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

Reviewer #1 (Public Review):

Summary: Inflammatory T cells have been recognized to play an important role in human COPD lung tissue and animal models of emphysema. The authors have previously identified that Th17 cells regulate chronic inflammatory diseases, including in mice exposed to smoke or nanoparticulate carbon black (nCB). Here, the authors interrogate the role of Tc17 cells using similar mouse models. Investigating let-7 miRNA, which induces antigen-presenting cells activation and T cell mediated Th17a inflammation, they show that the master regulator of Tc17/Th17 differentiation, RAR-related orphan receptor gamma t (RORγt), is a direct target of let-7 miRNA in T cells. Because RORγt expression is elevated in COPD patients and in mouse models of COPD, the authors generate a Let-7 overexpressing mouse in T cells and reduce RORγt expression and Th17 and Tc17 cell recruitment in nCB-exposed mice.

Strengths: The authors use previous a previously published RNA-seq dataset (GSE57148) from the lungs of control and COPD subjects to explore the involvement of Let-7 in emphysema. They further evaluate Let-7a expression by qPCR in lung tissue samples of smokers with emphysema and non-emphysema controls. Moreover, expression of Let-7a, Let-7b, Let-7d, and Let-7f in purified CD4+ T cells were inversely correlated with emphysema severity lungs. Similar findings were found in their mouse models (CS or nCB) in both lung tissue and isolated lung CD4+ and CD8+ T cells, with reduced let-7afd and let-7bc2 expression.

Using mice harboring a conditional deletion of the let-7bc2 cluster in all T cells (let-7bc2LOF) derived from the CD4+CD8+ double-positive stage, the authors show enhanced emphysema in nCB- or CS-exposed mice with enhanced recruitment of macrophages and neutrophils to the lung. While CD8+IL17a+ Tc17 cells and CD4+ IL17a+ Th17 cells were increased in nCB-exposed control animals, only let-7bc2LOF mice showed an increase in CD8+IL17a+ Tc17 cells. Further, unexposed let-7bc2LOF and let-7afdLOF mice expressed greater RORγt expression in both CD8+ and CD4+ T cells.

Generating a let-7 gain of function mouse with overexpression of let-7g in thymic double-positive-derived T cells, protein levels of RORγt were suppressed in CD8+ and CD4+ T cells of let-7GOF mice relative to controls. Let-7GOF mice treated with nCB showed similar lung alveolar distension as controls suggesting that increased let-7 expression does not protect the lung from emphysema. However, let-7GOF mice showed reduced lung Tc17 and Th17 cell populations and were resistant to the induction of RORγt after nCB exposure.

Weaknesses: Limited data is shown on the let-7afdLOF mice. Does this mouse respond similarly to nCB as the let-7bc2LOF.
Because the authors validate their findings from a previously published RNA-seq dataset in subjects with and without emphysema, the authors should include patient demographics from the data presented in Figure 1C-D.
To validate their mouse models, the absence of Let-7 or enhanced Let-7 expression needs to be shown in isolated T cells from exposed mice.
In Figure 3, the authors are missing the unexposed let-7bc2LOF group from all panels. This is again an issue in Figure 6 with the let-7GOF.
Because the GOF mouse enhances Let-7g within T cells, the importance of Let-7g should be determined in human subjects. Why did the authors choose to overexpress Let-7g, the rational is not clear?
The purity of the CD4+ and CD8+ T cells is not shown and the full gating strategy should be included.
The authors indicate that Tc17 and Th17 T cells were reduced in the GOF mouse, it remains unclear if macrophage or neutrophil recruitment is altered in GOF mice.

Reviewer #2 (Public Review):

Summary:
Let-7 family miRNAs are largely redundant in function, and originate from multiple genomic loci ("clusters"). Erice et al demonstrate that two individual clusters (let7afd and let7bc2) in mice regulate the generation of IL-17 producing CD8 T cells in vitro and in vivo in a model of emphysema. These cells also express higher levels of the IL-17-inducing transcription factor RORgt, encoded by Rorc, which the authors demonstrate to be a direct target of let-7. Since multiple let-7 family miRNAs are downregulated in T cells and lung tissue in emphysema, these data support a model in which reduced let-7 allows increased IL-17 production by T cells, contributing to disease pathogenesis.

Strengths:
The inclusion of miRNA and pri-miRNA expression data from sorted human lung T cells as well as mouse T cells from an emphysema model is a strength.

The study includes complementary loss of function and gain of function experimental systems to test the effect of altered let-7 function, though it should be noted that these involved different let-7 family members and did not yield simple, complementary results for all experimental outcomes.

The most important finding is that deletion of just one let-7 cluster ("Let7bc2") is sufficient to exacerbate emphysema in the nCB and CS models.

Weaknesses:
The functional analyses are unusually focused on IL-17 producing CD8 T cells, but it is not made clear whether these cells are an important player in emphysema pathogenesis in the nCB and CS models. The data shown reveal that they are far less numerous than IL-17-producing CD4 T cells. It is also notable that the Figure 1 expression data from human subjects used sorted CD4+ T cells. And as the author mentioned, prior work on let-7 showed that it regulated Th17 (CD4) responses.

Compared with Let7bc2 deletion, Let7afd deletion had a much larger effect on IL17 production by CD8 T cells in vitro, and it also had a larger effect on RORgt expression in untreated mice in vivo, especially in the lung. It would be valuable to more thoroughly characterize the let7afd mice. RORgt expression should be shown in the in vitro assays. In the results, the authors state that let7afdLOF mice "did not exhibit lung histopathology nor inflammatory changes" up to 6 months of age. Similarly, it is stated in the conclusion that "the let-7afdLOF mice ... did not exhibit changes in Tc17/Th17 subpopulations" in vivo. All these data should be shown, and if no baseline changes are apparent, then I also recommend challenging these mice with nCB and/or cigarette smoke.

This brings up the larger issue of redundancy among the let-7 family members and genomic clusters. This should be discussed, including some explanation of the relative expression of each mature family member in T cells, and how that maps to the clusters studied here (and those that were not investigated). It would also be helpful to explain the relationship between mouse Let7bc2 and human Let7a3b, since Let7bc2 is the primary focus of emphysema experiments in this manuscript.

This is especially important because the study of individual let-7 clusters is the core novelty of this body of work, as described in the first paragraph of the discussion. The regulation of let-7 expression has been reported before and its functional role has been investigated with a variety of tools.

Let7g overexpression caused a marked reduction in Rorgt expression in T cells at baseline and in the setting of nCB challenge, and it reduced the frequency of IL17+ producing CD8 T cells in the lung to baseline levels. Yet there was no change in the MLI measurement of histopathology. Is this a robust result? The responses in the experiment shown in Fig. 6C-D are quite muted compared to those shown in Figure 2. The latter also shows a larger number of replicates, and it is unclear whether the data in 6D include measurement from all of the mice tested (e.g. pooled from 2 small experiments) or only mice from one experiment.

Although RORgt is a great candidate to have direct effects on IL-17 expression, the mechanistic understanding of let-7 action on T cell differentiation and cytokine production is limited to this single target. As noted in the discussion, others have identified cytokine receptor targets that may play a role, but it is also likely others among the many targets of let-7 also contribute.

Reviewer #3 (Public Review):

Summary: The manuscript by Erice et al describes let-7 miRNA promotes Tc17 differentiation and emphysema by repressing the transcription factor RORgt. The authors found that overall expression of the let-7 miRNA clusters, let-7b/let-7c2 and let-7a1/let-30 7f1/let-7d are reduced in the lungs and T cells of mice with cigarette smoke-induced emphysema. They also found that the loss of the let-7b/let-7c2-cluster in T cells exaggerated cigarette smoke-induced emphysema. It appears that deletion of the let-7b/let-7c2-cluster lead to enhancement of IL-17-secreting CD8+ T cells (Tc17) in mice with emphysema. The opposite phenotype was observed when let-7 was overexpressed in T cells. They found a potential let-7 binding site in the 3' UTR of RORgt. They demonstrated a direct effect of let-7 on RORgt expression using let-7 mimic in a RORgt luciferase reporter assay. They have done an outstanding job of translating the finding of reduced let-7 expression in emphysema patients to a thorough delineation of its mechanism in a mouse model. Together, this study suggests an important role for let-7 miRNA in Tc17 cells in emphysema which appears to be mediated via repression of RORgt.

Strengths: This well written manuscript flows logically and the data supports the overall claim let-7 miRNA promotes Tc17 differentiation during emphysema. There are several strengths to this study including the use of conditional let-7 knock out animals to decipher the role of this miRNA in Tc17 cells in emphysema.

Weaknesses: There are no major weaknesses in this study. It would be interesting to see if knockdown RORgt could rescue enhanced Tc17 differentiation seen in let-7b/let-7c2-cluster-deficient T cells. The authors show no change in frequencies of Treg cells in let-7bc2LOF mice exposed to nCB. Do these Treg cells also express higher levels of RORgt and IL-17? The major question that was not addressed in this study is how let-7 expression is regulated in emphysema. The other recommendation is that the authors include the sequences of the let-7 mimic oligos used in the luciferase assay.