To explore the involvement of Mirlet7 in emphysema, we scrutinized the genomic locations and transcriptional annotation of Mirlet7 members frequently downregulated in lung T cells isolated from smoker’s lungs as well as mouse models of emphysema. This combined approach showed close linkage and high conservation of two Mirlet7 clusters encoded from long intergenic non-coding RNA (linc)-like precursors in humans and mice (Figure 1A). To shed light on whether these Mirlet7 clusters are downregulated in patients with COPD, we analyzed a published (GSE57148) lung RNA-seq dataset obtained from COPD (n = 98) and control (n = 91) subjects (Kim et al., 2015). Our analysis identified significant downregulation of the Mirlet7ahg and Mirlet7bhg gene cluster transcripts in COPD compared to control subjects (Figure 1B). We carried out quantitative PCR (qPCR) detection of MIRLET7A, which is encoded by both clusters, in lung tissue samples of smokers with emphysema and non-emphysema controls, detecting significant downregulation of MIRLET7A in emphysema samples relative to controls (Figure 1C). Because Mirlet7 has been shown to participate in IL-17⁺ T cell responses (Angelou et al., 2019; Guan et al., 2013; Newcomb et al., 2015), we next sought to determine if the expression pattern of Mirlet7ahg and Mirlet7bhg-derived Mirlet7 members are impaired in purified CD4⁺ T cells from emphysematous lungs. In support of our original hypothesis, the CD4⁺ T cell expression of Mirlet7a, Mirlet7b, Mirlet7d, and Mirlet7f were all inversely correlated with more severe emphysema distribution in the lungs as determined by Computed Tomography (CT) scan (Figure 1D).

Next, we elucidated Mirlet7b/Mirlet7c2 and Mirlet7a1/Mirlet7f1/Mirlet7d cluster expression (herein referred to as Mirlet7bc2 and Mirlet7afd, respectively) in murine models of CS- or nCB-induced emphysema, respectively (Figure 1E). Paralleling our observations in human COPD and emphysema, mice with CS- or nCB-induced emphysema exhibited reduced expression levels of pri-Mirlet7b/c2 and pri-Mirlet7a1/f1/d transcripts in the lung and from isolated lung CD4⁺ and CD8⁺ T cells (Figure 1F–H). Collectively, our expression results indicate suppression of Mirlet7bc2 and Mirlet7afd in the lung and T cells in human and pre-clinical models of emphysema.

To investigate the in vivo requirement of the Mirlet7bc2 within T cells, we generated conditional ready floxed mice (let7bc2^f/f). We then crossed let7bc2^f/f mice with CD4-Cre mice to generate let7bc2^f/f; CD4-Cre LOF mice (let7bc2^LOF) (Figure 2A). This approach allowed us to conditionally delete Mirlet7bc2 in all T cells derived from the CD4⁺CD8⁺ double-positive (DP) stage (Lee et al., 2001; Shi and Petrie, 2012). We confirmed that let7bc2^LOF mice exhibit robust conditional deletion of Mirlet7bc2 in DP thymocytes and peripheral T cells (Figure 2B and data not shown). Our let7bc2^LOF adult mice were born at the expected Mendelian frequency and did not show any overt histopathologic or inflammatory changes in lungs histopathology up to 1 year of age in comparison to let7bc2^f/f control mice (Figure 2—figure supplement 1A–D). Furthermore, quantification of major immune populations and T cell subsets by flow cytometry in let7bc2^LOF were comparable to control mice under baseline conditions and with moderate aging (Figure 2—figure supplement 1C, D).

We next exposed let7bc2^LOF and let7bc2^f/f control mice to nCB or CS and examined the lungs under the context of experimental emphysema. Histomorphometry measurements of mean linear intercept (MLI) from hematoxylin and eosin (H&E)-stained sections revealed that the enlargement of alveolar spaces sustained from either nCB or CS exposure was exaggerated in let7bc2^LOF mice relative to controls (Figure 2C–E). Chronic inflammation in emphysema is characterized by the recruitment of macrophages and neutrophils to the lung tissue and airways (Peleman et al., 1999; Senior and Anthonisen, 1998). Internally consistent with MLI measurements, let7bc2^LOF mice treated with nCB showed significantly increased airway infiltration of macrophages and neutrophils in BAL fluid as compared to wild-type control animals (Figure 2F). Concomitant with these findings, expression levels of Mmp9 and Mmp12, which are secreted by macrophages and neutrophils to degrade elastin and mediate alveolar damage, were elevated in airways of let7bc2^LOF mice exposed to CS versus controls (Figure 2G). As expected, let7bc2^LOF mice treated with nCB exhibit significantly less pri-Mirlet7b/c2 transcript expression in isolated lung T cells relative to wild-type control mice (Figure 2—figure supplement 2A and data not shown). Collectively, our data suggest that the Mirlet7bc2 cluster within T cells protects by dampening airway destruction and inflammation because the absence of this cluster worsens the severity of experimental emphysema in mice.

We sought to identify the T cell-intrinsic mechanisms that underlie the exaggerated inflammation observed in emphysematous let7bc2^LOF mice. We focused on the IL-17-mediated T cell response because it promotes neutrophil and macrophage recruitment in the lungs (Beringer et al., 2016; Veldhoen, 2017; Shan et al., 2012). Previously, we established the induction of CD4⁺IL17⁺ (Th17) cells along with CD4⁺IFNγ⁺ (Th1) cells in mice with chronic nCB exposure (You et al., 2015), however whether nCB similarly induces CD8⁺IL17A⁺ T cells (Tc17) or cytotoxic T cells (Tc1) had not been studied. The flow cytometric profiling of lung T cells revealed enriched proportions and counts of Tc1/Tc17 as well as Th1/Th17 cells in wild-type mice upon treatment with nCB (Figure 3A, B and Figure 3—figure supplement 1A). These findings suggests that nCB elicits both the type 17 and type 1T cell responses, consistent with CS and elastase pre-clinical models of emphysema (Zhang et al., 2019).

We next interrogated the regulatory role of Mirlet7bc2 in the type 17 and type 1 responses generated from exposure to nCB. Interestingly, let7bc2^LOF mice showed increased CD8⁺IL17A⁺ Tc17 cells relative to nCB control animals. In contrast, CD8⁺IFNγ⁺ and GZMA⁺ Tc1 populations remained unperturbed with absence of Mirlet7bc2, suggestive of a more refined regulatory role on Tc17 differentiation (Figure 3A, B). There were no significant differences in either Th1 or Th17 cells when comparing nCB-treated let7bc2^LOF to wild-type controls, indicating the Mirlet7bc2 was dispensable for their generation (Figure 3C). Regulatory T cells form a dynamic axis with Tc17/Th17 cells and act as a counterbalance to lung inflammation in emphysema (Duan et al., 2016; Jin et al., 2014). Therefore, we examined whether Tc17 cell alterations were driven by Mirlet7bc2 acting on regulatory T cells (Tregs). The let7bc2^LOF mice showed no significant difference in the Tregs subset relative to controls in our model (Figure 3D). Together, our data support the notion that deletion of Mirlet7bc2 is insufficient to provoke Tc17 cell generation under homeostatic conditions. However, under the context of chronic inflammation in emphysema, the loss of the Mirlet7bc2 cluster is intrinsic for the potentiation of T cells toward Tc17 differentiation.

We utilized the TargetScan predictive algorithm to identify putative Mirlet7 targets that are known to control the IL-17-mediated T cell response (Agarwal et al., 2015). This analysis revealed that the 3′UTR region of Rorc, encoding RORγt, contains an evolutionarily conserved and complementary motif for the Mirlet7 family (Figure 4A). Thus, we examined if Mirlet7bc2 loss in T cells would stimulate and enhance RORγt. Initially, we carried out flow cytometric quantification for RORγt in thymocyte, splenic, and lung T cells of naïve control and let7bc2^LOF mice up to 6 months of age. Our interrogation of RORγt mean fluorescent intensity (MFI) by flow cytometry showed induction of RORγt in single-positive CD8⁺ and CD4⁺ thymocytes, as well as peripheral splenic CD8⁺ and CD4⁺ T cells (Figure 4B). However, RORγt levels appeared unchanged in purified lung CD8⁺ T cells and CD4⁺ T cells of naive let7bc2^LOF mice, alluding to a compensatory effect in homeostatic lung T cells (Figure 4B). Since we and others have shown that miRNAs are frequently associated with stress-dependent phenotypes, we posited that emphysematous let7bc2^LOF T cells are poised toward induction of RORγt and production of IL-17⁺ subsets after challenge with nCB. Indeed, nCB-emphysematous let7bc2^LOF mice exhibited enhanced RORγt protein levels in both CD8⁺ and CD4⁺ T cells relative to control mice with emphysema (Figure 4C).

Because we had found that the Mirlet7afd cluster is downregulated in T cells isolated from COPD lungs in human and mice, and that the Mirlet7 family operates with some functional redundancy, we generated mice with conditional deletion of the Mirlet7afd in T cells (let7afd^LOF). The let7afd^LOF mice aged up to 6 months did not exhibit overt lung histopathology and inflammatory changes (Figure 4—figure supplement 1A–E). Of particular interest, ablation of Mirlet7afd enhanced levels of RORγt in thymic and peripheral T cells of mice (Figure 4D). Overall, this indicates that independent Mirlet7 clusters restrain RORγt expression levels from thymic development to peripheral T cells under homeostatic conditions. Next, we determined whether loss of Mirlet7afd in T cells likewise sensitizes mice toward induction of RORγt in nCB emphysema. Intranasal administration of nCB provoked increased RORγt expression in lung T cells of let7afd^LOF mice compared to control mice (Figure 4E), supporting overlapping functionality between the Mirlet7bc2 and Mirlet7afd in repression of RORγt within T cells.

To confirm that the Mirlet7 family negatively regulates Tc17 cell differentiation, at least in part, cell autonomously in CD8⁺ T cells, we purified naïve CD8⁺ T cells from let7bc2^LOF and control mice spleens and cultured these cells in vitro in the presence of Tc17 polarizing (TGFβ, IL-6, anti-IFNγ, IL-23, and IL-1β) or Tc1 polarizing (IL-2) conditions (Flores-Santibáñez et al., 2018). Our flow cytometric analysis confirmed the enhanced commitment of Mirlet7bc2 deficient CD8⁺ T cells toward Tc17 cells and IL-17A⁺ production relative to control CD8⁺ T cells (Figure 5A, B). Moreover, enhanced Tc17 cell differentiation mirrored the increased IL-17A detected in the supernatant from in vitro polarized cells as quantified by ELISA (Figure 5C). Parallel assessment of Tc1 differentiation did not detect a difference in CD8⁺IFNγ⁺ cells (Figure 5A and D). Altogether, these data recapitulated our in vivo findings that the Mirlet7bc2 cluster negatively regulates Tc17 response but is dispensable in Tc1 cells. Finally, to determine whether Tc17 differentiation is likewise controlled by Mirlet7afd, we cultured naive CD8⁺ splenocytes from let7afd^LOF and controls under Tc17 conditions. As we had observed with let7bc2^LOF, absence of Mirlet7afd in T cells further enhanced differentiation toward Tc17 cells as quantified by flow cytometry and ELISA (Figure 5F, G).

Next, we focused on Rorc as a potential direct target of Mirlet7, which could mechanistically mediate enhanced Tc17 differentiation in let7bc2^LOF mice. Toward this objective, we tested whether let7bc2^LOF or let7afd^LOF naive CD8⁺ T cells show elevated RORγt expression under either Tc0 or Tc17 differentiation conditions. In agreement with enhanced Tc17 differentiation, RORγt expression was differentially and significantly upregulated under both Tc0 and Tc17 differentiation conditions in Mirlet7 LOF cells relative to controls (Figure 5E, H). To determine whether Mirlet7 directly represses Rorc mRNA levels we cloned the 3′UTR of Rorc into luciferase constructs. These reporter assays with Mirlet7b expressing cells independently confirmed that Mirlet7b represses Rorc (Figure 5I, left). Furthermore, deletion of the putative Mirlet7-binding sequence (Figure 4A) abrogated repression by Mirlet7b (Figure 5I, right), thus confirming Rorc as a functional target. Overall, these in vitro experiments readily recapitulated an upstream regulatory role of Mirlet7 in Tc17 differentiation, mediated in part, via direct suppression of RORγt.

To explore a potential protective role of Mirlet7 in experimentally induced emphysema, we generated mice which allowed for selective induction of Mirlet7g in T cells using the published rtTA-iLet7 mice crossed to CD4-Cre (herein referred to as let7^GOF; Figure 6A; Angelou et al., 2019; Belteki et al., 2005; Pobezinskaya et al., 2019; Wells et al., 2017; Zhu et al., 2011). The rtTA-iLet7 mouse model has been utilized to promote ~two- to threefold rise in total Mirlet7 activity in T cells (Angelou et al., 2019; Wells et al., 2017; Wells et al., 2023; Angelou et al., 2019). Steady-state let7^GOF and control (rtTA-iLet7) mice were examined for compromised RORγt protein levels within thymocytes and peripheral T cells. Providing further evidence of Mirlet7-dependent regulation of Rorc, protein levels of RORγt were suppressed in CD8⁺ and CD4⁺ T cells of let7^GOF mice relative to controls (Figure 6B). To determine whether enforced expression of Mirlet7g offered protection from experimental emphysema, let7^GOF and control mice were treated with nCB and then examined for changes in lung pathology and T cell type 17 responses. The let7^GOF mice did not exhibit any signs of lung inflammation or pathologic remodeling at baseline (Figure 6C, D and data not shown). Histopathologic analysis revealed a comparable degree of lung alveolar distension via morphometric measurements of MLI in nCB-treated let7^GOF mice versus controls suggesting that enforced Mirlet7g expression is insufficient to protect the lung from emphysema (Figure 6C, D). On the other hand, evaluation of the IL-17⁺ response and RORγt levels in emphysematous lung T cells demonstrated that, in contrast to control nCB-treated mice, let7^GOF mice exhibited dampened lung Tc17 and Th17 cell populations and were resistant to the induction of RORγt after nCB exposure (Figure 6E, F). Taken together, our Mirlet7 LOF and GOF models demonstrate the necessity and sufficiency of Mirlet7 to act as a molecular brake to the type 17T cell response through the direct regulation of RORγt, further our data suggest that nCB- or CS-mediated suppression of this braking mechanism furthers inflammation and exacerbates emphysema severity (Figure 6G).

Conditional knockout-ready floxed for either the Mirlet7b/Mirlet7c2 and Mirlet7a1/Mirlet7f1/Mirlet7d cluster, respectively, were generated using CRISPR gene editing in an isogenic C57BL/6 genetic background and were sequence verified for rigor. Mice were PCR genotyped from ear samples with primers flanking loxP sites (Supplementary file 1). Mice were crossed to CD4-Cre obtained from JAX (Stock 022071) and genotyped by PCR. The R26-STOP-rtTA; Col1a1-tet0-Mirlet7g (rtTA-iLet7) mice were obtained from JAX (Stocks 023912 and 05670) and also PCR genotyped with established JAX primers (Belteki et al., 2005; Zhu et al., 2011). Control rtTA-iLet7 and Mirlet7g GOF mice were fed ad libitum with 200 mg/kg of doxycycline-containing chow (Bio-Serv S3888) at weaning age (Belteki et al., 2005; Zhu et al., 2011). Syngeneic littermates served as controls for all mouse experiments. All mice were bred in the transgenic animal facility at Baylor College of Medicine. All experimental protocols used in this study were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine animal protocol (AN-7389) and followed the National Research Council Guide for the Care and Use of Laboratory Animals.

Lung tissues were obtained from a total of 19 non-atopic current or former smokers with significant (>20 pack-years, one pack-year equals to smoking one pack of cigarettes per day each year) history of smoking who were recruited into studies from the chest or surgical clinics at Michael E. DeBakey Houston Veterans Affairs Medical Center hospitals (Supplementary file 2; Shan et al., 2009). Emphysema and non-emphysema control patients were diagnosed from CT scans according to the criteria recommended by the National Institutes of Health–World Health Organization workshop summary (Pauwels et al., 2001). Human single-cell suspensions were prepared from surgically resected lungs as previously described (Yuan et al., 2019; Grumelli et al., 2004). Briefly, fresh lung tissue was minced into 0.1 cm pieces in Petri dishes and treated with 2 mg/ml of collagenase D (Worthington) for 1 hr at 37°C. Digested lung tissue was filtered through a 40-μm cell strainer (BD Falcon) followed by red blood cell lysis using ACK lysis buffer (Sigma-Aldrich) for 3 min to yield a single-cell suspension. CD4⁺ T cells were selected from resultant suspensions by labeling with bead conjugated anti-CD4 for enrichment by autoMACs (Miltenyi Biotec). Studies were approved by the Institutional Review Board at Baylor College of Medicine and informed consent was obtained from all patients.

A publicly available RNA-seq dataset from a Korean cohort GSE57148 was selected for the analysis (Kim et al., 2015). The raw FASTQ files of paired end reads representing the transcriptome of control and cases were retrieved from the GEO database at the National Centre for Biological Information (NCBI) through accession number GSE57148 and analyzed with R package for differential expression.

To promote emphysema, mice were exposed to CS using our custom designed whole-body inhalation system (Morales-Mantilla et al., 2020). In total, mice were exposed to four cigarettes (Marlboro 100’s; Philip Morris USA) per day, 5 days a week, for 4 months as previously described (Shan et al., 2012).

Nanosized particulate carbon black was prepared and administered as previously described (You et al., 2015; Lu et al., 2015). Dried nCB nanoparticles were resuspended in sterile PBS to a concentration of 10 mg/ml. Fifty µl of reconstituted nCB (0.5 mg) were intranasally delivered to deeply anesthetized mice on a schedule of three times a week for 4 weeks (total delivered dose of 6 mg). Lung histomorphometry and airway inflammation were assessed 4 weeks after the final nCB challenge. For histomorphometric analysis, mice lungs were fixed with 10% neutral-buffered formalin solution via a tracheal cannula at 25 cm H₂O pressure followed by paraffin embedding and tissue sectioning and stained with H&E. MLI measurement of mouse lung morphometry was done as previously described (Shan et al., 2014; Morales-Mantilla et al., 2020). Briefly, this was done in a blinded fashion to mice genotypes from 10 randomly selected fields of lung parenchyma sections. Paralleled lines were placed on serial lung sections and MLI was calculated by multiplying the length and the number of lines per field, divided by the number of intercepts (Morales-Mantilla et al., 2020).

BALF was collected by instilling and withdrawing 0.8 ml of sterile PBS twice through the trachea. Total and differential cell counts in the BALF were determined with the standard hemocytometer and HEMA3 staining (Biochemical Sciences Inc, Swedesboro, NJ) using 200 μl of BALF for cytospin slide preparation (Morales-Mantilla et al., 2020; Lu et al., 2015).

Mouse lung tissue was cut into 2 mm pieces and digested with collagenase type D (2 mg/ml; Worthington) and deoxyribonuclease (DNase) I (0.04 mg/ml; Roche) for 1 hr in a 37°C incubator. Single-cell suspensions from lung digest, spleen, and thymus were prepared by mincing through 40-μm cell strainers then washing and resuspension in complete RPMI media. Mouse lung and spleen single-cell suspensions were additionally overlaid on Lympholyte M cell separation media (Cedarlane) as indicated in the manufacturer’s protocol to purify lymphocytes. For murine Mirlet7 expression studies, lung single-cell suspensions were labeled with anti-CD4⁺ or anti-CD8⁺ magnetic beads and separated by autoMACS (Miltenyi Biotec), or CD4⁺CD8⁺ DP cells purified from thymus single-cell suspensions by flow-cytometric sorting on FACS Aria (BD Biosciences).

CD8⁺ naive T cells were isolated from spleen using Mojosort Mouse CD8 Naïve T cell isolation Kit (Biolegend) and adjusted to a concentration of 1.0 × 10⁶ cells/ml. Purified cells were activated with plate-bound anti-CD3 (1.5 µg/mL) and complete RPMI media containing anti-CD28 (1.5 µg/ml) and β-mercaptoethanol (50 nM) for Tc0 polarization, or further supplemented with Tc1 [IL-2 (10 ng/ml)] or Tc17 [TGFβ (2 ng/ml), IL-6 (20 ng/ml), anti-IFNγ (10 µg/ml), IL-23 (20 ng/ml), and IL-1β (5 ng/ml)] polarization conditions for 72 hr (Flores-Santibáñez et al., 2018).

Supernatant was collected from in vitro polarized murine CD8⁺ T cells and centrifuged to remove cellular debris (Lu et al., 2015). Cytokine levels of IL-17A and IFNγ were quantified from collected supernatant using Mouse IL-17A Uncoated ELISA and Mouse IFN gamma Uncoated ELISA (Invitrogen) Kits, respectively, per the manufacturer’s instructions with colorimetric analysis by the Varioskan LUX microplate reader (Thermo Fisher).

Cells used for in vitro or in vivo cytokine analysis were stimulated with PMA (20 ng/ml; Sigma-Aldrich), Ionomycin (1 µg/ml; Sigma-Aldrich), and Brefeldin A (2 µg/ml; Sigma-Aldrich) for 4 hr prior to flow staining (Lu et al., 2015). For intracellular staining, cells were fixed and permeabilized using the Mouse FOXP3 Buffer Set (BD) per the manufacturer’s protocol. The fluorophore-conjugated antibodies used in this study were as follows: Live/Dead Fix Blue (Invitrogen), CD3 PerCPCy5.5 (Biolegend), TCRb PE/Cy7 (Biolegend), CD4 PB (Biolegend), CD4 AF700 (Biolegend), CD8 BV650 (Biolegend), CD25 BV421 (Biolegend), FOXP3 AF488 (Biolegend), ROR gamma T PE (Invitrogen), TCF1 AF647 (Cell Signaling Technologies), TCF1 PE (Biolegend), IFNγ AF647 (Biolegend), IL17A FITC (Biolegend), and IL17A PE (ebioscience). Samples were analyzed using BD LSR II flow cytometer (BD Biosciences) and FlowJo software (TreeStar).

RNA was isolated using miRNeasy (QIAGEN) or RNeasy Mini Kit (QIAGEN) in conjunction with the RNase-Free DNase (QIAGEN) according to the manufacturer’s instructions. cDNA of miRNAs and mRNAs was synthesized using TaqMan Advanced miRNA cDNA Synthesis Kit (Thermo Fisher) and High-Capacity cDNA Reverse Transcription Kit Real-Time PCR system (Applied Biosystems). 18S and snoRNA-202 were used to normalize mRNA and miRNA expression, respectively (Lu et al., 2015). Quantitative RT-PCR data were acquired on 7500 Real-Time PCR System or StepOne Real-Time PCR System (Applied Biosystems) with the following TaqMan probes: hsa-let-7a-5p [478575_mir], hsa-let-7b-5p [478576_mir], hsa-let-7d [478439_mir], hsa-let-7f [478578_mir], pri-let7a1/f1/d [44411114, arfvmhy], pri-let7b/c2 [4441114, areptx2], Mmp9 [Mm00442991], and Mmp12 [Mm00500554].

Genomic fragment containing the murine Rorc 3′UTR was cloned into psiCHECK2 luciferase reporter plasmid (Promega). This construct was also used to generate the Mirlet7 ʻseedʼ deletion mutant derivative using the QuikChange Multi Site Mutagenesis Kit (catalog 200514-5, Stratagene). 3T3 mouse embryonic fibroblasts were transfected using Oligofectamine (Invitrogen) with 100 ng of psiCheck-2 plasmid containing wild-type or mutant 3′UTR, along with the miRNA control or Mirlet7b duplex (Dharmacon) at a final concentration of 6 nM (Gurha et al., 2012; Supplementary file 1). Reporter activity was detected with the Dual-Luciferase Reporter Assay System (Promega).

Statistical analyses were performed using GraphPad Prism 10.0.1 software. Statistical comparison between groups was performed using the unpaired Student’s t-test, two-way analysis of variance with Tukey’s or Sidak’s correction, and Mann–Whitney test when indicated. A p-value less than 0.05 was considered statistically significant; ns indicates not significant. Statistical significance values were set as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Data are presented as means ± standard error of the mean. p-value and n can be found in the main and supplementary figure legends.