstrated that miR-10a negatively regulates Treg suppressive function in colitis by regulating transcriptional and metabolic programs,[6] and miR-10a suppresses IL-10 production in CD4⁺ T cells through targeting Blimp1,[7] highlighting its critical role in T cell responses. However, whether miR-10a also controls IL-22 expression and the metabolic mechanisms involved remain unclear.

Cellular metabolism is critical in regulating T cell fate and function.[8, 9] While glycolysis supports proliferation, mitochondrial oxidative phosphorylation (OXPHOS) more likely supports T cell suppressive function. The mitochondrial electron transport chain, including Complex III, is central to OXPHOS by transferring electrons from ubiquinol to cytochrome c and maintaining the proton gradient for ATP synthesis.[10] Here, we show that miR-10a deficiency in CD4⁺ T cells enhances IL-22 production under steady and inflammatory conditions, conferring resistance to Citrobacter rodentium infection. Mechanistically, miR-10a targets Uqcrq, a subunit of Complex III,[11] limiting mitochondrial oxidation and IL-22 expression. These findings reveal a novel link between miRNA-mediated metabolic control and cytokine regulation in CD4⁺ T cells.

Materials and Methods

Mice

C57BL/6J wild-type (WT) mice, B6.Cg-Tg(Cd4-cre)1Cwi/BfluJ mice, and B6.129S7-Rag1tm1Mom/J were purchased from Jackson Lab. MiR-10a^fl/fl mice were generated as described previously.[7] CD4^cremiR-10a^fl/fl and CD4^cremiR-10a^fl/+ mice were generated by crossing Cd4-cre mice and MiR-10a^fl/fl mice.[7] ] CD4^cremiR-10a^fl/+ littermates were used as WT controls for CD4^cremiR-10a^fl/fl mice in all experiments. All animals were maintained under specific pathogen-free conditions at the University of Texas Medical Branch or the Center for Comparative Medicine at Northwestern University. Age- and sex-matched co-housed mice were used for all in vivo studies. All procedures were approved by the Institutional Animal Care and Use Committees at the University of Texas Medical Branch and Northwestern University.

CD45Rb^hi T Cell Transfer-Induced Colitis Model

CD45Rb^hi T cells were purified by sorting the top 20% CD45Rb-expressing CD4⁺ T cells. CD45Rb^hi T cells (100K/mouse) were transferred into Rag1^−/− mice intravenously. Mice were sacrificed six weeks later.

Citrobacter Rodentium Infection Model

CD4^cremiR-10a^fl/fl and CD4^cremiR-10a^fl/+ mice were infected with Citrobacter Rodentium (Strain DBS100, 5 × 10⁸ CFU/mouse) by gavage. In some experiments, mice were given anti-IgG or anti-IL-22 antibody (20 mg/kg, every other day) intraperitoneally. Mice were sacrificed ten days later.

T Cell Isolation, Activation, and Differentiation

CD4⁺ T cells were isolated from spleens using anti-CD4 magnetic beads and activated by plate-bound anti-CD3 antibody and soluble anti-CD28 antibody. Th1-skewed conditions included IL-12 (20 ng/mL), and Th17-skewed conditions included IL-6 (50 ng/mL), TGF-β (1 ng/mL); anti-IFN-γ (5 μg/mL), and anti-IL-4 (5 μg/mL).

Quantitative Polymerase Chain Reaction

Total RNA was extracted from cells or intestinal tissues using TRIzol. RNA purity and concentration were determined using a BioTek Synergy microplate reader. For mRNA analysis, cDNA was synthesized from 250 ng of total RNA using the Bio-Rad iScript cDNA Synthesis Kit. For determining the expression of miR-10a in CD4⁺ T cells, reverse transcription was performed using the TaqMan MicroRNA Reverse Transcription Kit and a miR-10a-specific RT primer. Gene expression levels were determined by quantitative polymerase chain reaction (qPCR) using iTaq Universal SYBR Green Supermix (mRNA) or iTaq Universal Probes Supermix (miR-10a) on a Bio-Rad CFX96 Real-Time PCR System. βactin was used as an internal reference.

Flow Cytometry

T cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin for five hours, with Glogistop added for the last three hours. Cells were stained with live/dye and CD4, followed by permeabilization and fixation using Foxp3 /Transcription Factor Staining Buffer Set. Subsequently, cells were stained with transcription factors and cytokines. Cells were then fixed using 2% paraformaldehyde. For gating strategy, debris and doublets were excluded based on FSC/SSC and FSC-A/FSC-H gating. Live CD4⁺ T cells were identified as live/dead dye-negative CD4⁺ cells, and cytokine expression was analyzed within the CD4⁺ T cell population.

Hematoxylin and Eosin Staining

Intestinal tissues were cut longitudinally and Swiss-rolled. After fixation, dehydration, and embedding, tissues were cut into 5-μm sections, which were then transferred onto slides. Tissue slides were rehydrated and stained with Hematoxylin and Eosin as described previously.[12] The pathology scores were assessed.[13, 14]

Mitochondrial Oxidation Measurement

Splenic CD4⁺ T cells (200K/well) were seeded onto the poly-L-lysine–coated cell plates, and mitochondrial respiration parameters were assessed by measuring the oxygen consumption rate (OCR) using the Seahorse XF96 Analyzer with the Seahorse XF Cell Mito Stress Test Kit, according to the manufacturer’s instructions.

Luciferase assay

RAW264.7 cells were plated in 24-well plates and transfected with Psi-CHECK-2 reporter constructs containing either WT or mutant Uqcrq 3′UTR sequences using Lipofectamine 3000 in Opti-MEM. Cells were collected 24 hours later, and luciferase activity was quantified with the Dual-Luciferase Reporter Assay System according to the manufacturer’s instructions. Renilla luciferase activity was normalized to Firefly luciferase as an internal control for transfection efficiency.

CRISPR-Cas9 RNP Nucleofection

Cas9 ribonucleoproteins were formed by combining Alt-R S.p. Cas9 Nuclease V3 with sgRNAs and incubating for 10 min at room temperature.[15] Two sgRNAs were used to improve knockout efficiency, with two non-targeting sgRNAs as controls. CD4⁺ T cells were resuspended in 20 μL P3 nucleofector solution, mixed with the Cas9–sgRNA complexes, and nucleofected using the 4D-Nucleofector X Unit (Lonza) with program DN100.

Western Blot

Proteins were extracted from T cells using RIPA buffer, which contains protease/phosphatase inhibitors and PMSF. Following BCA-based quantification, equal amounts of protein were separated on SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked and incubated with anti-Uqcrq antibody (1:500) overnight at 4 °C and then with HRP-linked secondary antibodies for 2 hours at room temperature. Immunoreactive bands were detected by ECL reagents and imaged using a ChemiDoc imaging system. Membranes were then stripped with Restore PLUS buffer, re-blocked, and probed with an anti-β-actin antibody. Band intensities were quantified using ImageJ.

Statistical Analysis

Data were analyzed using GraphPad Prism 10 and are shown as mean ± SEM, with sample sizes indicated in the figure legends. Statistical significance was defined as p < 0.05. Two-group comparisons used two-tailed Student’s t-tests or Mann–Whitney U tests as appropriate. For three or more groups, one-way or two-way ANOVA was performed with Tukey’s or Sidak’s post-hoc tests.

Results

1. Deficiency of miR-10a in CD4⁺ T cells leads to a higher level of IL-22

To investigate whether miR-10a affects IL-22 in CD4⁺ T cells, we activated and cultured WT and miR-10a-deficient CD4⁺ T cells, isolated from WT CD4^cremiR10a^fl/+ littermate control mice and CD4⁺ T cell-specific miR-10a knockout CD4^cremiR-10a^fl/fl mice, under neutral conditions. We found that miR-10a-deficient T cells expressed a higher level of IL-22 (Figure 1A). Among T cells, Th1 and Th17 are major sources of IL-22.[2] We then activated and cultured WT and miR-10a-deficient CD4⁺ T cells under Th1 and Th17 conditions. Loss of miR-10a increased IL-22 at both mRNA and protein levels under either Th1 or Th17 conditions (Figure 1A–C). In addition, CD4^cremiR-10a^fl/fl mice showed a higher percentage of intestinal IL-22-producing CD4⁺ T cells compared to CD4^cre miR10a^fl/+ mice under steady conditions (Figure 1D–E).

Figure 1. Deficiency of miR-10a leads to a higher level of IL-22 in CD4⁺ T cells.

(A-C) Splenic CD4⁺ T cells were isolated from CD4^cremiR10a^fl/+ littermate control mice (WT) and CD4^cremiR-10a^fl/fl mice (miR-10a^ΔCD4) and activated with anti-CD3 and anti-CD28 antibodies under neutral, Th1, and Th17 conditions. (A) Il22 mRNA expression on day 2 (n = 3/group). IL-22 production by CD4⁺ T cells was analyzed by flow cytometry, with representative plots (B) and summarized frequencies (C).

(D-E) CD4^cremiR10a^fl/+ mice (WT) and CD4^cremiR-10a^fl/fl mice (miR-10a^ΔCD4) (n = 4/group) were sacrificed under steady conditions. IL-22 production by colonic CD4⁺ T cells was analyzed by flow cytometry, with representative plots (D) and summarized frequencies (E).

Data are presented as mean ± SEM. Results are representative of three independent experiments. Unpaired Student’s t-test (A-D); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

To investigate whether miR-10-deficient CD4⁺ T cells produce elevated levels of IL-22 under inflammatory conditions, we transferred WT and miR-10a-deficent CD45RB^hi CD4⁺ T cells into immunodeficient Rag1^−/− mice. Intestinal inflammation was assessed 5 weeks after cell transfer. Deficiency of miR-10a in CD4⁺ T cells protected mice against colitis, as evidenced by lower pathological scores (Figure 2A–B), reduced intestinal TNF-α levels (Figure 2C), and decreased frequencies of intestinal IFN-γ-producing T cells in Rag1^−/− recipients of miR-10a-deficient CD4⁺ T cells (Figure 2D and F). Of note, CD4⁺ T cell production of IL-22 was increased in Rag1^−/− recipients of miR-10a-deficient CD4⁺ T cells (Figure 2D–E). Furthermore, there was no difference in IL-17-producing Th17 cells and Foxp3⁺ Tregs between the two groups of recipients (Figure 2D, G, and H). IL-10-producing CD4⁺ T cells was increased in infected CD4^cremiR-10a^fl/fl mice compared to WT mice (Figure 2D and I).

Figure 2. MiR-10a-deficient CD4⁺ T cells induce less severe colitis in the CD45Rb^hi T cell transfer model.

Splenic CD4⁺ CD45Rb^hi T cells were isolated from CD4^cremiR10a^fl/+ littermate control mice (WT) and CD4^cremiR-10a^fl/fl mice (miR-10a^ΔCD4) (100K/mouse) were transferred into Rag1^−/− mice intravenously (n = 5/group). Mice were sacrificed after six weeks.

(A–B) Representative H&E-stained intestinal sections (A) and corresponding pathological scores (B).

(C) Colonic TNF-α mRNA expression.

(D-I) Colonic IL-22⁺, IFN-γ⁺, IL-17A⁺, Foxp3⁺, and IL-10⁺ in CD4⁺ T cells were analyzed by flow cytometry, with representative plots (D) and summarized frequencies (E-I).

Data are presented as mean ± SEM. Results are representative of three independent experiments. Mann–Whitney U test (B); Unpaired Student’s t-test (C, E-I); ns, not significant; *p < 0.05, **p < 0.01.

Taken together, miR-10a deficiency in CD4⁺ T cells leads to increased IL-22 production both in vitro and in vivo, under healthy and inflammatory conditions.

2. CD4⁺ T cell-specific miR-10a knock-out mice are resistant to C. rodentium infection

CD4⁺ T cell production of IL-22 is critical for host defense against Citrobacter rodentium (C. rodentium) infection.[2] We then orally infected WT CD4^cre miR10a^fl/+ mice and CD4⁺ T cell-specific miR-10a knockout CD4^cremiR-10a^fl/fl mice with C. rodentium.CD4^cremiR-10a^fl/fl mice developed less severe intestinal inflammation compared to WT mice (Figure 3A–B). Additionally, loss of miR-10a in CD4⁺ T cells reduced C. rodentium CFU in feces (Figure 3C) and liver (Figure 3D), indicating enhanced bacterial clearance in the colon and reduced dissemination to the liver. We found that the percentage of intestinal IL-22-producing CD4⁺ T cells was increased in infected CD4^cremiR-10a^fl/fl mice compared to WT mice (Figure 3E–F). Furthermore, CD4⁺ T cell production of IL-10 was also increased in the intestine of CD4^cremiR-10a^fl/fl mice, while there was no difference in intestinal Th1, Th17, and Treg between WT and CD4^cremiR-10a^fl/fl mice (Figure 3G–K).

Figure 3. CD4^cremiR-10a^fl/fl mice are resistant to Citrobacter rodentium infection.

CD4^cremiR10a^fl/+ mice (WT) and CD4^cremiR-10a^fl/fl mice (miR-10a^ΔCD4) were infected with Citrobacter Rodentium (5 × 10⁸ CFU/mouse) by gavage (n = 5/group). Mice were sacrificed on day 10.

(A–B) Representative H&E-stained intestinal sections (A) and corresponding pathological scores (B).

(C) Fecal C. rodentium burden (CFU/g) on days 7 and 10.

(D) Liver C. rodentium burden (CFU/liver) on day 10.

(E-F) IL-22 production by colonic CD4⁺ T cells was analyzed by flow cytometry, with representative plots (E) and summarized frequencies (F).

(G-K) Colonic IFN-γ⁺, IL-17A⁺, Foxp3⁺, and IL-10⁺ in CD4⁺ T cells were analyzed by flow cytometry, with representative plots (G) and summarized frequencies (H-K).

Data are presented as mean ± SEM. Results are representative of two independent experiments. Mann–Whitney U test (B); Unpaired Student’s t-test (C-D, F, H-K); ns, not significant; *p < 0.05, **p < 0.01.

3. Blocking IL-22 abrogates the resistance of CD4⁺ T cell-specific miR-10a knockout mice to C. rodentium infection

To investigate whether increased IL-22 production contributes to protection against C. rodentium infection in CD4⁺ T cell-specific miR-10a knockout CD4^cremiR-10a^fl/fl mice, we used anti-IL-22 antibody to block the IL-22 pathway in WT CD4^cre miR10a^fl/+ mice and CD4⁺ T cell-specific miR-10a knockout CD4^cremiR-10a^fl/fl mice, which were orally infected with C. rodentium. Anti-IgG antibody was used for control. We found that blocking IL-22 pathways abrogated the protection of miR-10a-deficient T cells against C. rodentium in CD4^cremiR-10a^fl/fl mice, as demonstrated by similar pathological scores and comparable C. rodentium loads in feces and liver between WT and CD4^cremiR-10a^fl/fl mice (Figure 4A–D).

Figure 4. IL-22 Blockade abolishes the enhanced resistance of miR–10a–deficient mice to Citrobacter rodentium infection.

WT CD4^cremiR10a^fl/+ mice and CD4^cremiR-10a^fl/fl mice were infected with Citrobacter Rodentium (5 × 10⁸ CFU/mouse) by gavage. Mice were given anti-IgG or anti-IL-22 antibody (20 mg/kg, every other day) intraperitoneally (n = 5/group). Mice were sacrificed on day 10.

(A–B) Representative H&E-stained intestinal sections (A) and corresponding pathological scores (B).

(C) Fecal C. rodentium burden (CFU/g) on day 10.

(D) Liver C. rodentium burden (CFU/liver) on day 10.

Data are presented as mean ± SEM. Results are representative of two independent experiments. Mann–Whitney U test (B); Unpaired Student’s t-test (C-D); ns, not significant; **p < 0.01.

4. Enhanced mitochondrial oxidation contributes to increased IL-22 in miR-10a-deficient T cells

Cellular metabolism plays a critical role in regulating T cell fate and function. To examine whether miR-10a influences T cell metabolism, we assessed key parameters of mitochondrial respiration. miR-10a-deficient CD4⁺ T cells exhibited a significantly higher oxygen consumption rate (OCR), including basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity, compared to WT CD4⁺ T cells (Figure 5A–B). However, there was no difference in glycolysis between WT and miR-10a-deficient T cells, as evidenced by similar extracellular acidification rates (ECAR) (Figure 5C). Furthermore, miR-10a-deficient CD4⁺ T cells showed a higher percentage of J-aggregates, indicating mitochondrial membrane potential (Figure 5D–E). These data suggest that miR-10a deficiency leads to enhanced mitochondrial oxidation, but not glycolysis, in CD4⁺ T cells.

Figure 5. Elevated mitochondrial oxidative metabolism mediates upregulated IL-22 in miR-10a-deficient T cells.

(A-B) The oxygen consumption rate (OCR) profile (A) and quantification of basal respiration, ATP-related respiration, maximum respiration, and spare respiration (B) in WT and miR-10a-deficient CD4⁺ T cells (200K/well, n = 6/group).

(C) Quantification of extracellular acidification rate (ECAR) in WT and miR-10a-deficient CD4⁺ T cells (200K/well, n = 6/group).

(D-E) Representative flow cytometry plots (D) and quantification of the percentage of J-aggregates in CD4⁺ T cells (n = 3/group).

(F-G) Splenic WT and miR-10a-deficient CD4⁺ T cells were activated and treated with or without Antimycin A (1 nM) under Th1 conditions for 5 days. IL-22 production by CD4⁺ T cells was analyzed by flow cytometry, with representative plots (F) and summarized frequencies (G) (n = 4/group).

(H) Splenic WT CD4⁺ T cells were activated and treated with or without Antimycin A (1 nM) for 2 days. MiR-10a expression in T cells.

Data are presented as mean ± SEM. Results are representative of two independent experiments. Unpaired Student’s t-test (B-C, E, and G); ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

To determine whether increased mitochondrial oxidation mediates IL-22 production in miR-10a–deficient T cells, we activated WT and miR-10a–deficient T cells with antimycin A, an inhibitor of mitochondrial Complex III. Antimycin A decreased IL-22 expression in both WT and miR-10-deficient T cells, and the difference in IL-22 levels between WT and miR-10a–deficient T cells was abolished under this treatment (Figure 5F–G). These findings suggest that mitochondrial oxidation is required for IL-22 production. To investigate whether antimycin A affects miR-10a in CD4⁺ T cells, we treated WT T cells with antimycin A and found that antimycin A did not affect T cell expression of miR-10a (Figure 5H).

5. miR-10a suppresses IL-22 in CD4⁺ T cells through targeting Uqcrq, a key subunit of the mitochondrial Complex III

We then investigated how miR-10a affects mitochondrial oxidation. Using miRmap for target prediction,[16] we identified a predicted miR-10a binding site within the 3′UTR of mouse Uqcrq mRNA, which encodes a critical subunit of mitochondrial Complex III. (Figure 6A). To validate whether Uqcrq is a direct target of miR-10a, we performed a luciferase reporter assay. Transfection with miR-10a precursor significantly reduced luciferase activity, whereas miR-10a inhibitor increased activity in the reporter vector containing the predicted seed sequence within the Uqcrq 3′UTR. No changes were observed when the seed sequence was mutated (Figure 6B). Additionally, both Uqcrq mRNA and protein levels were significantly increased in miR-10a–deficient T cells compared to WT T cells (Figure 6C–E). To further confirm the role of Uqcrq in regulating IL-22 expression, we introduced a CRISPR-Cas9 ribonucleoprotein (RNP) complex, consisting of Cas9 and a single-guide RNA targeting Uqcrq (sgUqcrq), into CD4⁺ T cells via nucleofection (Figure 6F). A non-targeting sgRNA (sgCtrl) was used as a negative control. Loss of Uqcrq decreased IL-22 expression in T cells (Figure 6G–I). In addition, knocking out Uqcrq also suppressed IL-10 production in T cells (Figure 6J). These data suggest that miR-10a suppresses IL-22 in CD4⁺ T cells through targeting Uqcrq.

Figure 6. MiR-10a regulates CD4⁺ T cell production of IL-22 via targeting Uqcrq.

(A) Predicted binding alignment between Mus musculus miR-10a and the 3′ UTR of Uqcrq.

(B) Luciferase activity of Uqcrq WT and Mut 3′ UTR reporters (n = 3/group).

(C) Relative Uqcrq mRNA expression in WT and miR-10a–deficient T cells.

(D–E) Representative immunoblots of Uqcrq and β-actin in WT and miR-10a–deficient T cells (n = 3/group) (D) and quantification of Uqcrq normalized to β-actin (E).

(F) Immunoblots of Uqcrq and β-actin in sgctrl and sgUqcrq CD4⁺ T cells.

(G) Il22 mRNA expression in sgctrl and sgUqcrq CD4⁺ T cells (n = 4/group).

(H-I) IL-22 production In sgctrl and sgUqcrq CD4⁺ T cells (n = 4/group) was analyzed by flow cytometry, with representative plots (H) and summarized frequencies (I).

(J) Il10 mRNA expression in sgctrl and sgUqcrq CD4⁺ T cells (n = 4/group).

Data are presented as mean ± SEM. Results are representative of two independent experiments. One-way ANOVA with Tukey’s multiple comparisons test (B); Unpaired Student’s t-test (C, E, G, I and J); *p < 0.05, **p < 0.01, ***p < 0.001.

Discussion

Previous studies have shown that individual microRNAs can directly target cytokine transcripts or regulate upstream transcription factors and signaling molecules, thereby regulating cytokine production[17]. For example, several miRNAs have been reported to control IFN-γ, IL-17, and IL-2 expression through direct targeting of cytokine mRNAs or modulation of lineage-defining transcription factors and associated pathways[18]. Our study identifies the role of miR-10a in controlling IL-22 production in CD4⁺ T cells and connects this regulation to mitochondrial metabolism. Although miR-10a has been mainly examined in the context of Treg biology and tissue repair,[6, 19] its influence on IL-22 has not been defined. Here, we show that miR-10a deficiency markedly increases IL-22 expression both in vitro and in vivo, under steady conditions, during colitis, and following Citrobacter rodentium infection. These findings indicate that miR-10a acts as a negative regulator of IL-22 in CD4⁺ T cells and contributes to maintaining intestinal homeostasis.

Although IL-22 production was increased in miR-10a-deficient T cells, Il22 mRNA does not contain a predicted miR-10a seed-matching site within its 3′UTR, suggesting that miR-10a does not directly regulate IL-22. Our data indicate that mitochondrial Complex III activity is required for IL-22 production, as antimycin A reduced IL-22 levels in both WT and miR-10a–deficient T cells. The comparable suppression observed following antimycin A treatment likely reflects the requirement for mitochondrial oxidative phosphorylation in supporting IL-22 production, thereby abolishing the difference between WT and miR-10a–deficient cells under inhibited conditions. Importantly, under basal conditions, miR-10a deficiency enhances mitochondrial oxidative capacity and augments IL-22 production, supporting a regulatory role for miR-10a upstream of mitochondrial metabolism. Mitochondrial oxidation is known to regulate T cell responses, so it is possible that this pathway influences other cytokines beyond IL-22. We further identified Uqcrq, a subunit of Complex III, as a direct target of miR-10a. Uqcrq expression was elevated in miR-10a-deficient T cells, and knocking out Uqcrq significantly decreased IL-22 production in T cells. These data support the central mechanism that miR-10a limits IL-22 production by restraining mitochondrial oxidative metabolism. Bioinformatic analysis indicates that the miR-10a binding site validated in the murine Uqcrq 3′UTR is not strictly conserved in the human UQCRQ transcript. However, the human UQCRQ 3′UTR contains multiple seed-like complementary regions (e.g., UGGGA-containing sequences). Whether these seed-like complementary regions in the human UQCRQ 3′UTR mediate functional regulation by miR-10a requires further investigation.

Our previous study demonstrated that miR-10a also suppresses IL-10 production in CD4⁺ T cells by targeting Blimp1.[7] Here, we found that miR-10a regulation of mitochondrial oxidation also affects IL-10 production. Together, these findings suggest that miR-10a controls IL-10 and IL-22 through shared and distinct mechanisms. Mice lacking CD4⁺ T cell-specific miR-10a showed lower bacterial loads and reduced intestinal pathology in the C. rodentium infection model, and this protection was abolished when IL-22 was blocked. IL-10 is also important in suppressing excessive immune response in the intestine.[20, 21] We cannot exclude the importance of enhanced CD4⁺ T cell IL-10 production in protecting against intestinal inflammation in mice lacking CD4⁺ T cell-specific miR-10a. However, our data support the conclusion that increased IL-22 in miR-10-deficient T cells is indispensable for protection against C. rodentium infection. Other major effector populations, including Th1, Th17, and Foxp3⁺ Tregs, were unchanged, suggesting that miR-10a deficiency preferentially enhances IL-22 and IL-10 without broadly altering T cell differentiation.

However, several questions remain to be investigated. The precise link between Complex III activity and IL-22 production remains unclear. Mitochondrial oxidation, ROS, metabolite-driven signaling, or changes in chromatin accessibility are all possible contributors. It will also be important to determine whether miR-10a regulates IL-22 in other cell types, such as ILCs. Given that IL-22 supports epithelial repair and host defense, targeting the miR-10a-Uqcrq axis may offer therapeutic potential for the treatment of intestinal inflammation and infection.

In summary, our study identifies miR-10a as a key negative regulator of IL-22 production in CD4⁺ T cells through mitochondrial function, thereby regulating intestinal homeostasis.

Data Availability:

The data supporting the findings of this study are available from the corresponding author upon reasonable request.