Downregulation of Let-7 miRNA promotes Tc17 differentiation and emphysema via de-repression of RORγt

Environmental air irritants including nanosized carbon black (nCB) can drive systemic inflammation, promoting chronic obstructive pulmonary disease (COPD) and emphysema development. The let-7 family of miRNAs is associated with IL-17-driven T cell inflammation, a canonical signature of lung inflammation. Recent evidence suggests the let-7 family is downregulated in patients with COPD, however, whether this repression conveys a functional consequence on emphysema pathology has not been elucidated. Here we show that overall expression of the let-7 miRNA clusters, let-7b/let-7c2 and let-7a1/let-7f1/let-7d, are reduced in the lungs and T cells of smokers with emphysema as well as in mice with cigarette smoke (CS)- or nCB-elicited emphysema. We demonstrate that loss of the let-7b/let-7c2-cluster in T cells predisposed mice to exaggerated CS- or nCB-elicited emphysema. Furthermore, ablation of the let-7b/let-7c2-cluster enhanced CD8+IL17a+ T cells (Tc17) formation in emphysema development in mice. Additionally, transgenic mice overexpressing let-7 in T cells are resistant to Tc17 and CD4+IL17a+ T cells (Th17) development when exposed to nCB. Mechanistically, our findings reveal the master regulator of Tc17/Th17 differentiation, RAR-related orphan receptor gamma t (RORγt), as a direct target of let-7 miRNA in T cells. Overall, our findings shed light on the let-7/RORγt axis with let-7 acting as a molecular brake in the generation of Tc17 cells and suggests a novel therapeutic approach for tempering the augmented IL-17-mediated response in emphysema.

downregulated in patients with COPD, however, whether this repression conveys a functional consequence on emphysema pathology has not been elucidated.Here we show that overall expression of the let-7 miRNA clusters, let-7b/let-7c2 and let-7a1/let-7f1/let-7d, are reduced in the lungs and T cells of smokers with emphysema as well as in mice with cigarette smoke (CS)-or nCB-elicited emphysema.We demonstrate that loss of the let-7b/let-7c2-cluster in T cells predisposed mice to exaggerated CS-or nCB-elicited emphysema.Furthermore, ablation of the let-7b/let-7c2-cluster enhanced CD8 + IL17a + T cells (Tc17) formation in emphysema development in mice.Additionally, transgenic mice overexpressing let-7 in T cells are resistant to Tc17 and CD4 + IL17a + T cells (Th17) development when exposed to nCB.Mechanistically, our findings reveal the master regulator of Tc17/Th17 differentiation, RAR-related orphan receptor gamma t (RORγt), as a direct target of let-7 miRNA in T cells.Overall, our findings shed light on the let-7/RORγt axis with let-7 acting as a molecular brake in the generation of Tc17 cells and suggests a novel therapeutic approach for tempering the augmented IL-17-mediated response in emphysema.

INTRODUCTION
Chronic obstructive pulmonary disease (COPD) ranks as the third leading cause of mortality and is projected to account for over a billion deaths by the end of the twenty-first century (GBD Chronic Respiratory Disease Collaborators, 2020; Findings from the Global Burden of Disease Study 2017Study , 2019;;Laniado-Laborín, 2009).Currently, there are no treatment options to reverse emphysema, the most clinically significant variant of COPD, which often is progressive despite smoking cessation (Bhavani et al., 2015;Anthonisen et al., 2002).
Inhalation of fine particulate matter smaller than 2.5 microns (PM2.5)found in outdoor and indoor air pollution as well as tobacco smoke are risk factors for COPD development (Adeloye et al., 2022;Eisner et al., 2010;Hu et al., 2010).We have previously shown that nano-sized carbon black (nCB), a noxious chemical constituent of PM2.5 found in the lungs of smokers, activates macrophages and dendritic cells orchestrating a pathogenic T cell-dependent inflammatory response and emphysema in mice (W.Lu et al., 2015;You et al., 2015;Shan et al., 2009;C.-Y. Chang et al., 2022).
Research over the last decade has pointed to the importance of dysfunctional inflammatory T cells in human COPD lung tissue and animal models of emphysema (Grumelli et al., 2004;Xu et al., 2012;Williams et al., 2021).Aberrant T cells are implicated in impaired host defense, exaggerated inflammation, and loss of self-tolerance in COPD (Williams et al., 2021;Chen et al., 2023;Hogg et al., 2004;Maeno et al., 2007;Xu et al., 2012).In this regard, we and others have demonstrated the role and pathogenicity of activated IFN-γ and IL-17-secreting subsets of CD4 + and CD8 + T lymphocytes including Th1, Th17, and Tc1 cells in clinical isolates and in mice with COPD (W.Lu et al., 2015;You et al., 2015;Shan et al., 2009;S.-H. Lee et al., 2007;Kheradmand et al., 2023).The IL-17-secreting Th17 cells are particularly important as they promote the destruction of lung epithelium and recruitment of macrophages and neutrophils which then release proteolytic enzymes such as matrix metalloproteinases (MMPs) involved in the degradation of the lung structural matrix (Barnes, 2016;Hoenderdos & Condliffe, 2013).We previously demonstrated that intranasal inhalation of nCB in mice is sufficient to induce emphysema by stimulating lung T cell activation by dendritic cells and macrophages.Moreover, we found that genetic ablation of IL-17A can attenuate nCB-or cigarette smoke-induced alveolar destruction and airway inflammation (Shan et al., 2012;You et al., 2015).More recently, IL-17A and IL-17F secreting CD8 + T cell (Tc17) subpopulation has been shown to play a critical role in the pathogenesis of several autoimmune and inflammatory disorders (Globig et al., 2022;Huber et al., 2013;Srenathan et al., 2016).Both Th17 and Tc17, require the fate-deterministic transcription factor RAR-related orphan receptor gamma t (RORγt, encoded by Rorc) for differentiation and production of IL-17A (Ivanov et al., 2007).RORγt is the best-studied positive transcriptional regulator of IL-17A and IL-17F (Ivanov et al., 2006).In accordance with the importance of IL-17A transcription, RORγt expression has also been reported to be elevated in COPD patients and in mouse models of COPD (Chu et al., 2011;Li et al., 2015).However, the upstream pathophysiologic mechanisms that contribute to the induction of RORγt and differentiation of Tc17 cells in COPD have not been well elucidated.
We previously reported that miR-22 inhibits HDAC4, promoting antigen-presenting cell activation (APC) in the lungs and inducing Th17-mediated emphysema in response to CS or nCB in mice (W.Lu et al., 2015).Additional miRNAs that control APC and/or T cell driven IL-17A + inflammation have been identified by others including the let-7 miRNA family (Mai et al., 2012;Angelou et al., 2020).MicroRNA expression-based studies have shown frequent downregulation of members of the let-7 miRNA family, including let-7a, let-7b, let-7c, let-7d, let-7e, and let-7f in human emphysematous lung tissue and in murine models of emphysema, but the mechanism(s) of action remain ill-defined (Christenson et al., 2013;Pottelberge et al., 2011;Conickx et al., 2017;Izzotti et al., 2009).Let-7 microRNA genes are encoded across eight loci either as single genes or as polycistronic clusters which have confounded their analysis in vivo (Rodriguez et al., 2004).
Previous studies used Lin28b transgenic overexpression in T cells to block the maturation and processing of the let-7 miRNA family.They showed an inhibitory role of let-7 family in Th17-driven response in the murine model of experimental autoimmune encephalomyelitis (EAE) attributed in part to regulation of IL-1 receptor 1 and IL-23 receptor (Angelou et al., 2020).
Here we found that let-7 miRNA, notably the let-7a3/let-7b and let-7a1/let-7f1/let-7d clusters, are suppressed in the T cells isolated from lungs of emphysema patients.Consistently, the analogous murine let-7b/let-7c2-(let-7bc2) and let-7a1/f1/d1-(let-7afd) clusters were similarly downregulated in pre-clinical emphysema models.We engineered mouse models with the specific loss-of-function (LOF) mutations of the let-7bc2-or let-7afd-clusters (let-7bc2 LOF and let-7afd LOF , respectively) in T cells as well as an inducible let-7g gain-of-function (GOF) (let-7 GOF ) model to determine the T cell-intrinsic role of let-7 miRNA in emphysema pathogenesis.Deletion of let-7 miRNA in T cells worsened alveolar damage elicited by inhalation of CS or nCB, and increased infiltration of immune cells in the airways, including IL-17-producing CD8 + T (Tc17) cells.
Mechanistically, we found that let-7 controls type 17 differentiation by directly targeting the lineage-determining transcription factor, RORγt.In support of this conclusion, let-7 GOF mice were resistant to nCB-mediated induction of RORγt and Tc17 responses.Thus, we show a previously unappreciated role for let-7 miRNA as a repressor of RORγt and a molecular brake to the IL-17mediated T cell inflammation in emphysema.

RESULTS
The let-7bc2-and let-7afd-clusters are downregulated in lungs and T cells in COPD.
To explore the involvement of let-7 in emphysema, we scrutinized the genomic locations and transcriptional annotation of let-7 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 Let-7 clusters encoded from long intergenic non-coding RNA (linc)-like precursors in humans and mice (Figure 1A).To shed light on whether these Let-7 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 Let-7a, which is encoded by both clusters, in lung tissue samples of smokers with emphysema and non-emphysema controls, detecting significant downregulation of Let-7a in emphysema samples relative to controls (Figure 1C).Because Let-7 has been shown to participate in IL-17 + T cell responses (Angelou et al., 2020;Guan et al., 2013;Newcomb et al., 2015), we next sought to determine if the expression pattern of Mirlet7ahg and Mirlet7bhg-derived Let-7 members are impaired in purified CD4 + T cells from emphysematous lungs.In support of our original hypothesis, the CD4 + T cell expression of  were all inversely correlated with more severe emphysema distribution in the lungs as determined by CT scan (Figure 1D).
Next, we elucidated let-7a1/let-7f1/let-7d-and let-7b/let-7c2-clusters expression (herein referred to as let-7afd and let-7bc2 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-let7a1/f1/d and pri-let7b/c2 transcripts in the lung and from isolated lung CD4 + and CD8 + T cells (Figure 1F-H).Collectively, our expression results indicate suppression of let-7afd and let-7bc2-clusters in the lung and T cells in human and pre-clinical models of emphysema.
We confirmed that let-7bc2 LOF mice exhibit robust conditional deletion of the let-7bc2-cluster in DP thymocytes and peripheral T cells (Figure 2B and data not shown).Our let-7bc2 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 let-7bc2 f/f control mice (Figure 2-figure supplement 1A-C).Furthermore, quantification of major immune populations and T cell subsets by flow cytometry in let-7bc2 LOF were comparable to control mice under baseline conditions and with moderate aging (Figure 2-figure supplement 1C,D).
We next exposed let-7bc2 LOF and let-7bc2 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 & Anthonisen, 1998).Internally consistent with MLI measurements, let-7bc2 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, let-7bc2 LOF mice treated with nCB exhibit significantly less pri-let7b/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 suggests that the let-7bc2-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.
The let-7bc2 miRNA cluster negatively regulates T C 17 inflammation in emphysema.
We sought to identify the T cell-intrinsic mechanisms that underlie the exaggerated inflammation observed in emphysematous let-7bc2 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).These findings suggests that nCB elicits both the type 17 and type 1 T cell responses, consistent with CS and elastase pre-clinical models of emphysema (Zhang et al., 2019).
We next interrogated the regulatory role of the let-7bc2-cluster in the type 17 and type 1 responses generated from exposure to nCB.Interestingly, let-7bc2 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 the let-7bc2-cluster, 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 let-7bc2 LOF to wild-type controls, indicating the let-7bc2-cluster 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 the let-7bc2-cluster acting on regulatory T cells (Tregs).The let-7bc2 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 the let-7bc2-cluster is insufficient to provoke Tc17 cell generation under homeostatic conditions.However, under the context of chronic inflammation in emphysema, the loss of let-7bc2-cluster is intrinsic for the potentiation of T cells towards Tc17 differentiation.
The let-7 family directly inhibits RORγt expression governing Tc17 differentiation in emphysema.
We utilized the TargetScan predictive algorithm to identify putative let-7 miRNA 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 let-7 miRNA family (Figure 4A).Thus, we examined if let-7bc2cluster 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 let-7bc2 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 let-7bc2 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 let-7bc2 LOF T cells are poised towards induction of RORγt and production of IL-17 + subsets after challenge with nCB.Indeed, nCB-emphysematous let-7bc2 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 let-7afd-cluster is downregulated in T cells isolated from COPD lungs in human and mice, and that the let-7 family operates with some functional redundancy, we generated mice with conditional deletion of the let7afd-cluster in T cells (let-7afd f/f ; CD4-Cre).The let-7afd f/f ; CD4-Cre (let-7afd LOF ) mice aged up to 6-months did not exhibit overt lung histopathology and inflammatory changes (Figure 4-figure supplement 1A-F).Of particular interest, ablation of the let-7afd-cluster enhanced levels of RORγt in thymic and peripheral T cells of mice (Figure 4D).Overall, this indicates that independent let-7 clusters restrain RORγt expression levels from thymic development to peripheral T cells under homeostatic conditions.
Next, we determined whether loss of let-7afd-cluster in T cells likewise sensitizes mice towards induction of RORγt in nCB-emphysema.Intranasal administration of nCB provoked increased RORγt expression in lung T cells of let-7afd LOF mice compared to control mice (Figure 4E), supporting overlapping functionality between the let-7bc2-and let-7afd-clusters in repression of RORγt within T cells.
To confirm that the let-7 family negatively regulates Tc17 cell differentiation, at least in part, cell autonomously in CD8 + T cells, we purified naïve CD8 + T cells from let-7bc2 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 let-7bc2-cluster deficient CD8 + T cells towards 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 Figure 5D).
Altogether, these data recapitulated our in vivo findings that the let-7bc2-cluster negatively regulates Tc17 response but is dispensable in Tc1 cells.Finally, to determine whether Tc17 differentiation is likewise controlled by the let-7afd-cluster, we cultured naive CD8 + splenocytes from let-7afd LOF and controls under Tc17 conditions.As we had observed with let-7bc2 LOF , absence of the let-7afd-cluster in T cells further enhanced differentiation towards Tc17 cells as quantified by flow cytometry and ELISA (Figure 5F,G).
Next, we focused on Rorc as a potential direct target of let-7, which could mechanistically mediate enhanced Tc17 differentiation in let7bc2 LOF mice.Towards this objective, we tested whether let-7bc2 LOF or let-7afd LOF naïve 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 let-7 LOF cells relative to controls (Figure 5E,H).To determine whether let-7 directly represses Rorc mRNA levels we cloned the 3'UTR of Rorc into luciferase constructs.These reporter assays with let-7b expressing cells independently confirmed that let-7b represses Rorc (Figure 5I, left).Furthermore, deletion of the putative let-7 binding sequence (Figure 4A) abrogated repression by let-7b (Figure 5I, right), thus confirming Rorc as a functional target of let-7 miRNA.Overall, these in vitro experiments readily recapitulated an upstream regulatory role of let-7 in Tc17 differentiation, mediated in part, via direct suppression of RORγt.

Enforced expression of let-7 tempers RORγt T cell expression levels in experimental
emphysema.
To explore a potential protective role of let-7 miRNA in experimentally-induced emphysema, we generated mice which allowed for selective induction of let-7 activity in T cells using the published rtTA-iLet7 mice crossed to CD4-Cre (herein referred to as let-7 GOF ; Figure 6A) (Angelou et al., 2020;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 ~2-3-fold rise in total let-7 activity in T cells (Angelou et al., 2020;Wells et al., 2017Wells et al., , 2023;;Angelou et al., 2020).Steadystate let-7 GOF and control (rtTA-iLet7) mice were examined for compromised RORγt protein levels within thymocytes and peripheral T cells.Providing further evidence of let-7-dependent regulation of Rorc, protein levels of RORγt were suppressed in CD8 + and CD4 + T cells of let-7 GOF mice relative to controls (Figure 6B).To determine whether enforced expression of let-7 offered protection from experimental emphysema, let-7 GOF and control mice were treated with nCB and then examined for changes in lung pathology and T cell type 17 responses.The let-7 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 let-7 GOF mice versus controls suggesting that enforced let-7 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, let-7 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 let-7 LOF and GOF models demonstrate the necessity and sufficiency of let-7 miRNA to act as a molecular brake to the type 17 T cell response through the direct regulation of RORγt, further our data suggests that nCB-or CS-mediated suppression of this braking mechanism furthers inflammation and exacerbates emphysema severity (Figure 6G).

DISCUSSION
MiRNA expression-based studies of COPD patients and mice exposed to CS have reported downregulation of let-7 miRNA expression in lung tissues (Conickx et al., 2017;Christenson et al., 2013b;Schembri et al., 2009).We and others explored the consequence of loss of let-7 expression/activity with synthetic oligonucleotides, sponges, lentiviral antisense knockdown, or via ectopic delivery of Lin28b (Polikepahad et al., 2010;Viswanathan et al., 2008;Piskounova et al., 2011), but studies pinpointing the role of individual let-7 clusters as potential drivers of lung inflammation and COPD within T cells remained elusive.In the present study, we established that the let-7 miRNA family members encoded by the let-7bc2-and let-7afd-clusters are downregulated in T cells from lungs of emphysema patients and emphysematous mice that were exposed to CS or nCB.Correspondingly, we demonstrated that in vivo genetic ablation of let-7bc2-cluster further sensitized mice to lung tissue destruction and emphysema upon treatment with nCB or CS.Mechanistically, our studies suggests that let-7bc2-cluster prevents the emergence of CD8 + T cell differentiation into Tc17 cells during emphysema in part, by directly silencing of Rorc.
Tc17 cells are vital for defense against viral, fungal and bacterial infections and they have also been associated with inflammation in various human diseases such as multiple sclerosis, inflammatory bowel disease, and cancer (Huber et al., 2013;Globig et al., 2022;Corgnac et al., 2020).In accordance with the potential pathogenic role of Tc17 cells as drivers of COPD, several studies detected increased cell numbers in airways and tissues of COPD patients as well as lungs of smoke-exposed animal models (Y.Chang et al., 2011;Zhou et al., 2020;Duan et al., 2013).
Other researchers also detected increased Tc17 subpopulations in tissues of COPD patients with infectious microbial exacerbations.In our earlier work to define the adaptive T cell immune responses in nCB induced COPD, we predominantly focused on the pathogenic role of Th17 cells, but did not examine Tc17 cells (You et al., 2015).Here we expand upon our prior observations, revealing that chronic exposure to nCB and elicitation of emphysema mice orchestrates the emergence and accumulation of Tc 17 cells which may act in parallel with Th17 cells to promote tissue damage.
Prior research has shown the importance of both transcriptional and post-transcriptional regulatory control of RORγt expression in T cells (Ciofani et al., 2012;Donate et al., 2013;Medvedev et al., 1997).Altogether, our in vivo studies establish let-7 as a new important link associated with regulation of RORγt and lung Tc17 differentiation in COPD.Our data also showed that in vivo conditional genetic ablation of individual let-7 clusters in T cells stimulates a rise in RORγt protein expression in single-positive thymocytes and peripheral CD8 + and CD4 + T cells while enforced let-7 activity leads to partial repression of RORγt in T cells.Despite these alterations in RORγt expression in our let-7 T cell LOF mice, the mice did not exhibit spontaneous gross phenotypes in thymus, spleens, or lungs at baseline nor did they exhibit changes in Tc17/Th17 subpopulations.This may be due to the subtle and modest expression thresholding of RORγt detected in mice and/or residual let-7 expression in T cells.On the other hand, and in agreement with our Tc17 and experimental emphysema data, we observed enhanced RORγt expression in lungs of let-7 LOF mice after treatment with nCB.We corroborated the importance of let-7 activity in Tc17 differentiation of ex vivo cultured CD8 + T cells, as well as in the direct posttranscriptional control of RORγt, suggesting that this defect, is in part, direct and cell autonomous.We did not ascertain whether deletion of let-7afd-cluster is an equally or more effective modulator of experimentally induced emphysema than the let-7bc2-cluster.Nonetheless, we predict that under different cell stress contexts, the functions of let-7 clusters do not fully overlap due to differential thresholding of mRNAs.
Prior studies have elucidated the relative and absolute quantities of individual let-7 family members in murine thymocytes and peripheral T cells which range from ~2-30% (Pobezinskaya et al., 2019;Pobezinsky et al., 2015).Moreover, the same group reported that all let-7 miRNAs are coordinately downregulation following antigen stimulation through the T cell receptor (Wells et al., 2017).Another recent study discerned a role for the lncRNA, CCAT1 (colon cancerassociated transcript 1) as a molecular decoy or sponge in human bronchial epithelial cells which drives downregulation of let-7c following cigarette smoke extract exposure (L.Lu et al., 2017).
Thus, it seems likely that complex synergistic transcriptional and post-transcriptional mechanisms contribute to downregulation of let-7 activity in emphysematous T cells.
It is also important to note that let-7 has been reported to exert potent effects by titrating the levels of multiple gene targets in mechanisms that contribute to Th17 inflammatory response and influence diverse set of processes including T cell activation, proliferation, differentiation, and cell homing (Angelou et al., 2020;Beachy et al., 2012;Bronevetsky et al., 2016;Pobezinskaya et al., 2019;Pobezinsky et al., 2015;Wells et al., 2017).A particular feature of these studies has been the utilization of Lin28b transgenic mice to block maturation and activity of entire let-7 family to promote a let-7 LOF function phenotype (Angelou et al., 2020;Piskounova et al., 2011;Pobezinskaya et al., 2019;Wells et al., 2023;Zhu et al., 2011).Furthermore, Lin28b was recently reported to also influence transcriptome-wide ribosome occupancy and global miRNA biogenesis (Tan et al., 2019).Thus, it is likely that Lin28b transgenic overexpression could give rise stronger phenotypes than we observed in our single cluster let-7 LOF mice.Nonetheless, unbiased omicsbased methods will be useful to determine if other gene targets beyond RORγt synergistically potentiate the in vivo Tc17-response and emphysema phenotype in context of deletion of let-7bc2cluster.
Tc17 cells play a major role in microbial infections, providing a potent anti-viral response (Hamada et al., 2009;Yeh et al., 2010), while viral infection has been an established factor in COPD exacerbations (Hewitt et al., 2016;Wedzicha, 2004).It will be interesting to determine whether loss of let-7bc2-or let-7afd-cluster activity in the T cell compartment contributes to COPD disease susceptibility in the context of viral exposure.Our experiments with let-7 GOF were partially successful in limiting the emergence of Tc17 and Th17 in nCB-elicited emphysema.Let-7 GOF mice exhibited a reduction in RORγt expression levels and type 17 responses but were not protected from alveolar remodeling following nCB exposure.A potential limitation of the let-7 GOF transgenic model is that it expresses only the let-7g sequence which may render it less potent than the corresponding two mature forms transcribed from the let-7bc2-cluster.Additional studies will be required to ascertain whether other interventions that enhance let-7 activity in T cells are successful in preventing or reversing COPD.

Mice
Conditional knockout-ready floxed let-7bc2 and let-7afd mice 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 Table 1).
The let-7bc2 flox/flox ; CD4-cre and let-7afd flox/flox ; CD4-cre mice were PCR genotyped.The R26-STOP-rtTA; Col1a1-tet0-let-7 (rtTA-iLet7) mice were obtained from JAX Jax Stocks 023912 and 05670 and then bred to CD4-Cre were PCR genotyped with established JAX primers (Belteki et al., 2005;Zhu et al., 2011).Control rtTA-iLet7 and the let-7 GOF mice were fed ad libitum with 200mg/kg of doxycycline-containing chow (Bio-Serv S3888) at weaning age primers (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.

Human emphysema tissue samples and T cell isolation
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 Table 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., 2020;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 hour 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 minutes 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.

Human lung transcriptome data
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.

Cigarette smoke exposure model of pulmonary emphysema
To promote emphysema, mice were exposed to cigarette smoke 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, five days a week, for four months as previously described (Shan et al., 2012).

nCB exposure model of pulmonary emphysema
Nano-sized particulate carbon black was prepared and administered as previously described (You et al., 2015;W. 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 four weeks (total delivered dose of 6 mg).Lung histomorphometry and airway inflammation were assessed four weeks after the final nCB challenge.For histomorphometric analysis, mice lungs were fixed with 10% neutralbuffered formalin solution via a tracheal cannula at 25-cm H2O pressure followed by paraffin embedding and tissue sectioning and stained with hematoxylin and eosin.Mean linear intercept (MLI) measurement of mouse lung morphometry were 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 ten 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;W. Lu et al., 2015).

Cell isolation from murine lung tissue
Mouse lung tissue were 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 hour 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 let-7 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 + double positive cells purified from thymus single-cell suspensions by flow-cytometric sorting on FACS Aria (BD Biosciences).

ELISA
Supernatant was collected from in vitro polarized murine CD8 + T cells and centrifuged to remove cellular debris (W.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 (ThermoFisher).
Representative flow cytometric gating and quantification strategy for detection of lung Th1/Th17 and Tc1/Tc17 cell populations is shown in Supplementary Figure 3.Samples were analyzed using BD LSR II flow cytometer (BD Biosciences) and FlowJo software (TreeStar).

RNA Isolation and Quantitative RT-PCR
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 were synthesized using TaqMan Advanced miRNA cDNA Synthesis Kit (ThermoFisher) 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 (W.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-

Luciferase reporter assays
Genomic fragment containing the murine Rorc 3ʼUTR was cloned into psiCHECK2 luciferase reporter plasmid (Promega).This construct was also used to generate the let-7 ʻseedʼ deletion mutant derivative using the QuikChange Multi Site Mutagenesis Kit (catalog 200514-5, Stratagene).3T3 mouse embryonic fibroblasts (MEFs) were transfected using Oligofectamine (Invitrogen) with 100 ng of psiCheck-2 plasmid containing wild-type or mutant 3ʼUTR, along with the miRNA control or let-7b duplex (Dharmacon) at a final concentration of 6 nM (Gurha et al., 2012) (Supplementary Table 1).Reporter activity was detected with the Dual-Luciferase Reporter Assay System (Promega).

Statistical analysis
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 (ANOVA) with Tukey's or Sidak's correction, and Mann-Whitney Test when indicated.