Perivascular Fibrosis Is Mediated by a KLF10-IL-9 Signaling Axis in CD4+ T Cells

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Perivascular fibrosis is a hallmark of advanced vascular disease states often associated with elevated blood pressure, vascular stiffness, adverse vascular remodeling, and end-organ dysfunction such as in the heart and kidney.^1,2 Continuous excessive stress and inflammation during hypertension increase the amount of perivascular connective tissue and collagen deposition that is characteristic of pathological perivascular fibrosis.³ Indeed, hypertension remains a serious public health problem that contributes to considerable global cardiovascular disease and premature death worldwide⁴ by increasing the risk of ischemic heart disease, stroke, and chronic kidney disease.⁵ Early management and therapeutic intervention may greatly reduce the occurrence or postpone the development of chronic fibrotic diseases, especially hypertensive cardiovascular, kidney, and aortic disease. However, therapeutic targets that specifically control perivascular fibrosis are not well defined.

Accumulating studies revealed that perivascular fibrosis is greatly dependent on perivascular inflammation and immune cells in hypertension.^6–9 Ang II (angiotensin II) contributes to vascular damage and end-organ damage via promoting T cell activation and proliferation.¹⁰ The importance of T cells in the progression of perivascular fibrosis has been highlighted recently through the prevention of aortic fibrosis and stiffening in Rag1−/− mice, which was reversed by adoptive transfer of T cells after Ang II infusion.⁶ Furthermore, Ang II induced T cell-derived microRNA-214 mediated perivascular fibrosis, vascular stiffening, and dysfunction by regulating T cell activation and chemotaxis in hypertension.¹¹ Despite recent data suggesting the role of T cells in the progression of perivascular fibrosis, the underlying mechanisms remain poorly understood.

KLF10 (Kruppel-like factor 10), a TF (transcription factor) that belongs to the kruppel-like zinc-finger family, was found to be expressed in T cell subsets. Our previous studies demonstrated that KLF10 regulates both CD4+CD25− T cell effectors and CD4+CD25+ regulatory T cells, thereby regulating atherosclerotic lesion formation in a murine atherosclerosis model.¹² Furthermore, we also found that KLF10 in CD4+ T cells regulate obesity, insulin resistance, and fatty liver in the presence of a high fat diet.¹³ Given the prominent role of CD4+ T cells in mediating a range of Ang II-associated vascular diseases, we hypothesized that KLF10 in CD4+ T cells may contribute to the regulation of vascular remodeling. Herein, we find that CD4-targeted KLF10 deficient (TKO) mice develop marked acceleration of perivascular fibrosis, but not interstitial fibrosis, through distinct mechanisms involving CD4+ T cell release of IL (interleukin)-9 and activation of specific fibroblast and myofibroblast subsets.

Methods

Data Availability

The data, methods used in the analysis, and materials used to conduct the research of this study are available from the corresponding author upon reasonable request. Major Resources Table and detailed methods are provided in the Supplemental Material.

Results

KLF10 Expression Is Increased in CD4+ T Cells After Ang II Treatment

Given the importance of transcriptional control of leukocytes in hypertension,^14–18 dysregulated TFs were examined from a published dataset of hypertensive patients with or without left ventricular (LV) remodeling in peripheral blood mononuclear cells (PBMCs). Multiple TFs were found dysregulated in hypertensive patients with or without LV remodeling compared with control individuals (Figure 1A). However, only 2 TFs (KLF10 and STAT3) were found commonly dysregulated in hypertensive patients with or without LV remodeling compared with controls (Figure 1B). The increased expression of KLF10 was further confirmed in an independent dataset of uncontrolled hypertensive subjects (Figure 1C). Considering the important regulatory role of KLF10 in CD4+ T cells in atherosclerosis,¹² obesity, and insulin resistance,¹³ we sought to investigate if Klf10 expression was altered in T cells in a murine model of hypertension. RNA-sequencing from Ang II-treated mice revealed Klf10 to be one of the top TFs significantly increased in splenic CD3+ T cells (Figure 1D). This was confirmed by RT-qPCR analysis of isolated CD3− cells, CD3+, and CD4+ T cells from the spleen after 28 days of Ang II treatment in which Klf10 expression increased in response to Ang II in CD3+ T cells and even higher in CD4+ T cells (Figure 1E). Because Ang II treatment is known to impact vascular remodeling,¹⁹ co-staining of KLF10 and CD4+ T cells was performed in cross-sections of the descending aorta. Higher counts of CD4+KLF10+ cells were observed after Ang II treatment compared with the PBS infused group (Figure 1F). To identify whether Ang II can increase Klf10 in T cells, CD4+ T cells were isolated from C57BL/6 mice and treated with increasing amounts of Ang II. Klf10 expression was found to be significantly up-regulated by Ang II in a dose-dependent manner (Figure 1G). Taken together, KLF10 expression increased in PBMCs in hypertensive patients, and in CD4+ T cells after Ang II treatment in mice, highlighting a potential role of Klf10 in CD4+T cell in Ang II-induced vascular remodeling.

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KLF10 Deficiency in CD4+ T Cells Impairs the Function of Hypertension-Related Organs and Triggers Perivascular Fibrosis Independent of Blood Pressure

To evaluate the role of KLF10 in CD4+ T cells in Ang II-induced vascular remodeling, CD4-targeted KLF10 deficient (Klf10^fl/flCD4^Cre+, [TKO]) and CD4-Cre (Klf10^+l+CD4^Cre+, [Cre]) control mice¹³ were infused with PBS or Ang II for 28 or 42 days (Figure 2A). Aortic blood pressure increased in the Ang II treatment groups compared with PBS controls; however, no difference was found between TKO and Cre control mice in both 28 days and 42 days (Figure 2B). Considering Ang II-induced hypertension can cause tissue remodeling and multiorgan dysfunction, we further evaluated the function of heart, aorta, and kidney in these mice. After 28 days of Ang II infusion, hearts of TKO mice demonstrated impaired LV global longitudinal strain (Figure 2C, Table S3); furthermore, aortas of TKO mice developed increased abdominal pulse-wave velocity (PWV, Figure 2D) and decreased circumferential strain (Circ Strain, Figure 2E) compared with Cre control mice, while there were no differences before Ang II infusion (Figure S1A and S1B). Similarly, following 28 days of Ang II infusion, TKO mice demonstrated smooth muscle hypercontractility compared with Cre control mice, and both smooth muscle hypercontractility and impaired smooth muscle relaxation manifested after 42 days of Ang II infusion (Figure S1C and S1D). Ang II infusion also resulted in a marked increase of albuminuria and KIM-1 (kidney injury molecule-1) levels as detected in the urine of TKO mice (Figure 2F and 2G), while no statistical difference was observed under basal conditions compared with control mice (Figure S1E and S1F). Altogether, our results suggest end organ dysfunction and likely tissue remodeling occurred after Ang II infusion in mice deficient of Klf10 in CD4+ T cells, effects that were independent of blood pressure differences between TKO and Cre control mice.

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As a cardiovascular damage inducer, chronic Ang II infusion can induce significant hypertension, cardiomyocyte hypertrophy, and fibrosis in mice.^20,21 To identify histopathologic changes, we first evaluated the hearts and found no histopathologic difference in total fibrosis, interstitial fibrosis, and myocyte cross-sectional area between Cre and TKO groups after Ang II infusion (Figure S1G and S1H). However, perivascular fibrosis in the heart was markedly increased in TKO mice after Ang II treatment compared with Cre mice (Figure 2H). Similarly, increased perivascular fibrosis was also observed in the kidneys of TKO mice (Figure 2I). Furthermore, severe aortic perivascular fibrosis and adventitial collagen deposition were detected in Ang II treated TKO mice by Masson trichrome staining and Sirius red staining (Figure 2J and 2K). No differences in fibrosis or collagen were observed between TKO and Cre before Ang II treatment (Figure S1I). Analogous perivascular phenotypes were also found in female mice (Figure S1J). Collectively, our results indicate that mice deficient of Klf10 in CD4+ T cells develop accelerated perivascular fibrosis compared with Cre control mice in aortas, hearts, and kidneys after Ang II infusion.

KLF10-Deficient CD4+ T Cells Release IL-9 That Mediates Perivascular Fibrosis

To elucidate possible mediators involved with the observed multi-organ perivascular fibrosis, we first examined the expression of angiotensin II receptors in CD4+ T cells and fibroblasts; however, no differences were observed between the TKO and Cre control groups (Figure S2A and S2B). To identify potential mediators in the circulation, we performed plasma cytokine profiling from Ang II-treated TKO and Cre mice (Figure S2C). Among a panel of inflammatory markers, IL-9 was the only significantly increased cytokine after Ang II treatment in both male and female TKO mice compared with controls (Figure 3A; Figure S2D). In concordance, Il9 expression was also upregulated in PBMCs, hearts, kidneys, and aortas (Figure 3B). Considering that Ang II treatment is known to increase the accumulation of leukocytes and T cells in tissues to mediate vascular dysfunction, hypertension, and end-organ inflammation,^10,22,23 we sought to determine the role of CD4+IL9+ cells in vascular inflammation. Flow cytometry from single-cell suspensions of the thoracic aorta showed that the percentage of CD4+IL9+ cells and CD4+IL4+ cells increased in TKO mice after Ang II treatment, while no differences were observed in other cell types compared with Cre control including CD8+IL9+ cells (Figure 3C, Figure S2E through S2G). Similar changes were found in the spleen (Figure 3D, Figure S2H and S2I). Consistently, immunofluorescence imaging also captured the increase of CD4+IL9+ cells in the periadventitial tissue (Figure 3E; Figure S2J). Although the percentage of CD4+IL4+ T cells increased in TKO mice, there were no differences of released IL-13, IL-5, and IL-4 in both male and female TKO mice compared with Cre controls (Figure S2K and S2L). To confirm whether KLF10 deficiency in CD4+ T cells affected the production of IL-9, CD4+ T cells were isolated from Ang II-treated TKO and Cre mice (Figure 3F). CD4+ T cells from TKO mice had higher mRNA expression for Il9 and released more IL-9 protein in supernatants compared with Cre controls (Figure 3G; Figure S2M), while no differences were observed in secreted IL-5 and IL-4 (Figure S2N). Similarly, CD4+ T cells isolated from TKO or Cre mice and subsequently treated with Ang II ex vivo for 1 day (Figure 3H) demonstrated increased Il9 expression and had a higher level of IL-9 protein release in supernatants from TKO CD4+ T cells compared with Cre CD4+ T cells (Figure 3I).

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To further assess the role of IL-9 in the development of perivascular fibrosis, recombinant mIL9 was administrated in Ang II-treated Cre mice (Figure 3J). Systemic delivery of mIL9 markedly impaired LV global longitudinal strain (Figure 3K, Table S4), increased PWV, and decreased Circ Strain (Figure 3L) in Cre mice. In addition, the ratio of albumin and creatinine, and the value of KIM-1 increased after treating with mIL9 (Figure 3M). Importantly, histopathologic analyses confirmed that the mIL9 treated group developed severe perivascular fibrosis and increased adventitial collagen deposition (Figure 3N and 3O). Taken together, deficiency of Klf10 in CD4+ T cells increased IL-9, and ectopic delivery of IL-9 mediated the perivascular fibrosis phenotype, effectively phenocopying the perivascular fibrosis observed in Klf10-deficient CD4+ T cell mice.

KLF10 Binds to the IL-9 Promoter and Interacts With HDAC1 to Inhibit IL-9 Transcription

Previous studies have demonstrated that KLF10 could bind to target gene promoters to regulate transcription.^24,25 Given the increased expression of IL-9 in TKO mice and in TKO CD4+ T cells, we explored potential underlying mechanisms of this regulation. As shown in Figure 4A, there are 4 putative KLF10 DNA-binding sites in the Il9 promoter sequence.^24,26 Using chromatin immunoprecipitation assays with KLF10 antibodies in CD4+ T cells and the designated primer pairs directed for each putative-binding site, we found that Ang II markedly increased the KLF10 enrichment to these sites (Figure 4B). Overexpression of mouse KLF10 by transfecting overexpression plasmid in a heterologous HEK293 T cell was confirmed by Western blot (Figure 4C). It was shown that overexpression of mouse KLF10 in HEK293 T cell significantly inhibited the transcriptional activity of a mouse IL9 promoter reporter (Figure 4D). In addition, 5’ truncations of the IL-9 promoter demonstrated that successive deletions of the putative KLF10-binding sites increased transcriptional activity of the IL-9 promoter, suggesting that KLF10 mediates transcriptional repression (Figure 4E). KLF10 has been reported to interact with HDAC1 (histone deacetylase 1) to facilitate transcriptional repression.^24,25 Consistently, we found that KLF10 recruited HDAC1 in T cells, especially after Ang II treatment (Figure 4F). Next, we assessed the role of HDAC1 on IL-9 promoter in the presence of KLF10 by performing luciferase reporter assays (Figure 4G). Depletion of HDAC1 with siRNAs demonstrated elevated luciferase activity among the WT and 3 mutant IL-9 promoters (Figure 4G and 4H). Collectively, these findings suggest that KLF10 interacts with HDAC1 and binds to the proximal IL-9 promoter to inhibit IL-9 transcriptional activity.

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Transcriptomic Changes Involved in Ang II-Induced Perivascular Fibrosis

To ascertain the underlying global transcriptomic changes involved in perivascular fibrosis, we performed RNA-sequencing and gene ontology pathway analysis on stripped (perivascular adventitial tissue removed) (Figure S3) and nonstripped aorta (perivascular adventitial tissue remained intact) (Figure S4) from Cre or TKO mice after Ang II treatment. Comparison of stripped aortas between Cre and TKO mice revealed that most differentially regulated genes are related to metabolic processes but not to fibrotic process (Figure S3D through S3F). Considering the pathophysiological changes of fibrosis occurred in the perivascular adventitial tissue, we next focused our bioinformatic analysis on the nonstripped aortas. In contrast to Ang II-treated Cre mice, nonstripped aortas of TKO mice manifested pathways involved in assembly and muscle contraction (‘Muscle contraction’, ‘Myofibril assembly’), ion transport, and sarcomere organization (Figure 5A, Figure S4, S4E, and S4F). To identify transcriptomic differences derived from the vessel and adventitial tissues, we compared overlapping and unique differentially expressed genes (DEGs; FDR<0.05) from the stripped and nonstripped aortic TKO versus Cre contrasts (Figure 5B; Figure S5). From a total of 48 overlapping DEGs between TKO and Cre aortas, gene set enrichment analyses revealed several pathways involved in fibrosis, junctional signaling, and ion-related pathways (Figure 5C). Notably, from the 748 unique DEGs derived from the nonstripped aorta, calcium signaling emerged as a dominant pathway in the TKO group (Figure 5D), and the significantly regulated calcium signaling pathway genes were shown in the heatmap (Figure 5E). As such, we performed von Kossa staining, which revealed higher calcium deposition in the adventitial tissues from Ang II-treated TKO mice compared with Cre controls (Figure 5F). Furthermore, calcium flux assays in isolated aortic fibroblasts (Figure S6A through S6D) from C57BL/6 mice were performed to detect the effect of IL-9 on intracellular calcium levels. Consistent with previous reports,^27,28 we found that Ang II as a profibrotic agonist increased intracellular calcium; however, IL-9 further augmented this intracellular calcium flux in the presence of Ang II (Figure 5G, Video S1). Collectively, comparative RNA-seq transcript profiling identified several pathways associated with periadventitial remodeling and that the calcium signaling pathway is dominantly upregulated in nonstripped aortas of TKO mice after Ang II treatment. In addition, IL-9 and Ang II cooperatively increase calcium flux in aortic fibroblasts ex vivo.

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TKO Fibroblasts Display an Activation Signature and IL-9 and Ang II Treatment Recapitulate the Phenotype in Control Fibroblasts

Given that calcium is involved in particularly critical signaling pathways for fibroblast and myofibroblast differentiation,^27,29 we isolated and cultured aortic fibroblasts from Ang II-treated Cre control and TKO mice (Figure 6A). Immunofluorescence staining showed that the expression of Col1a1 and α-SMA increased significantly in aortic fibroblasts of TKO mice compared with those from Cre mice (Figure 6B, Figure S6A). RNA-sequencing of those TKO and Cre fibroblasts revealed upregulation of myofibroblast marker-related genes (Cnn1, Acta2, etc) and fibroblast activation signature genes (Col1a1, Fn1, etc) in aortic fibroblasts from TKO mice (Figure 6C, Figure S6B through S6E). Furthermore, calcium signaling related genes were also increased in TKO fibroblasts (Figure 6C). To further assess whether these fibrotic-related changes were mediated by IL-9, primary aortic fibroblasts were isolated from C57BL/6 mice, and supernatants from WT or KO CD4+ T cells were added to the fibroblasts grown in culture with and without anti-IL-9 antibodies (Figure 6D). Supernatants from KO CD4+ T cells increased the expression of multiple fibrotic genes including Col1a1, Col3a1, Col8a1, Acta2, Angptl1, Fmod, and Mmp9, whereas anti-IL-9 antibodies reversed their expression (Figure 6E). Conversely, treatment of fibroblasts with recombinant IL-9 or Ang II increased the expression of Col1a1, Col3a1, Col5a1, Acta2, and Mmp9, and treatment with IL-9 plus Ang II together demonstrated additive effects (Figure 6F). Immunofluorescence also confirmed that IL-9, Ang II, or IL-9 plus Ang II additively increased the expression of Col1a1 and α-SMA (Figure 6G). Notably, there were no differences in expression of Il9r in any of the CD4+ T cell, fibroblast, or aortic tissue RNA-seq datasets (Figure S6F). Altogether, fibroblasts from TKO mice exhibit an activated fibroblast signature along with myofibroblast-related genes, which is recapitulated in C57BL/6 fibroblasts treated with IL-9 and Ang II.

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Considering KLF10 is a putative regulator of TGF (transforming growth factor)-β signaling in specific disease states,^12,13 we evaluated the expression of TGF-β isoforms in plasma. There were no differences in TGF-β1 or TGF-β2 in plasma between KO and Cre control mice treated with Ang II (Figure S7A). Despite a modest reduction in TGF-β3 in the plasma, there were no differences in expression levels for TGFβ1-3 between KO and Cre control CD4+ T cell supernatants (Figure S7A and S7B). In addition, examination for mediators of the TGF-β signaling pathway in our 3 RNA-seq datasets and 1 published T cell dataset showed no impact of KLF10 on TGF-β signaling in vivo supported by pathway analyses from these 4 different RNA-seq datasets (Table S5). These findings suggest that the KLF10-mediated effects in CD4+ T cells on fibroblasts were likely independent of TGF-β signaling.

Single-Cell RNA Sequencing Revealed Fibroblast Heterogeneity and Activation Signatures Induced in TKO Aortas

To further understand the differences in phenotypic heterogeneity in fibroblasts during the progression of perivascular fibrosis between TKO and control mice in response to Ang II treatment, we performed single-cell RNA-sequencing (scRNA-seq) of the descending aorta (Figure 7A). A total of 53 346 cells from our 4 samples met quality control metrics (Figure S8A) and were integrated and analyzed. The Seurat package³⁰ was used for integration, clustering, and marker analysis (detailed in extended methods). We classified the cells in 10 major aortic cell types based on identified marker genes in each cluster (Figures S8B, S8C, S9A through S9D and Table S6). The Uniform Manifold Approximation and Projection with the labeled clusters are shown in Figure 7B; Figure S9B. The percentage and the number of aortic cells from TKO and Cre aorta in each cell type are shown in Figure 7C, Figure S9E, and Table S7. Considering the important biological role of fibroblasts in perivascular fibrosis, fibroblasts in the integrated dataset were identified by established markers such as Pdgfrα (platelet-derived growth factor receptor-α)^31,32 (Figure S10A). Notably, we found that both the percentage and the number of fibroblasts increased in Ang II-treated TKO aortas compared with Cre controls (Figures 7C; Figure S9E and Table S7). Differential expression analysis between TKO and Cre aortas in the fibroblast clusters revealed a total of 627 DEGs. Pathway enrichment analysis of these DEGs highlighted several pathways associated with fibrosis in the TKO aorta including ECM (extracellular matrix), ECM organization, and collagen fibril and extracellular fibril organization (Figure 7D). Considering the important biological role of fibroblasts in the production of the enriched ECM,³³ we assessed for relative changes in fibroblast activation signature genes, collagen family genes, and MMPs (matrix metalloproteinases) between TKO and Cre aorta controls, and found that many were upregulated in the Ang II-treated TKO group (Figure S10B).

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Further subsetting and cluster analysis³⁰ was performed in the fibroblast group and 9 fibroblast subcluster (FBS) were identified based on the unique enriched markers (Figure 7E, Figures S10C, S10D, S11, Table S8). Specifically, FBS_1 and FBS_2 showed high expression of Pdgfa, a canonical fibroblast marker that was found in perivascular regions rarely overlapping with αSMA³⁴; FBS_3 and FBS_4 were enriched in lymphocyte markers; FBS_5 was an EC-like fibroblast cluster that highly expressed Cdh5 and Kdr,^35,36 suggesting potential endothelial-mesenchymal transition during fibrosis^37,38; FBS_6 highly expressed Col4a5, a collagen marker that was found to be related to a matrix fibroblast signature with endoplasmic reticulum stress activation^39,40; FBS_7 highly expressed Mmps, also considered as matrix fibroblasts highly expressing Mmp3^40,41; FBS_8 highly expressed Col8a1, Col11a1, Angptl1, and Cthrc1 and was considered as a group of collagen-related pathological fibroblasts that are activated^42–45; and FBS_9 highly expressed Lyz2 and Ccl6, considered as a group of inflammation-related activated fibroblasts³¹ (Figure S11). A differential abundance analysis for the conditions in each fibroblast subcluster showed a decrease in FBS_2 (log2FC=−1.18, adj. pval=0.04), while there was an increase in the number of cells in FBS_8 (log2FC=1.64, adj. pval=0.01) from the aorta of Ang II-treated TKO mice compared with Cre control (Figure 7F, Figure S12A, and Table S9). Interestingly, the increased FBS subclusters (FBS_8) in TKO mice were highly associated with pathological or disease enriched fibrosis-related genes (Figure 7G, Table S10). A combined gene score using a previously defined fibrosis-related gene list (Table S11) showed that FBS_8 had the highest score among all the FBS subclusters, suggesting the FBS_8 was a key fibrosis-driven subcluster (Figure 7H). In contrast, a combined gene score using a group of canonical fibroblast genes (Table S11) showed that FBS_2 had the highest score, suggesting that FBS_2 may be the canonical type of fibroblast (Figure S12B).

We next performed RNA velocity analysis, a method which calculates both spliced and unspliced mRNA counts to predict potential future directionality and speed of cell state transitions.⁴⁶ Higher proportions of unspliced to spliced mRNA highlights the active transcription state within a given cell cluster. Using this analysis, we obtained a trajectory of the different FBS subclusters states (Figure 7I). FBS_2 appeared to be the initial state of FBS in the stream plot (Figure 7I), which was consistent with our previous observation on the enriched markers of canonical FBS genes in FBS_2. Following the paths from FBS_2 in the stream plot, we observed FBS_8 as the most progressed state from FBS_2 (Figure 7I).

Together with the observation of a higher percentage of cells in FBS_8 in TKO samples and fibrosis-related gene markers for this subcluster, FBS_8 is likely the most important cluster contributing to the perivascular fibrosis in TKO mice. Furthermore, k-means cluster analyses confirmed FBS_8 is completely different with FBS_1-5 and FBS_9 (Figure 7J). Further pathway analysis found that FBS_8 is highly involved in ECM and structure organization (Figure 7K). Since Col8a1 is one of the unique collagen-related genes in FBS_8, we assessed its expression by co-staining with PDGFRα in the aorta. The Immunofluorescence staining result showed that Col8a1+PDGFRα+ cells were located in the perivascular area and the expression of Col8a1 increased in Ang II treated-TKO group (Figure 7L). Interestingly, we also found that Col8a1 increased in the aortic fibroblasts from TKO mice after overlapping transcripts between the fibroblast upregulated DEGs from the scRNA-seq dataset and the upregulated DEGs from the isolated fibroblast bulk RNA-seq dataset (Figure 7M). Taken together, scRNA-seq of aortas from Cre and TKO mice after Ang II treatment revealed fibroblast heterogeneity with activated signatures most prominent among FBS_8, and induction of pathways associated with robust ECM and perivascular fibrosis.

Neutralization of Endogenous IL-9 Reversed the Ang II-Induced Perivascular Fibrosis and Ameliorated Injury of Hypertension-Related Organs

Considering the crucial profibrotic role of IL-9 in Ang II-treated perivascular fibrosis and the elevated plasma levels observed in the TKO mice, we hypothesized that perivascular fibrosis might be rescued by administration of anti-IL-9 neutralizing monoclonal antibodies (mAbs) in Ang II-treated TKO mice (Figure 8A). TKO mice treated with anti-IL9 mAbs showed improved LV global longitudinal strain (Figure 8B, Table S12), decreased PWV, and increased Circ Strain (Figure 8C) compared with the IgG treated Ang II group, while no difference was observed in PBS groups. In addition, the ratio of albumin/creatinine and the value of KIM-1 decreased after treatment with IL-9 mAb in Ang II group (Figure 8D). Furthermore, histopathology analyses revealed significantly decreased perivascular fibrosis in the aorta, heart, and kidney in the anti-IL-9 mAb treatment group, and reduced aortic adventitial collagen was also observed in the anti-IL-9 mAb-treated Ang II group compared with the IgG-treated Ang II group (Figure 8E and 8F and Figure S13A through S13D). Moreover, the increased calcium deposition in the perivascular adventitia was reversed by treatment with anti-IL-9 mAb in Ang II-treated TKO mice (Figure S13E).

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To further understand the transcriptomic changes associated with the rescue of perivascular fibrosis with anti-IL-9 mAbs, RNA-seq differential expression analysis was performed from the anti-IL9 mAbs versus IgG groups in nonstripped aortas from TKO mice after Ang II treatment (Figure S13F through S13I). By analyzing the overlap between upregulated genes in nonstripped aortas from Ang II-treated TKO versus Cre mice with the downregulated transcripts in the anti-IL-9 mAb-treated TKO aortas, we identified potential genes involved with reversal of perivascular fibrosis, including Alox15 and Hp (Figure S12J).^47–49 We also compared the significant downregulated transcripts from the nonstripped aortic anti-IL-9 mAb treatment with the significant upregulated transcripts from aortic fibroblasts of Ang II treated TKO versus Cre mice to identity transcripts uniquely changed in fibroblasts after anti-IL-9 mAb rescue (Figure 8G). Indeed, the profibrotic genes Col8a1, Mmp2, Fmod, and Angptl1, which were identified as significantly increased in TKO aortas and in isolated fibroblasts by both bulk RNA seq and scRNA seq (Figure 7M), were all decreased after treatment with anti-IL-9 mAbs (Figure 8G). In addition, these genes were observed as overlapping from the upregulated genes from scRNA-seq with the downregulated genes from anti-IL-9 mAb RNA-seq dataset (Figure 8H).

To further determine if anti-IL-9 mAbs could provide therapeutic benefit outside of the context of genetically modified mice, we treated C57BL/6 mice with Ang II and with or without anti-IL-9 mAbs. Treatment with anti-IL9 mAbs showed decreased PWV and increased Circ Strain compared with the IgG treated control group (Figure 8I). Furthermore, histopathology analyses revealed significantly decreased perivascular fibrosis and reduced aortic adventitial collagen in the aorta in the anti-IL-9 mAb treatment group compared with the IgG control group (Figure 8J and 8K).

Collectively, neutralization of endogenous IL-9 ameliorated the Ang II-induced dysfunction of hypertension-related organs and reversed perivascular fibrosis not only in TKO mice by suppressing the expression of profibrotic genes but also in C57BL/6 mice.

Discussion

Accumulating studies reveal the critical role of T cells not only in hypertension and hypertensive cardiovascular injury but also in interstitial and perivascular fibrosis.^{6,10,11,19,50–53} A variety of cell surface receptors, enzymes, and TFs associated with T cells have been implicated in the development of hypertension and target organ damage.^11,21,23 However, the molecular events in T cells specifically driving perivascular fibrosis in response to Ang II remain poorly understood. In this study, we found that Ang II induced Klf10 expression in CD4+ T cells in vitro and in vivo, suggesting the potential involvement of KLF10 in Ang II-mediated end organ damage and possibly fibrosis. We found that Ang II-treated TKO mice developed adverse cardiac remodeling reflected by impaired global longitudinal strain,⁵⁴ worse arterial stiffness (evaluated by PWV and Circ Strain),⁵⁵ and more kidney injury (evaluated by the levels of albuminuria and KIM-1 expression).²³ Notably, the TKO mice exhibited marked perivascular fibrosis in hearts, aortas, and kidneys. However, we found that those functional and histopathologic changes were independent of blood pressure. These findings build upon a broader role for KLF10 in CD4+ T cells in a range of chronic inflammatory disease states including atherosclerosis, insulin resistance, and fatty liver disease.^12,13

A prior report considered that hypertension, vascular remodeling, and subsequent end-organ damage not only can be induced by mechanical forces but also can result from other forms of mediators independent of arterial pressure,⁵⁶ which suggests that blood pressure alone is not sufficient to predict end organ damage in hypertension. Indeed, abnormalities in aortic stiffness and PWV are typically associated with hypertensive subjects well before phenotypic manifestations of cardiac hypertrophy or other end-organ damage. Furthermore, more accurate clinical diagnostics are needed to detect early end organ damage in hypertensive patients.⁵⁷ Surprisingly, we did not find any differences in cardiac hypertrophy or interstitial fibrosis between Ang II-induced TKO and Cre control mice. Considering cardiac hypertrophy results from sustained pressure overload or heart injury,⁵⁸ the lack of difference in blood pressure is consistent with the findings of similar extent of cardiac hypertrophy between TKO and Cre mice. The lack of difference in blood pressure between TKO and Cre also allowed us to leverage novel molecular insights into perivascular fibrosis.

Consistent with a prior finding,¹¹ Ang II treatment increased the expression of the profibrotic cytokine IL-9 in both groups. Surprisingly, IL-9 was the only cytokine detected much higher in the TKO mice after Ang II treatment compared with Cre mice, which suggested that IL-9 played a crucial role in this perivascular fibrosis phenotype. IL-9 was reported in the regulation of immune responses and involved in the pathogenesis of various inflammatory diseases,⁵⁹ including a profibrotic role in lung inflammation and fibrosis,⁶⁰ liver fibrosis,⁶¹ and kidney fibrosis.⁶² In cardiovascular disease, IL-9 has been implicated in the pathogenesis of atherosclerosis (exerting proatherosclerotic effects)⁶³ and in heart failure (aggravating isoproterenol-induced heart failure).⁶⁴ To confirm the profibrotic role for IL-9 in Ang II-induced perivascular fibrosis, we systemically administrated recombinant murine IL-9 in Ang II-treated Cre mice and found that it increased perivascular fibrosis and induced dysfunction in heart, kidney, and aortas, which effectively phenocopied that observed in Ang II-treated TKO mice. Importantly, both the end organ injury and perivascular fibrosis were rescued by administration of anti-IL-9 neutralizing monoclonal antibodies in Ang II-treated TKO mice. Future studies evaluating the kinetics of IL-9 in plasma, CD4+ T cells, or PBMCs of hypertensive human subjects with increasing severity of end organ injury will be informative. Taken together, blocking IL-9 may be an attractive novel therapeutic strategy for treatment of perivascular fibrosis or hypertensive-associated end organ damage.

The main cellular sources of IL-9 are T cell subsets, including Th2, Treg, and from recently identified Th9 cells.⁵⁹ Consistently, flow cytometry also found that the percentage of CD4+IL9+ and CD4+IL4+ T cells increased in the aortic tissue and spleen in Ang II-infused TKO mice compared with Cre mice, while there were no differences in CD4+IL17+, CD4+FoxP3+, or CD4+ IFN-γ+ T cells. Although the percentage of CD4+IL4+ cells increased in TKO mice, there were no differences of released IL-13, IL-5, or IL-4 in both male and female TKO mice compared with Cre controls. In addition, using flow cytometry, we found there was no difference in the percentage of CD8+IL-9+ cells between Ang II treated Cre and TKO mice in both aorta and spleen. Furthermore, we found increased transcript and protein expression of IL-9 in both isolated CD4+ T cells from Ang II-infused TKO mice in vivo and Ang II-treated primary TKO CD4+ T cells in vitro, which suggested that KLF10 may directly regulate IL-9 expression in CD4+ T cells. Unlike many other TFs, KLF10 is primarily known to repress transcription of targeted genes. For example, KLF10 can negatively regulate cardiac MCP-1 expression by blinding to the MCP-1 promoter with HDAC1.²⁴ Moreover, KLF10 can reduce acetylated histone H4 on the C/EBPα promoter and inactivate C/EBPα transcription.⁶⁵ In our study, we found that KLF10 can bind to the IL-9 promoter and interact with HDAC1 to inhibit Il9 transcription. These findings suggest that the Ang II-mediated increase in KLF10 expression in CD4+ T cells may serve a protective, counter-regulatory mechanism to limit perivascular fibrosis. However, this increase is likely not sufficient to inhibit IL-9 release completely and can still trigger perivascular fibrosis. As a pro-fibrotic cytokine, IL-9 could also be regulated by multiple factors. As a tonic repressor of IL-9, KLF10 once deleted in CD4+ T cells triggers more IL-9 production and subsequently perivascular fibrosis. Further exploration of the interaction between KLF10, IL-9, and the promoters of other relevant targets may provide insights for the role of KLF10 more broadly in health and disease.

Ang II treatment, as a pathological stress, increases intracellular calcium to stimulate fibroblasts to differentiate into myofibroblasts, which can further induce fibrosis by secreted ECM proteins, MMPs, and others.⁶⁶ In our study, we found that fibrosis related signaling, ECM-related pathways, and calcium signaling were particularly upregulated in Ang II-treated TKO mice compared with Cre control mice by RNA-seq. Given that calcium homeostasis is important in pulmonary fibroblasts⁶⁷ and cardiac fibroblasts,^28,66 we isolated aortic fibroblasts and found myofibroblast markers, fibroblast activation signature genes, and calcium signaling genes to be highly expressed from Ang II-treated TKO fibroblasts by RNA-seq. In particular, we show for the first time that IL-9 can also stimulate increased calcium flux and fibrosis phenotypes in isolated primary fibroblasts under basal and Ang II treatment. Thus, the IL-9 mediated perivascular fibrosis may be dependent in part on hyperactivation of calcium signaling in fibroblasts.

Fibroblasts are important cells involved in the maintenance of tissue integrity and tissue repair in response to injury.⁶⁸ They can differentiate into different phenotypes including an ECM-producing contractile phenotype that further contributes to the secretion and accumulation of ECM and MMPs leading to the progression of fibrosis in a range of fibrotic diseases. An appreciation of fibroblast heterogeneity within disease-specific conditions developed only recently with the advent of newly developed approaches using single-cell transcriptome analysis. By using scRNA-seq, TKO aortic fibroblasts subsets exhibited higher activation markers including Col1a1, Col8a1, and Col1a2. Furthermore, 9 identified subpopulations of fibroblasts displayed different aspects of fibroblast activation in TKO aortas, which demonstrated their relevance to perivascular fibrosis. In addition, in the Ang II-treated TKO aortas, the percentage of fibroblasts increased and the number and the percentages of specific FBS clusters were more abundant especially FBS_8. FBS_8 shows high expression of collagen-related genes including: Cthrc1, a potential marker for activated fibroblasts in the heart and lungs^44,69,70; Tnc, a marker involved in stimulating collagen-related gene expression, myofibroblast transformation,⁷¹ and perivascular inflammation and fibrosis⁷²; and Ddah1, a marker related to perivascular or adventitial fibrosis and vascular remodeling.⁷³ Using RNA velocity analyses, FBS_8 represented one of the most advanced fibroblast clusters, while FBS_2 appeared as an earlier state reflecting less activation. Indeed, our results showed that FBS_8 is highly involved in the ECM and structure organization pathway and highly enriched for fibrosis-related genes, whereas FBS_2 was enriched for most of the canonical fibroblast genes. Col8a1 was found to be a unique gene in FBS_8, and Col8a1+ PDGFRα+ cells were located in the perivascular area and the expression of Col8a1 increased in the Ang II treated-TKO group. Thus, we consider the FBS_8 subcluster as the key fibroblast subcluster contributing to the perivascular fibrosis in the Ang II treated-TKO group. In addition, the majority of the other increased genes in the TKO group are known to contribute to the development of fibrosis,^42,74–78 including Col1a1, Mmp2, Angptl1, Comp, Fmod, and Acta2—all decreased after administration of anti-IL-9 mAbs. Collectively, this anti-IL-9 therapeutic strategy demonstrated reduced end-organ injury and perivascular fibrosis in part by downregulating these fibrosis-related gene signatures.

In summary, KLF10 is upregulated in PBMCs of hypertensive patients and in peripheral CD3+ T cells and CD4+ T cells in Ang II-treated mice. Administration of Ang II in TKO mice triggered perivascular fibrosis, multiorgan dysfunction in heart, kidney, and aorta, and release of IL-9 from CD4+ T cells. These functional and histopathologic differences were independent of blood pressure between Ang II-treated TKO and Cre mice. Mechanistically, in response to Ang II treatment, KLF10 bound to the IL-9 promoter and interacted with HDAC1 to inhibit Il9 transcription. Ectopic IL-9-activated calcium flux, induced fibroblast activation and differentiation, increased production of collagen and ECM, thereby promoting the progression of perivascular fibrosis and inducing target organ dysfunction. Importantly, IL-9 neutralizing antibodies potently rescued perivascular fibrosis, and target organ dysfunction in Ang II-treated TKO and C57BL/6 mice. RNA-seq and scRNA-seq revealed the presence of fibroblast heterogeneity and marked myofibroblast activation in nonstripped aortas from Ang II-treated TKO mice. These results indicate that the KLF10-IL-9 signaling axis in CD4+ T cells tightly regulate the processes of Ang II-induced pathological perivascular fibrosis and end organ damage and provide novel therapeutic opportunities for the treatment of hypertensive-associated disease.

Article Information

Acknowledgments

We thank Dr Lay-Hong Ang and Aniket P. Gad for their help with immunofluorescence imaging (Harvard Digestive Disease Center, NIH P30DK034854), and Dr Sudeshna Fisch (Department of Medicine, Brigham and Women’s Hospital Harvard Medical School) for assistance with echocardiography.

Supplemental Material

Expanded Methods

Table S1–S12

Figures S1–S13 and Figure legends

Video S1

Major Resources Table

References 11–13,23,30,46,63,79–91

Footnotes