Caveolin-1 restrains pathogenic T follicular helper cell response in primary Sjögren’s syndrome

Caveolin-1 restrains pathogenic T follicular helper cell response in primary Sjögren’s syndrome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Caveolin-1 restrains pathogenic T follicular helper cell response in primary Sjögren’s syndrome Xiang Lin, Sulan Yu, Meiling Wu, Weizhen Zeng, Weiwei Fu, Yacun Chen, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3230861/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract T follicular helper (Tfh) cells play a central role in humoral autoimmunity, including primary Sjögren’s syndrome (pSS). However, targeting Tfh cells is challenging in clinical management. Previous studies suggested inducible T­cell co­stimulator (ICOS) directed Tfh cell motility in engaging bystander B cells. Here, we identified a novel function of caveolin-1 (Cav-1) in restraining Tfh cell motility, in which Icos transcription was repressed by peroxisome proliferator-activated receptor alpha (PPARα), unexpectedly, independence of lipid metabolism. In the context of autoimmunity, Cav-1 and PPARα expressions were decreased in CD4+ T cells from pSS patients and mice with experimental SS (ESS), while Cav-1 deficiency significantly exacerbated Tfh cell response and ESS pathology. Importantly, pharmaceutical activation of PPARα with fenofibrate effectively ameliorated ESS in mice with acute or chronic inflammation. These results revealed an unrecognized role of Cav-1/PPARα axis in Tfh cell tolerance, suggesting PPARα as a promising target in the treatment of humoral autoimmunity. Health sciences/Rheumatology/Rheumatic diseases/Connective tissue diseases/Sjögren's disease Biological sciences/Immunology/Autoimmunity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights - Caveolin-1 deficiency exacerbates ESS development in mice - Caveolin-1/PPARα axis inhibits Tfh cell response - PPARα represses Icos transcription in Tfh cells independence of lipid metabolism - Pharmaceutical activation of PPARα suppresses Tfh response in ESS and humanized mice Introduction Caveolin-1 (Cav-1), a scaffold protein involved in assembling caveolae components and lipid domains, is structurally conserved in mammalian cells 1 . Early studies showed that mice deficient for Cav-1 were vial, but resistant to diet-induced obesity 2 , rendering its close involvement in diabetic development in both humans and mice 3 . Our recent studies have identified the versatile functions of Cav-1 in cell fate decisions, which critically regulated the nitric oxide-mediated matrix metalloproteinases activity 4 , vascular endothelial growth factor 5 , 6 and integrin expression 7 pathways in neuronal differentiation, neuronal regeneration, hepatocyte and endothelial cell responses. Consequently, these findings further extend the important features of Cav-1 in the pathogenesis of multiple sclerosis 7 , ischemia/reperfusion injuries in stroke 4 , 8 and liver diseases 9 , 10 , respectively. However, the role of Cav-1 in autoimmune pathogenesis remains largely unclear. Primary Sjögren’s syndrome (pSS) is a common autoimmune disease characterized by exocrinopathy involving the lacrimal and salivary glands (SG), leading to the clinical manifestations of severe dry-eye and dry-mouth symptoms 11 . Highly activated CD4 + T cell signature, including Th17 and T follicular helper (Tfh) cells, has been reported both in the inflammatory SG and peripheral blood of pSS patients 12 . We previously established a mouse model of experimental Sjögren’s syndrome (ESS), which recapitulated the key features of human pSS 13 . Tfh cells were found to play critical roles in pSS development, as revealed by the findings that mice deficient for Tfh cells exhibit markedly attenuated autoantibody production and disease pathology upon ESS induction 14 . Tfh cells are a subset of CD4 + T cells that provide B-cell help during germinal center (GC) reactions and promote plasma cell differentiation in adaptive immune response 15 , 16 . Tfh cells are characterized by the phenotypes of transcription factor Bcl-6 and surface markers of PD-1, ICOS and CXCR5, among which mostly drive and stabilize Tfh cell positioning in the B cell follicles 17 . Through CD40 and cytokine productions (i.e. IL-21) 18 , Tfh cells facilitate high-affinity B cell selection and antibody production. Thus, stringent control of Tfh cells is critical for maintaining immune tolerance and optimal humoral response 16 . Although impaired regulatory function of certain immune cell subsets may contribute to the hyperactive Tfh cell response 19 , the checkpoints that maintain Tfh cell tolerance were not fully understood. Although the knowledge of Cav-1 in immune response is limited, emerging studies have reported its involvement in fine-tuning TCR and BCR signaling. Cav-1-/- B cells exhibited reduced T cell-independent humoral immune response, but had normal response to T cell-dependent antigens 20 , 21 . This is coincided with the findings that Cav-1 deficiency did not affect CD4 + T cell expansion upon viral infection 22 . Nonetheless, it is not known whether and how Cav-1 could regulate effector CD4 + T cell subsets in pSS pathogenesis. In this study, we demonstrated that Cav-1 deficiency did not attenuate, but rather exacerbated ESS pathology and Tfh cell responses in mice. Cav-1-/- CD4 + T cells highly expressed ICOS and thus exhibited increased motility toward B cell follicles. Mechanistic studies revealed that peroxisome proliferator-activated receptor alpha (PPARα) served as a potent transcription repressor of ICOS during Tfh polarization, independent of lipid metabolism, while PPARα expression was markedly decreased in the absence of Cav-1. Consistently, Cav-1 ablation or PPARα antagonism enhanced human Tfh cell differentiation. In the context of autoimmunity, Cav-1 levels were positively correlated with PPARα, but negatively correlated with ICOS expression in the circulating CD4 + T cells from ESS mice and pSS patients. By contrast, fenofibrate, an FDA-approved PPARα agonist, effectively suppressed human Tfh cells both in vitro and in vivo . This pharmaceutical activation of PPARα significantly ameliorated the disease pathology in established ESS mice with acute or chronic inflammation. Thus, our work has revealed an unrecognized role of Cav-1/PPARα axis in Tfh cell dysregulation and pSS pathogenesis, while targeting PPARα may serve as a promising approach in treating Tfh cell dysregulation in autoimmunity. Results Caveolin-1 deficiency exacerbates ESS pathology. Although previous studies suggested ameliorated EAE pathology in Cav-1-/- mice 7 , unexpectedly, Cav-1 deficiency significantly exacerbated ESS development in mice upon disease induction, as revealed by accelerated salivary hypofunction and higher titer of autoantibodies compared with wildtype (WT) ESS mice (Fig. 1 A-B). This was associated with enhanced IgG deposition on the salivary epithelium (Fig. 1 C). In long-term observation, at acute-chronic stage, massive lymphocytic foci, together with much severe tissue damage were observed in Cav-1-/- ESS mice, whereas age-matched WT mice exhibited only mild lymphocytic infiltration along ESS progression. Confocal imaging analyses also indicated the presence of Bcl-6 + CD4 + Tfh cells and GC-like B cells within the infiltrating aggregates, suggesting ectopic lymphoid structure formation in the inflamed SG tissues from Cav-1-/- ESS mice (Fig. 1 D). Quantitatively, Cav-1-/- ESS mice exhibited higher histological scores, larger infiltrating area (Fig. 1 E), and increased apoptotic epithelial cell counts when compared with WT mice (Fig. 1 F). Thus, Cav-1 deficiency promoted ESS development in mice. Given the varied abundance of Cav-1 proteins in lymphoid and non-lymphoid organs 23 , it is not clear whether Cav-1 expression in hematopoietic origin would be responsible for the ESS pathogenesis. Thus, we generated chimera mice with Cav-1 deficiency in the immune cells by bone marrow transfer, while the genotype in the lymphoid organ was validated upon mouse sacrifice (Fig. 1 G). Consistently, WT recipients reconstructed with Cav-1-/- bone marrows showed higher autoantibody levels (Fig. 1 H) and histopathological changes (Fig. 1 I-J), which was comparable with those mice of global Cav-1 deletion. Together, these results suggest the importance of Cav-1 in regulating immune responses during ESS development. Caveolin-1 constrains ICOS expression and follicular migration of Tfh cells. Previous studies, including our findings, identified a central role of Th17 cells in both EAE and ESS pathogenesis 7 , 13 , 24 . However, phenotypic analyses showed that Cav-1 deficiency did not increase Th17 cell frequencies and counts upon ESS development (Fig. 2 A). This was consistent with previous findings in Cav-1-/- EAE mice 7 , 25 . Instead, we detected significantly increased Tfh cells in Cav-1-/- ESS mice, which was associated with their follicular displacement in the germinal centers (Fig. 2 A, Fig. S1 A). This may explain the expanded GC areas and GC B cells (Fig. S1 A-B). Thus, we sought to determine Cav-1 expression levels in CD4 + T cells during ESS development. Upon ESS induction, we observed a transient increase of Cav-1 in CD4 + T cells at disease onset, followed by persistent downregulation during ESS progression (Fig. 2 B). This was negatively correlated with increased Tfh cell responses 19 . Since Cav-1-/- B cells could respond to T cell-dependent antigens 20 , we next investigated whether Cav-1 deficiency in CD4 + T cells would be sufficient to mount humoral dysregulation. WT or Cav-1-/- CD4 + T cells were co-transferred with WT B cells into SG-antigen immunized immunodeficient NOD- scid IL2Rg null (NSG) mice. Notably, Cav-1 deficiency in CD4 + T cells markedly promoted autoreactive B cell response (Fig. 2 C-D), resulting in elevated autoantibodies production and IgG deposition on the salivary epithelium (Fig. 2 E, Fig. S1 C). These data suggest that Cav-1 deficiency in CD4 + T cells might promote Tfh cell response in vivo . To validate this finding, WT or Cav-1-/- CD4 + T cells were purified and cultured for Tfh polarization. Indeed, Cav-1-/- CD4 + T cells exhibited higher capacity of Tfh cell differentiation (Fig. 2 F). A previous structural biology study implicates a central role of scaffolding domain of Cav-1 (CSD) as a docking site in signaling transduction 26 . Cavtratin, a cell-permeable peptide of CSD, binds to caveolin-binding motifs 27 . Herein, it dose-dependently restrained Cav-1-/- CD4 + T cell response to Tfh polarization (Fig. 2 F). Collectively, these data indicate the role of endogenous Cav-1 in regulating Tfh cell differentiation. We next determined the featured molecules in Cav-1-/- Tfh cells, in which PD-1 and ICOS, but not CXCR5 expressions were found markedly increased (Fig. S1 D). However, we did not observe the elevation of CD40 ligand and representative cytokine productions, in particular IL-17 and IL-21 in Cav-1-/- CD4 + T cells under the polarization conditions (Fig. S1 E). Moreover, Cav-1 deficiency-mediated Tfh cell response was also found stable in long term, as revealed by flow cytometric analysis of BrdU-incorporated WT or Cav-1-/- donor CD4 + T cells in the recipient ESS mice over 10 weeks post adoptive transfer (Fig. S1 F). ICOS-ICOSL plays an indispensable role of in Tfh cell motility 28 . Notably, flow cytometric analysis showed comparable ICOSL levels between WT and Cav-1-/- B cells from ESS mice (Fig. S1 G). Thus, we hypothesized that increased ICOS expression in Cav-1-/- CD4 + T cells might largely strengthen their follicular migration. Consistent with the previous finding 28 , anti-ICOS activating antibody rapidly induced WT Tfh cell polarization, as revealed by pseudopod protrusion and persistent movement (Fig. 2 G-H). Quantitatively, Cav-1-/- Tfh cells showed higher shape index with augmented pseudopod extension (Fig. 2 G) upon ICOS ligation, and thus resulted in increased centroid speed (Fig. 2 I) and cell displacement in random directions (Fig. 2 H, Fig. S1 H). This was validated in ICOSL-mediated transwell assay in response to CXCL13 (Fig. S1 I). Consistently, ICOS expression was significantly increased in CD4 + T cells obtained from draining cervical lymph nodes of Cav-1-/- ESS mice, accompanied with enhanced ICOS + CD4 + T cells present in the lymphocytic foci of SG (Fig. S1 J-K). We further testified Cav-1-/- CD4 + T cell motility in vivo , in particular those at T-B borders, by two-photon intravital imaging analysis. In this adoptive transfer model, CD4 + T cell zone could be clearly distinguished in the spleen at 72h post transfer (Fig. 2 J). Consistent with the findings in vitro , Cav-1-/-CD4 + T cells exhibited more polarized status, as reflected by less sphericity in morphology (Fig. 2 K). By analyzing the cell tracks, we observed markedly increased motility of Cav-1 deficient CD4 + T cells in situ (Fig. 2 K), as reflected by enhanced cell displacement (Fig. S1 L) and velocity (Fig. 2 M) by quantification, in particular those at the T-B borders (Supplementary Video 1–2). Since the follicular homing of CD4 + T cells, as the consequence of Tfh cell motility, was defined as the fundamental feature to mount B-cell response in vivo , we next evaluated their follicular homing capacity in the draining cervical lymph nodes under the identical conditions. CFSE-labelled WT and Qtracker-labeled Cav-1-/- naïve CD4 + T cells were co-transferred into WT ESS mice with active disease, while the draining cervical lymph nodes were analyzed after 72h. As expected, Cav-1-/- CD4 + T cells exhibited overt follicular displacement when compared with those WT counterparts (Fig. 2 N). Together, these results demonstrated that Cav-1 critically restrained Tfh cell migratory capacity toward B cell follicles. To further assess the B-cell help functions other than T cell motility, we sorting-purified WT or Cav-1-/- Tfh cells from ESS mice, and co-cultured with cognate WT B cells for 72h. Interestingly, Cav-1 deficiency did not obviously affect the effector molecules of T-B interactions, as revealed by comparable plasmacytic differentiation and autoantibody productions (Fig. 2 O). This was in line with the findings of CD40 ligand and cytokines above mentioned. Together, our data suggest that increased ICOS expression in Cav-1-/- CD4 + T cells were responsible for enhanced humoral autoimmunity and ESS pathology. To validate this notion, we performed anti-ICOS blocking antibodies treatment in Cav-1-/- mice upon ESS induction. The efficacy was validated by reduced intrafollicular ICOS + CD4 + T cell counts, which was associated with similar Tfh cell numbers and follicular homing coefficient to those in WT ESS mice (Fig. S2 A-C). Consequently, the reduced Tfh cell response upon ICOS blockage led to decreased plasma cell numbers and anti-SSA IgG levels in Cav-1-/- ESS mice (Fig. S2 D-E). Therefore, these data indicate a critical role of Cav-1 in ICOS expression and Tfh cell response. Impaired PPAR α expression in CD4 + T cells contributes to caveolin-1-mediated Tfh cell response. Cav-1 is mainly distributed at the plasma membrane and cytoplasm, but not in the nucleus in endothelial cells and B cells 21 , 26 , Similarly, this was also observed in CD4 + T cells (Fig. S3 A-B). Thus, we reasonably addressed the question that enhanced Icos transcription in CD4 + T cells could be indirectly regulated by Cav-1. We first performed RNA-seq analysis of WT and Cav-1-/- Tfh cells for transcriptome comparison. Notably, peroxisome proliferator-activated receptors (PPARs) signaling pathway was significantly affected by the absence of Cav-1 (Fig. 3 A). PPARs are a family of transcription factors including PPARα, PPARβ/δ and PPARγ 29 . Real time-PCR analysis revealed that Cav-1 deficiency mainly affected the transcription of PPARα, while PPARγ and PPARδ were comparable with WT Tfh cells (Fig. 3 B). Indeed, significantly reduced protein levels of PPARα were also found in Cav-1-/- CD4 + T cells (Fig. 3 C). This was similar to the recent findings in hepatocytes 30 . To investigate whether impaired PPARα expression contributes to Tfh cell response, we first performed genetic ablation of PPARα in CD4 + T cells by CRISPR/Cas9. Interestingly, PPARα-/- CD4 + T cells phenocopied Cav-1 deficiency and showed significantly augmented Tfh cell differentiation, regardless of cavtratin treatment (Fig. 3 D), suggesting that PPARα served as the downstream of Cav-1 in CD4 + T cells. This could be also achieved by the treatment of GW6471, a selective PPARα antagonist, which markedly promoted Tfh cell differentiation in dose dependent manner (Fig. 3 E). Functional studies using transwell assay further validated enhanced ICOS-mediated T cell motility upon PPARα antagonism (Fig. 3 F). Given an intense search for PPARs ligands in the past decades, selective agonists were reported to activate differential PPAR members 31 . Thus, based on the binding affinity, 8-hydroxyeicosapentaenoic (8-HEPE), 15-deoxy-D 12 , 14 -prostaglandin J2 (15-Deoxy) and GW0742 32 were used to differentially activate PPARα, PPARβ/δ and PPARγ respectively. In contrast to PPARα deficiency, PPARα agonist effectively suppressed Tfh cell differentiation, while no difference was observed in the presence of 15-Deoxy and GW0742 (Fig. 3 G). To avoid the possible off-target effect, we adopted fenofibrate, the pharmaceutical agonist of both human and murine PPARα 33 , which dose dependently suppressed both WT and Cav-1-/- Tfh differentiation in culture (Fig. 3 H). Thus, these data suggest that PPARα would be responsible for Cav-1 downstream signal and serves as negative regulator of Tfh cell response. In the context of ESS development, similar to Cav-1 expression in CD4 + T cells, we also observed a transient increase of PPARα, but progressively decreased during disease development (Fig. 3 I-J), in particular at disease chronic stages (Fig. 3 K). However, PPARγ was not correlated with Cav-1 and PPARα expressions in CD4 + T cells (Fig. 3 I, Fig. S3 C-D). Consequently, treatment with PPARα antagonist GW6471 accelerated ESS development and Tfh cell responses (Fig. 3 L-M). Together, these results demonstrated the functional importance of Cav-1/PPARα axis in restraining Tfh cell response. PPAR α represses ICOS transcription in Tfh cells. A previous study suggested downregulation of lipid metabolic processes as a major consequence of Cav-1 deficiency, among which PPARα was responsible 30 . Indeed, we observed significantly lower levels of lipid droplets in Cav-1 deficient CD4 + T cells (Fig. 4 A), in particular in the effector population (Fig. S4 A). Thus, we sought to investigate whether lipid metabolism would be involved in Cav-1/PPARα axis-mediated ICOS expression in Tfh cells. We first monitored the transcriptional regulation of Icos during Tfh polarization. Notably, the mRNA levels of ICOS were significantly increased as early as 16h in Cav-1-/- CD4 + T cells, while protein levels at 48h upon Tfh differentiation (Fig. 4 B). Since lipid metabolism-mediated energy generation consisted of several rate-limiting steps 34 , in which fatty acid β-oxidation (FAO) produced acetyl-CoA and entered mitochondrial tricarboxylic acid (TCA) cycle, we next determined whether Icos transcription was altered during this process. We first measured the mitochondrial respiration from fatty acids by using palmitic acid (16:0, PA) as sole extracellular substrate 35 . After 48h Tfh polarization, as expected, Cav-1 deficiency significantly decreased the basal and maximal respiratory capacity (Fig. 4 C, Fig. S4 B). Accordingly, ACADM protein levels, the representative enzyme of FAO initiation, were found significantly decreased in the absence of Cav-1. Moreover, succinyl-CoA synthetase (SDH), and aconitase (ACO2), the key regulatory enzymes of TCA cycle, were also significantly decreased at 48h in Cav-1-/- CD4 + T cells (Fig. 4 D). This was associated with decreased cellular fatty acid content, as reflected by free fatty acid assay (Fig. 4 E). However, this was not seen at the early stages upon Tfh polarization. FAO and cellular fatty acid content were minimal or undetectable in both WT and Cav-/- CD4 + T cells at 16h in culture, while there was no obvious difference of enzyme expression levels. Together, these results validated that Cav-1 deficiency indeed impaired FAO process. However, the rapid Icos transcription in Cav-1-/- CD4 + T cells, prior to energy status transition, suggests its transcriptional regulation in relatively direct manner. To validate this notion, we next looked into the target genes of PPARα in CD4 + T cells, which were previously identified for fatty acid transportation and β-oxidation through carnitine palmitoyltransferase (CPT) system 34 . Cpt1a, a highly conserved PPARα target gene and metabolic regulator, catalyzes the long chain fatty acids from acyl-CoA to carnitine for translocation across the mitochondrial membranes 36 . Indeed, PPARα agonist significantly increased Cpt1a expression 37 . Thus, we first determined the expression levels of Cpt1a during Tfh differentiation. Consistent with the findings of the lipid metabolism above, Cpt1a expression was elevated in WT CD4 + T cells at 48h under Tfh differentiation, which was found much lower in the Cav-1-/- counterparts. However, there was no obvious difference of Cpt1a level at early phase upon Tfh polarization (Fig. 4 F). We next performed Cpt1a overexpression in CD4 + T cells to restore, at least in part, the PPARα deficiency-mediated lipid metabolism. Surprisingly, overexpression of Cpt1a did not restrain, but rather promoted Tfh cell differentiation in PPARα-/- CD4 + T cells (Fig. 4 G). Similar findings were also observed following irreversible Cpt1a antagonism by etomoxir, as the etomoxir treatment did not phenocopy PPARα or Cav-1 deficiency, but rather suppressed Tfh cell development (Fig. 4 H), which may attribute to globally constrained energy generation and requirement. This notion was further supported by fatty acid-free culture conditions, in which Cav-1-/- CD4 + T cell retained higher capacity of ICOS expression and Tfh differentiation (Fig. 4 I, Fig. S4 C). In this context, nutrient addition by BSA further gave rise to both WT and Cav-1-/- Tfh cell differentiation. Thus, these results demonstrate that impaired lipid metabolism in Cav-1-/- CD4 + T cells would not affect the increased Icos transcription under Tfh polarized conditions. Additionally, we also determined the glucose oxidation of WT and Cav-1-/- CD4 + T cells under Tfh polarization conditions. Using glucose as a substrate, we detected minimal oxygen consumption rates (OCR) at 16h, but markedly increased at 48h in both WT and Cav-1-/- CD4 + T (Fig. S4 D). Among the glucose oxidation cascade, the phospho-PFK2 to PFK2 ratio represents the glycolytic rate 38 , in which a higher ratio leads to increased gluconeogenesis 39 . Consistent with the OCR findings, using glucose as the sole extracellular source of carbon, the ratio of p-PFK2:PFK2 was found comparable in WT and Cav-1-/- CD4 + T cells during Tfh differentiation (Fig. S4 E), as well as the expressions of SDH and ACO2. These data suggest that glucose oxidation might not be involved in Cav-1-mediated Tfh response. PPARα is also recognized to limit inflammatory responses via transcriptional repression 29 . This is achieved by a conserved mechanism that PPARs could form heterodimer with 9-cis-retinoic acid receptor (RXR), which binds to peroxisome proliferator response element (PPRE) at the promoter region of target genes 40 . Indeed, in the transcriptomic screening analysis, both PPAR and RXR binding activities were found significantly reduced in Cav-1-/- CD4 + T cells (Fig. 4 J). Thus, we next assessed the binding capacity of PPARα to Icos promoter. ChIP-PCR analysis indicated that PPARα rapidly bound to Icos promoter region 12h upon Tfh polarization, an effect could be largely abrogated by selective antagonist (Fig. 4 K). This was further supported by interfering the intranuclear translocation. Time series imaging showed that intranuclear translocation of PPARα initiated at 30 min and persisted for hours (Fig. 4 L). Early studies have suggested a role of COX-1 in the nuclear translocation of PPARs 41 , 42 . We also validated this finding in CD4 + T cells, as SC-560, a selective COX-1 inhibitor effectively retained PPARα in the cytoplasm (Fig. 4 M). Importantly, PPARα agonist-mediated Icos trans-repression was abolished by SC-560 (Fig. 4 N). Conversely, selective RXR antagonist HX531 augmented, while RXR agonist LG100754 43 inhibited ICOS and Tfh cell differentiation in vitro (Fig. 4 O, Fig. S4 F). Thus, these data suggest that PPARα could serve as a repressor of Icos transcription. Cav-1/PPARα axis critically regulates human Tfh cells. We next sought to investigate whether Cav-1/PPARα axis also operates in human subjects. Purified CD4 + T cells from healthy donors were transduced with GFP-incorporated plasmids for Cav-1 deletion. Similar to the murine system, Cav-1-/- hCD4 + T cells also exhibited strong capacity towards Tfh differentiation, as well as increased ICOS expression (Fig. 5 A-B). In addition, PPARα expression levels were also significantly decreased upon Cav-1 deficiency (Fig. 5 C). Thus, we evaluated the effects of fenofibrate in suppressing Tfh cell responses in dose dependent manner, which yielded an IC50 of 7.024 µM (Fig. 5 D). Upon RNA-seq analysis, fenofibrate significantly reduced Icos transcript copies at 16h post stimulation (Fig. 5 E), which strongly supported our findings in mice. Consistently, human PPARα also rapidly bound to Icos promoter region under Tfh polarization, revealed by ChIP-PCR analysis, which was largely abolished upon PPARα antagonism by GW6471 (Fig. 5 F). Consequently, blockage of intranuclear entrance of PPARα by SC-560 prevented the transcriptional repression of Icos (Fig. 5 G). Functional assay further verified the ICOS-mediated migration could be inhibited by fenofibrate (Fig. 5 H). To validate this phenotype under disease conditions, we analyzed the circulating Tfh (cTfh) cells from pSS patients. By measuring the protein expression of each patient, we first detected strongly positive correlation between PPARα and Cav-1 in CD4 + T cells (Fig. 5 I). Interestingly, patients with higher frequencies of cTfh cells exhibited relatively lower levels of Cav-1 and PPARα in CD4 + T cells, rendering a negative correlation between Cav-1 with ICOS expression (Fig. 5 J-K). Thus, these results demonstrate Cav-1/PPARα axis as a negative regulator in human Tfh cells, while targeting PPARα may be a promising approach in treating Tfh cell dysregulation. Pharmaceutical activation of PPARα ameliorates ESS development Fenofibrate has a similar affinity for both murine and human PPARα with high efficacies 33 . We found that fenofibrate had a half maximal inhibitory concentration of 8.39 µM in suppressing murine Tfh cell differentiation (Fig. S5 ). This prompted us to explore the therapeutic potential of this pharmaceutical PPARα agonist on mice with established ESS. Previous studies reported that oral administration of 100 mg/kg body weight fenofibrate was sufficient to induce global PPARα activation and downstream signaling pathways in vivo 44 , 45 . Thus, we first treated ESS mice with fenofibrate at disease onset (Fig. 6 A), as diagnosed by reduced saliva secretion and elevated autoantibodies. Fenofibrate effectively ameliorated salivary hypofunction and decreased serum levels of anti-SSA IgG, although anti-SSA IgM levels were not altered (Fig. 6 B-C). We next sought to determine the therapeutic potential of fenofibrate treatment at disease chronic stages. As revealed by mild lymphocytic infiltrations in the SG, ESS mice immunized for 20wk were treated with fenofibrate for 10wk. Notably, histopathological findings showed significantly reduced tissue damages and inflammation in SG of the fenofibrate-treated ESS mice, as evidenced by diminished lymphocytic infiltration and few apoptotic epithelial cells (Fig. 6 D). Phenotypic analyses further showed that fenofibrate treatment significantly suppressed Tfh cell responses, in particular GC-Tfh cell counts in the lymphoid tissues (Fig. 6 E-G). This was consequently associated with restrained GC area, GC B cell and plasma cell counts (Fig. 6 G-H). A recent study reported a humanized mouse model by transplanting PBMCs in NSG mice 46 . Thus, we adopted this method by dividing PBMCs from each pSS patient into two groups for paired analysis, followed by PBS or fenofibrate treatments. Although NSG mice xenografted with PBMCs from pSS patients exhibited higher frequencies of Tfh cells than those from healthy donors, fenofibrate administration effectively suppressed human Tfh cells in matched control mice (Fig. 6 I). Together, these results provide strong evidence to support the notion that targeting PPARα could be a promising therapeutic approach in pSS and related autoimmune disorders with dysregulated Tfh cell responses. Discussion Tfh cells play a central role in the pathogenesis of humoral autoimmunity 47 . However, the mechanism underlying the Tfh cell-intrinsic tolerance was not fully understood. In this study, we for the first time showed that Cav-1/PPARα axis negatively regulated Tfh cell migration capacity, in which PPARα rapidly bound to the promoter region of Icos upon Tfh polarization. This was further exemplified by the Cav-1 deletion and PPARα activation in human CD4 + T cells. In the context of autoimmunity, negative correlation between Cav-1 and ICOS in CD4 + T cells was observed in pSS patients. Importantly, pharmaceutical activation of PPARα effectively ameliorated the disease pathology of both ESS mice at acute and chronic stages. This study provided a previously unrecognized role of Cav-1/PPARα axis in Tfh cell response, and suggested the therapeutic potential of targeting PPARα in treating autoimmune patient cohorts with Tfh cell dysregulation. Currently, the investigation regarding how Cav-1 modulates Th cell response is limited. Early study reported that Cav-1 was dispensable in T cell development 22 . Although Cav-1 could optimize TCR-induced membrane raft polarity in CD8 + T cells, as supported by impaired IFN-gamma and NFAT-dependent transcription in Cav-1-/- CD8 + T cells, the TCR-mediated expansion and cytokine production was comparable between WT and Cav-1-/- CD4 + T cells 22 , suggesting the differential function of Cav-1 in T cell subsets. Cav-1 was reported to modulate regulatory T (Treg) cell response under differential conditions. At steady state, WT and Cav-1-/- mice exhibited comparable Treg cells. However, in an adoptive transfer model of murine graft-versus-host disease (GVHD), Cav-1-/- donor CD4 + T cells elicited superior suppressive function than those WT compartments 48 , suggesting that endogenous deficiency of Cav-1 may sensitize the T cells to acquire Treg phenotype in the microenviroment shaped by GVHD. In the present study, we did not observe statistical differences of Treg cell frequencies between WT and Cav-1-/- upon ESS induction. In contrast to Treg cells, recent studies, including our findings reported that Th1 and Th17 cell counts were comparable between WT and Cav-1-/- mice with active EAE 7 , 25 , while the remission of EAE mainly owed to the altered tight junction remodeling in the blood-brain barrier, and thus decreased immune cell transmigration towards spinal cord. Notably, early studies demonstrated that transfer of myelin-specific Tfh cells did not induce EAE development 49 , which may explain the differential outcomes between EAE and ESS mice upon Cav-1 deficiency. Moreover, although NFAT was reported to orchestrate CD4 + T cell responses 17 , in our transcriptome screening, we did not detect obvious change of NFAT activity in Cav-1-/- CD4 + T cells, which was consistent with the previous study in Cav-1 deficient T cells 22 . Previous studies, including our findings, reported a variety of signaling molecules affected by Cav-1 50 , including lipid metabolism, nitric oxide 9 and vascular endothelial growth factor pathways 5 . In several studies, altered PPARs and FAO pathways from Gene Ontology were highlighted in both immune cells (i.e. macrophages 51 ) and tissue cells (i.e. hepatocytes 30 ). However, few RNA-seq analysis has been done in CD4 + T cells. In this study, we also observed differential expressed genes related to PPARs pathway and nitrogen metabolism between WT and Cav-1-/- Tfh cells. Extensive studies have reported PPAR isoforms as lipid sensors and regulators of lipid metabolism. Indeed, in the absence of Cav-1, we detected significantly decreased lipid droplet in effector T cells, impaired FAO cascade and reduced PPARα target gene Cpt1a was detected in CD4 + T cells. However, the lipid metabolism was a rate-limiting process during the relative long-term culture of T cell differentiation, and thus did not explain the rapid Icos transcription in Cav-1-/- CD4 + T cells at the initiation stage of Tfh polarization. This was further validated under lipid-free culture conditions, regardless of exogenous lipids supplementation. Another pivotal function of PPARs is the inhibition of inflammatory gene expression. Notably, the expression levels of PPARα and PPARγ were diverse in naïve and activated, among which PPARγ was mostly studied 29 . Ligand-mediated PPARγ activation could induce T cell apoptosis, inhibit T cell activation, and suppress Th1 cytokines 52 . By contrast, PPARα activation did not affect T cell fate, but selectively regulate the Th1 differentiation in indirect manner, showing higher IFN-γ but lower IL-2 transcription in CD4 + T cells deficient for PPARα 53 . In this study, we for the first time showed that PPARα could bind to Icos promoter for repression. Since the nuclear localization signal within the PPARα domain enables the cytoplasmic and nuclear shuttling 54 , we also showed that blockade of nuclear transcription of PPARα could effectively prevent ICOS repression. Together, our results suggested that Cav-1/PPARα axis served as a checkpoint in Tfh cell tolerance, rather than a metabolic regulator. Although altered Cav-1 expression levels have been reported in various diseases, including metabolic disorders, cerebrovascular disease, and cancer, however, investigation of the Cav-1 in autoimmune pathogenesis is scarce. Reduced Cav-1 expression has been reported in the skin tissues and dermal fibroblasts isolated from patients with systemic sclerosis 55 . In this study, we observed decreased Cav-1 expression in CD4 + T cells from both ESS mice and pSS patients, which was positively correlated with PPARα, but negatively correlated with ICOS expression. Although the relationship between Cav-1 and PPARα has been reported in other cell types, including cancer cells and hepatocytes, the underlying mechanism of how Cav-1 regulates PPARα was not clear. Recent finding suggested that lower bile acid levels in Cav-1-/- mice may account for insufficient FXRα activation, a critical regulator of PPARα in hepatocytes 30 . This may be coincided with the comorbidity of liver involvement in pSS, as biliary cirrhosis was commonly seen in pSS, together with reduced bile acid levels 56 . Nonetheless, how Cav-1 regulates PPARα in CD4 + T cells requires further investigation. In contrast to Cav-1, emerging evidence has reported the role of PPARs in autoimmune disorders. Agonists of PPARγ have been reported to interfere pro-inflammatory cytokines produced by various cell types, showing beneficial effects on mouse models of autoimmune thyroid diseases, multiple sclerosis and systemic lupus erythematosus 57 . PPARα is well-conserved between humans and mice, as pharmaceutical agonists could selectively activate both human and murine PPARα 33 , which potentiates the clinical application from drug screening using mouse models including experimental cholestatic liver disease and autoimmune dry eyes 57 . Fenofibrate has been clinically prescribed for decades, showing good tolerance and safety in long-term usage in humans 58 . Here, we demonstrated that fenofibrate effectively ameliorated established ESS mice, in particular under the chronic inflammatory conditions. Together, our findings suggest that targeting Cav-1 or PPARα may serve as a promising strategy for autoimmune disorders with Tfh cell dysregulation, while the expression levels of Cav-1 or PPARα may be considered as factors in patients partition and cohort stratification. Declarations Acknowledgement This work was funded by grants through National Key Research and Development Program of China (2023YFE0203100), General Research Fund, Hong Kong Research Grants Council (17116521 and 27111820), Health and Medical Research Fund (19201121 and 20212601), Hong Kong Research Grants Council Area of Excellence Scheme 2016/2017 (No. AoE/P-705/16). We thanked the staff of Faculty Core Facility, Li Ka Shing Faculty of Medicine, the University of Hong Kong for their kind support. Author Contributions XL and JS conceptualized and supervised this study. XL designed the experiments. XL and JS provided research agents and animals. XL, JS and YF received the research fund. SY, MW and XL conducted the animal experiments and human samples from healthy donors. YC and JX assisted the animal experiments. YF and XL oversaw the conduct of patient recruitment and clinical investigations. Patient recruitment and screening were done by PHL, WZ and YF. Patient samples were analyzed by WF, WZ and PHL. XL constructed and JS revised the manuscript. Competing Interests The authors have declared that no conflict of interest exists. References Rothberg, K.G. , et al. Caveolin, a protein component of caveolae membrane coats. Cell 68 , 673-682 (1992). Razani, B. , et al. 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Supplementary Files Cav1methods5.docx Materials and Methods S1.pdf S2.pdf S3.pdf S4.pdf S5.pdf S6.pdf SupplFigurelegend.docx Supplemental Figure Legend WTT.mp4 Two-Photon confocal microscopy analysis of wild type CD4+ T cell motility Cav1KOT.mp4 Two-Photon analysis of Cav-1-/- CD4+ T cell motility Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3230861","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":233630697,"identity":"b86c5da0-0bf2-47ff-8ffb-aa8ef55fa6a7","order_by":0,"name":"Xiang Lin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIiWNgGAWjYBACPmYgkQDE/BJsDAyMDQmEtbDBtEjOIFoLjGFwg2gt7LzHJB7U3LHbfLstTYJxRxoxDuNLNkg49ix5251jxyQYz+QQo4XH8EEC2+FksxvpbRKMbRVEaTE4kPDvcLLxDBK0GD5IbDtsZyCRBnRYG3EOMzZI7DucIHEjLdkisY0I7/PznzGT/PHtsD3/jDTDGx/bkglrgYHEBhCZQLwGBgZ7UhSPglEwCkbBCAMAQcA1J66EZaAAAAAASUVORK5CYII=","orcid":"","institution":"The University of Hong Kong, Hong Kong, China","correspondingAuthor":true,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Lin","suffix":""},{"id":233630698,"identity":"4b9b4cee-5233-46de-9336-3dba08a318ed","order_by":1,"name":"Sulan Yu","email":"","orcid":"","institution":"The University of Hong Kong, Hong Kong, China","correspondingAuthor":false,"prefix":"","firstName":"Sulan","middleName":"","lastName":"Yu","suffix":""},{"id":233630699,"identity":"aff04162-7f63-4395-8004-4c7db9ac1fd6","order_by":2,"name":"Meiling Wu","email":"","orcid":"","institution":"The University of Hong Kong, Hong Kong, China","correspondingAuthor":false,"prefix":"","firstName":"Meiling","middleName":"","lastName":"Wu","suffix":""},{"id":233630700,"identity":"f7f88cdc-bab9-4964-a047-0d7cf0177a50","order_by":3,"name":"Weizhen Zeng","email":"","orcid":"","institution":"Department of Ophthalmology, Peking University Third Hospital, Beijing, China","correspondingAuthor":false,"prefix":"","firstName":"Weizhen","middleName":"","lastName":"Zeng","suffix":""},{"id":233630701,"identity":"1835bcea-4780-4aea-894e-7650832446db","order_by":4,"name":"Weiwei Fu","email":"","orcid":"","institution":"Department of Gastroenterology, Peking University Third Hospital, Beijing, China","correspondingAuthor":false,"prefix":"","firstName":"Weiwei","middleName":"","lastName":"Fu","suffix":""},{"id":233630702,"identity":"c17345ea-071a-4340-b3c3-4f853af7a613","order_by":5,"name":"Yacun Chen","email":"","orcid":"","institution":"The University of Hong Kong, Hong Kong, China","correspondingAuthor":false,"prefix":"","firstName":"Yacun","middleName":"","lastName":"Chen","suffix":""},{"id":233630703,"identity":"7ced1372-8df7-40fc-9c3e-811843bffb31","order_by":6,"name":"Jing Xie","email":"","orcid":"","institution":"The University of Hong Kong, Hong Kong, China","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Xie","suffix":""},{"id":233630704,"identity":"555b40b4-6c74-42e3-aac9-17a1e1529733","order_by":7,"name":"Philip Li","email":"","orcid":"","institution":"The University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Philip","middleName":"","lastName":"Li","suffix":""},{"id":233630705,"identity":"b47cd82e-892a-451d-b2a3-f98e987ba018","order_by":8,"name":"Yun Feng","email":"","orcid":"","institution":"Department of Ophthalmology, Peking University Third Hospital, Beijing, China","correspondingAuthor":false,"prefix":"","firstName":"Yun","middleName":"","lastName":"Feng","suffix":""},{"id":233630706,"identity":"8a7f9154-6ded-458e-9063-efaad6c9b630","order_by":9,"name":"Jiangang Shen","email":"","orcid":"","institution":"School of Chinese Medicine, LKS Faculty of Medicine, The University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Jiangang","middleName":"","lastName":"Shen","suffix":""}],"badges":[],"createdAt":"2023-08-03 10:13:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3230861/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3230861/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":43307514,"identity":"cbe40deb-f733-48a3-8451-b85ab17368d5","added_by":"auto","created_at":"2023-09-18 16:16:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":884092,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHematopoietic deficiency of Cav-1 exacerbated ESS development in mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Salivary flow rates of WT and Cav-1-/- ESS mice were determined overtime (n = 5; mean ± SEM); *P \u0026lt;0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Serum levels of autoantibodies against the SSA and M3R epitopes in WT and Cav-1-/- ESS mice, while dashed line indicated the levels of naïve controls (n = 5; mean ± SEM); **P \u0026lt;0.01; ***P \u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Immunofluorescence staining for IgG (red) deposition on the acini (AQP-5, green) in the salivary gland of WT and Cav-1-/- ESS mice (n = 80; mean ± SEM); ****P \u0026lt;0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eTissue damages were detected by apoptotic cells (TUNEL, cyan) and massive lymphocytic infiltrations (CD45, yellow) in the salivary glands (AQP-5, purple) of Cav-1-/- ESS mice 20wk post immunization, in which ectopic germinal center-like Bcl-6+CD19+ B cells and Bcl-6+CD4+Tfh-like cells were observed in the B cell follicle (white, gated by yellow dashed line) and T-B boarders.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E-F)\u003c/strong\u003e Quantification of histological score, infiltrating area and TUNEL+ cell counts in Figure 1D, (each dot represented one mouse, mean ± SEM), **P \u0026lt;0.01; ***P \u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G) \u003c/strong\u003eDiagram of chimeric mouse model by bone marrow transfer, while the genotype of the lymph nodes of recipient mice were validated upon sacrifice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H) \u003c/strong\u003eAutoantibodies against the SSA epitope in the sera of chimeric ESS mice 3wk post immunization, while the dashed line indicated the levels of naïve controls (n = 4; mean ± SEM); ns, not significant; **P \u0026lt;0.01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I) \u003c/strong\u003eHistopathological changes of salivary gland of chimeric ESS mice were assessed 20wk post immunization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J) \u003c/strong\u003eQuantification of histological score in Figure 1I (n=5; mean ± SEM); **P \u0026lt;0.01.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3230861/v1/e2306c5d848a260c80e56896.jpg"},{"id":43308641,"identity":"744cc4e0-9d79-4567-8f0a-f0be277295c7","added_by":"auto","created_at":"2023-09-18 16:24:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":875968,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCav-1 deficiency promoted Tfh cell response.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eFlow cytometric analyses of Th1, Th17, Tfh and Treg cells in the draining cervical lymph nodes (CLN) of naïve control, WT and Cav-1-/- ESS mice (n = 5; mean ± SEM); ns, not significant; **P \u0026lt;0.01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eCav-1 protein levels in CD4+ T cells from ESS mice were analyzed by Western blot during disease progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Flow cytometric profiles of CD138+ plasma cells in the immunodeficient NSG mice. NSG mice were co-transferred with WT CD19+ B cells and CD4+ T cells from WT or Cav-1-/- mice, followed by immunization for ESS induction (n = 4; mean ± SEM); **P \u0026lt;0.01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eELISpot assay of SSA-reactive B cells in the spleen of recipient NSG mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E) \u003c/strong\u003eQuantification of the MFI of IgG deposition per acinus in the SG of recipient NSG mice (141–150 acini from 6 mice per group; mean ± SEM); ***P \u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e Flow cytometric profiles of WT or Cav-1-/- CD4+ T cells under Tfh polarization for 3 days, in the absence or presence of cavtratin (Cavt).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G) \u003c/strong\u003ePolarized states of Tfh cells purified from WT or Cav-1-/- mice, treated with isotype IgG control or anti-ICOS activating (a) antibody (n = 121-163; mean ± SEM); *P \u0026lt;0.5; ***P \u0026lt;0.01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H)\u003c/strong\u003e x–y displacement (μm) plots of individual WT or Cav-1-/- Tfh cell traces, with starting positions realigned at the same origin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I)\u003c/strong\u003e The centroid velocity of WT or Cav-1-/- Tfh cells, treated with isotype control or anti-ICOS (a) antibody (n = 48-76; mean ± SEM); **P \u0026lt;0.01; ***P \u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J)\u003c/strong\u003e Two-photon live imaging of CD4+ T cells (green) and CD19+ B cells (red) in the spleen of recipient NSG mice. SG-antigen immunized NSG mice were co-transferred with WT CD19+ B cells and CD4+ T cells from WT or Cav-1-/- mice for 4 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(K) \u003c/strong\u003eSphericity of WT or Cav-1-/- CD4+ T cell in the spleen of recipient NSG mice in the period of 30 min (n=195, only those cells lasted at least 24 min were included; mean ± SEM); ****P \u0026lt;0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(L-M)\u003c/strong\u003e x-y-z displacement (μm) plots of individual WT (yellow) or Cav-1-/- (magenta) CD4+ T cell traces for 30 min, with starting positions overlayed and realigned at the same origin, while the velocity of WT or Cav-1-/- CD4+ T cells were summarized (n = 152-184; mean ± SEM); ****P \u0026lt;0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(N) \u003c/strong\u003eNaïve CD4+ T cells from WT or Cav-1-/- mice were labeled with CFSE (green) or Q-tracker (red) respectively, and co-transferred into in ESS mice with active disease for 4 days, while their distributions in the draining CLN were shown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(O)\u003c/strong\u003e WT or Cav-1-/ CD4+ Tfh cells were co-cultured with WT CD19+ B cells from ESS mice for 3 days, while Blimp-1+CD138+ plasma cells were analyzed by flow cytometry and anti-SSA IgG levels in the supernatant were measured by ELISA (n = 4; mean ± SEM).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3230861/v1/af8c74cd5cf21d1f466fb91c.jpg"},{"id":43307516,"identity":"fc72688c-f430-4ee2-a868-78f3ec756770","added_by":"auto","created_at":"2023-09-18 16:16:41","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":686231,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpaired PPARα expression in CD4+ T cells contributes to Cav-1-mediated Tfh cell response\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e KEGG pathway enrichment of RNA-seq analysis between WT and Cav-1-/- Tfh cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e mRNA expression of PPARα, PPARγ and PPARδ in WT and Cav-1-/- Tfh cells (n = 6; mean ± SEM); ***P \u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e PPARα expression levels in WT and Cav-1-/- Tfh cells from ESS mice were analyzed by flow cytometry (n=20; mean ± SEM); ****P \u0026lt;0.0001, while dashed line indicated the MFI of isotype control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e Purified CD4+ T cells from WT mice were transduced with GFP-incorporated plasmids for PPARα deletion, followed by Tfh polarization for 3 days, while GFP+Tfh cell differentiation was analyzed by flow cytometry (n = 4; mean ± SEM); ***P \u0026lt;0.001\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e CD4+ T cells were purified and polarized into Tfh cells in the absence or presence of GW6471 for 3 days while the phenotypic analysis was performed (n = 3; mean ± SEM); ***P \u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F) \u003c/strong\u003eCD4+ T cells were from \u003cstrong\u003e(E) \u003c/strong\u003ewere subjected to transwell assay toward recombinant CXCL13 in the bottom chamber (n = 4; mean ± SEM); **P \u0026lt;0.01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G) \u003c/strong\u003eFlow cytometric profiles of Tfh cells in the absence or presence of 8-Hepe, 15-Deoxy or GW0742.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H)\u003c/strong\u003e WT or Cav-1-/- CD4+T cells were cultured toward Tfh differentiation in the presence of fenofibrate (Feno) at various concentrations, while Tfh cell counts were analyzed by flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I)\u003c/strong\u003e mRNA expression of PPARα and PPARγ in CD4+ T were analyzed at different time points during ESS development (n = 4; mean ± SEM).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J) \u003c/strong\u003eCav-1 and PPARα protein levels in the CD4+ T cells were analyzed by Western blot at different time points during ESS development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(K)\u003c/strong\u003e PPARα expression levels in CD4+ T cells from naïve and ESS mice were analyzed by flow cytometry (n = 7; mean ± SEM); **P \u0026lt;0.01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(L) \u003c/strong\u003eESS mice were treated with vehicle or GW6471, while the salivary flow rates were analyzed over time (n = 5; mean ± SEM); *P \u0026lt;0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(M)\u003c/strong\u003e Flow cytometric analysis of CD4+ICOS+PD1+ Tfh cells from ESS mice treated with or without GW6471.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3230861/v1/7dc9d5be3601c1a4d882d1fb.jpg"},{"id":43307518,"identity":"673340c2-29e1-4435-af2e-a0b41504e0a7","added_by":"auto","created_at":"2023-09-18 16:16:41","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":727832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePPARα represses ICOS transcription in Tfh cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Lipid droplet staining by BODIPY in CD4+ T cells from WT or Cav-1-/- ESS mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e mRNA (left panel) and protein (right panel) levels of ICOS from Tfh cells at different time points (n = 5; mean ± SEM); *P \u0026lt;0.05, **P \u0026lt;0.01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e CD4+ T cells from WT or Cav-1-/- mice were cultured under Tfh polarization conditions, while mitochondrial oxygen consumption rate (OCR) levels at 16h and 48h were determined with palmitic acid as sole substrate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e Protein expressions of ACADM, SDH, ACO2 and β-actin in WT and Cav-1-/- CD4+ T cells 48h post Tfh polarization were analyzed by Western blot.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E-F)\u003c/strong\u003e Free fatty acid content and mRNA expression of \u003cem\u003eCpt1a\u003c/em\u003e in the WT or Cav-1-/- CD4+ T cells at 16h and 48h post Tfh polarization were analyzed by ELISA (n = 8) and q-PCR (n = 6), respectively (Mean ± SEM); **P \u0026lt;0.01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e WT and PPARα-/- CD4+ T cells with or without \u003cem\u003eCpt1a\u003c/em\u003e gene overexpression (Tg) were cultured under Tfh polarization for 3 days, while ICOS+PD1+ phenotype was analyzed by flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H)\u003c/strong\u003e CD4+ T cells from WT mice were cultured under Tfh polarization conditions, in absence or presence of GW7647, pioglitazone (PIO) or etomoxir (Eto) for 3 days, while Tfh cells were analyzed by flow cytometry and enumerated (n = 5; mean ± SEM); **P \u0026lt;0.01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I) \u003c/strong\u003eCD4+ T cells from WT or Cav-1-/- mice were cultured with fatty acid free medium X-VIVO toward Tfh differentiation, in absence or presence of bovine serum albumin (BSA) for 3 days, while ICOS expression was analyzed by flow cytometry (n = 4; mean ± SEM); ***P \u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J)\u003c/strong\u003e CD4+ T cells from WT mice were cultured under Tfh polarization conditions for 16h, while the binding activities of various transcription factors were assessed. The 20 mostly differentially active transcription factors were listed in the heatmap.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(K)\u003c/strong\u003e CD4+ T cells from WT mice were cultured under Tfh polarization conditions for 16h, while anti-PPARα ChIP-PCR assay was performed to reveal the binding sites in the\u003cem\u003e Icos\u003c/em\u003e promoter (n = 3; mean ± SEM); *P \u0026lt;0.05, **P \u0026lt;0.01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(L) \u003c/strong\u003eCD4+ T cells (red) from WT mice were cultured under Tfh polarization conditions, while nuclear expression of PPARα (green) was determined by confocal microscopy at different time points (n=368-443; mean ± SEM); **P \u0026lt;0.01; ***P \u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(M) \u003c/strong\u003eCD4+ T cells from WT mice were cultured under Tfh polarization conditions for 5h, in the absence or presence of SC560, while nuclear translocation of PPARα was quantified by confocal microscopy (n=30; mean ± SEM); **P \u0026lt;0.01; ***P \u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(N) \u003c/strong\u003eCD4+ T cells from WT or Cav-1-/- mice were cultured under Tfh polarization conditions for 16h, in the absence or presence of GW7647 and SC560, while \u003cem\u003eIcos\u003c/em\u003eexpression levels were determined by q-PCR (n = 4; mean ± SEM); ***P \u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(O) \u003c/strong\u003eCD4+ T cells from WT mice were cultured under Tfh polarization conditions for 3 days, in the absence or presence of HX531 or LG100574, while ICOS expression was determined by flow cytometry (n = 3; mean ± SEM); *P \u0026lt;0.05; **P \u0026lt;0.01.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3230861/v1/26f507fb61ff3dda432a58cf.jpg"},{"id":43307520,"identity":"83700588-db51-47cd-b578-645d90d71253","added_by":"auto","created_at":"2023-09-18 16:16:41","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":561284,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCav-1/PPARα axis critically regulates human Tfh cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003ePurified CD4+ T cells from healthy donors were transduced with GFP-incorporated plasmids for Cav-1 deletion, followed by Tfh polarization for 3 days, while Tfh cell differentiation was analyzed by flow cytometry (n = 4; mean ± SEM); **P \u0026lt;0.01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B-C) \u003c/strong\u003eUpon Tfh polarization for 3 days,\u003cstrong\u003e \u003c/strong\u003eprotein levels of ICOS (n = 15) and PPARa (n = 8) in CD4+ T cells were determined by flow cytometry (mean ± SEM); ***P \u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003ePurified CD4+T cells from healthy donors were cultured toward Tfh differentiation for 3 days in the presence of Feno at various concentrations, while Tfh cell counts were analyzed by flow cytometry (n = 3; mean ± SEM).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e Purified CD4+T cells from healthy donors were cultured toward Tfh differentiation for 16h and paired-end RNA-seq analyzed was performed. The transcription reads, revealed by FPKM of \u003cem\u003eIcos\u003c/em\u003e gene were shown (n = 3; mean ± SEM); **P \u0026lt;0.01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e Under culture condition described in (E), anti-PPARα ChIP-PCR assay was performed to reveal the binding sites in the\u003cem\u003eIcos\u003c/em\u003e promoter (n = 3; mean ± SEM); *P \u0026lt;0.05, ***P \u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e Purified CD4+T cells from healthy donors were cultured toward Tfh differentiation for 16h, in the absence or presence of GW7647 and SC560, while \u003cem\u003eIcos\u003c/em\u003eexpression levels were determined by q-PCR (n = 4; mean ± SEM); *P \u0026lt;0.05; **P \u0026lt;0.01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H)\u003c/strong\u003e Purified CD4+T cells from healthy donors were cultured toward Tfh differentiation for 3 days, in the absence or presence of Feno treatment, while T cells were subjected to transwell assay toward recombinant CXCL13 in the bottom chamber (n = 4; mean ± SEM); **P \u0026lt;0.01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I)\u003c/strong\u003e Correlation between Cav-1 and PPARa expression levels in the CD4+ T cells in the peripheral blood of pSS patients was analyzed by Pearson’s correlation coefficient (n = 24).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J)\u003c/strong\u003e Representative flow cytometric analysis of circulating Tfh cells from pSS patients, while the expression levels of Cav-1 and PPARa were determined.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(K)\u003c/strong\u003e Correlation between Cav-1 and ICOS expression levels in the CD4+ T cells in the peripheral blood of pSS patients was analyzed by Pearson’s correlation coefficient (n = 24).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3230861/v1/6b965c13f6ed1f8392f281d7.jpg"},{"id":43308644,"identity":"71847946-7d18-4567-8ff3-7ed957a668e7","added_by":"auto","created_at":"2023-09-18 16:24:41","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":714556,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePharmaceutical activation of PPARα ameliorated ESS development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eDiagram of Feno intervention strategies on ESS mice at acute or chronic stages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B-C) \u003c/strong\u003eESS mice at acute stage were treated with PBS vehicles or Feno, while salivary function and serum levels of anti-SSA antibodies were assessed (n = 6; mean ± SEM); **P \u0026lt;0.01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D-E) \u003c/strong\u003eESS mice at chronic stage were treated with PBS vehicles or Feno, while glandular damages, as characterized by lymphocytic infiltration (magenta) and TUNEL (green) staining were assessed by confocal microscopy. 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16:32:42","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":705894,"visible":true,"origin":"","legend":"","description":"","filename":"S6.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3230861/v1/bed5db6afc296e8f0c9adc0b.pdf"},{"id":43308647,"identity":"c9fec600-e1e1-4bbc-a20b-dce7ca3ab065","added_by":"auto","created_at":"2023-09-18 16:24:42","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":16387,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Figure Legend\u003c/p\u003e","description":"","filename":"SupplFigurelegend.docx","url":"https://assets-eu.researchsquare.com/files/rs-3230861/v1/c935701fb509375f703cf5f2.docx"},{"id":43307529,"identity":"74a13308-c87b-4f90-abc0-e2d80f3ef80b","added_by":"auto","created_at":"2023-09-18 16:16:42","extension":"mp4","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":7407666,"visible":true,"origin":"","legend":"Two-Photon confocal microscopy analysis of wild type CD4+ T cell motility","description":"","filename":"WTT.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3230861/v1/a9d86c5ce4d3702914ca547d.mp4"},{"id":43307530,"identity":"63da25a3-ab54-4671-90a1-ea109eebf2e6","added_by":"auto","created_at":"2023-09-18 16:16:42","extension":"mp4","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":8214483,"visible":true,"origin":"","legend":"Two-Photon analysis of Cav-1-/- CD4+ T cell motility","description":"","filename":"Cav1KOT.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3230861/v1/f776f46b5a917b0a0463aa95.mp4"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Caveolin-1 restrains pathogenic T follicular helper cell response in primary Sjögren’s syndrome","fulltext":[{"header":"Highlights","content":"\u003cp\u003e- Caveolin-1 deficiency exacerbates ESS development in mice\u003c/p\u003e\n\u003cp\u003e- Caveolin-1/PPARα axis inhibits Tfh cell response\u003c/p\u003e\n\u003cp\u003e- PPARα represses \u003cem\u003eIcos\u003c/em\u003e transcription in Tfh cells independence of lipid metabolism\u003c/p\u003e\n\u003cp\u003e- Pharmaceutical activation of PPARα suppresses Tfh response in ESS and humanized mice\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eCaveolin-1 (Cav-1), a scaffold protein involved in assembling caveolae components and lipid domains, is structurally conserved in mammalian cells \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Early studies showed that mice deficient for Cav-1 were vial, but resistant to diet-induced obesity \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, rendering its close involvement in diabetic development in both humans and mice \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Our recent studies have identified the versatile functions of Cav-1 in cell fate decisions, which critically regulated the nitric oxide-mediated matrix metalloproteinases activity \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, vascular endothelial growth factor \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and integrin expression\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e pathways in neuronal differentiation, neuronal regeneration, hepatocyte and endothelial cell responses. Consequently, these findings further extend the important features of Cav-1 in the pathogenesis of multiple sclerosis \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, ischemia/reperfusion injuries in stroke \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e and liver diseases \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, respectively. However, the role of Cav-1 in autoimmune pathogenesis remains largely unclear.\u003c/p\u003e \u003cp\u003ePrimary Sj\u0026ouml;gren\u0026rsquo;s syndrome (pSS) is a common autoimmune disease characterized by exocrinopathy involving the lacrimal and salivary glands (SG), leading to the clinical manifestations of severe dry-eye and dry-mouth symptoms \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Highly activated CD4\u0026thinsp;+\u0026thinsp;T cell signature, including Th17 and T follicular helper (Tfh) cells, has been reported both in the inflammatory SG and peripheral blood of pSS patients \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. We previously established a mouse model of experimental Sj\u0026ouml;gren\u0026rsquo;s syndrome (ESS), which recapitulated the key features of human pSS \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Tfh cells were found to play critical roles in pSS development, as revealed by the findings that mice deficient for Tfh cells exhibit markedly attenuated autoantibody production and disease pathology upon ESS induction \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Tfh cells are a subset of CD4\u0026thinsp;+\u0026thinsp;T cells that provide B-cell help during germinal center (GC) reactions and promote plasma cell differentiation in adaptive immune response \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Tfh cells are characterized by the phenotypes of transcription factor Bcl-6 and surface markers of PD-1, ICOS and CXCR5, among which mostly drive and stabilize Tfh cell positioning in the B cell follicles \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Through CD40 and cytokine productions (i.e. IL-21) \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, Tfh cells facilitate high-affinity B cell selection and antibody production. Thus, stringent control of Tfh cells is critical for maintaining immune tolerance and optimal humoral response \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Although impaired regulatory function of certain immune cell subsets may contribute to the hyperactive Tfh cell response \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, the checkpoints that maintain Tfh cell tolerance were not fully understood.\u003c/p\u003e \u003cp\u003eAlthough the knowledge of Cav-1 in immune response is limited, emerging studies have reported its involvement in fine-tuning TCR and BCR signaling. Cav-1-/- B cells exhibited reduced T cell-independent humoral immune response, but had normal response to T cell-dependent antigens \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. This is coincided with the findings that Cav-1 deficiency did not affect CD4\u0026thinsp;+\u0026thinsp;T cell expansion upon viral infection \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Nonetheless, it is not known whether and how Cav-1 could regulate effector CD4\u0026thinsp;+\u0026thinsp;T cell subsets in pSS pathogenesis. In this study, we demonstrated that Cav-1 deficiency did not attenuate, but rather exacerbated ESS pathology and Tfh cell responses in mice. Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T cells highly expressed ICOS and thus exhibited increased motility toward B cell follicles. Mechanistic studies revealed that peroxisome proliferator-activated receptor alpha (PPARα) served as a potent transcription repressor of ICOS during Tfh polarization, independent of lipid metabolism, while PPARα expression was markedly decreased in the absence of Cav-1. Consistently, Cav-1 ablation or PPARα antagonism enhanced human Tfh cell differentiation. In the context of autoimmunity, Cav-1 levels were positively correlated with PPARα, but negatively correlated with ICOS expression in the circulating CD4\u003csup\u003e+\u003c/sup\u003e T cells from ESS mice and pSS patients. By contrast, fenofibrate, an FDA-approved PPARα agonist, effectively suppressed human Tfh cells both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. This pharmaceutical activation of PPARα significantly ameliorated the disease pathology in established ESS mice with acute or chronic inflammation. Thus, our work has revealed an unrecognized role of Cav-1/PPARα axis in Tfh cell dysregulation and pSS pathogenesis, while targeting PPARα may serve as a promising approach in treating Tfh cell dysregulation in autoimmunity.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCaveolin-1 deficiency exacerbates ESS pathology.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAlthough previous studies suggested ameliorated EAE pathology in Cav-1-/- mice\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, unexpectedly, Cav-1 deficiency significantly exacerbated ESS development in mice upon disease induction, as revealed by accelerated salivary hypofunction and higher titer of autoantibodies compared with wildtype (WT) ESS mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). This was associated with enhanced IgG deposition on the salivary epithelium (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In long-term observation, at acute-chronic stage, massive lymphocytic foci, together with much severe tissue damage were observed in Cav-1-/- ESS mice, whereas age-matched WT mice exhibited only mild lymphocytic infiltration along ESS progression. Confocal imaging analyses also indicated the presence of Bcl-6\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e Tfh cells and GC-like B cells within the infiltrating aggregates, suggesting ectopic lymphoid structure formation in the inflamed SG tissues from Cav-1-/- ESS mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Quantitatively, Cav-1-/- ESS mice exhibited higher histological scores, larger infiltrating area (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), and increased apoptotic epithelial cell counts when compared with WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Thus, Cav-1 deficiency promoted ESS development in mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven the varied abundance of Cav-1 proteins in lymphoid and non-lymphoid organs \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, it is not clear whether Cav-1 expression in hematopoietic origin would be responsible for the ESS pathogenesis. Thus, we generated chimera mice with Cav-1 deficiency in the immune cells by bone marrow transfer, while the genotype in the lymphoid organ was validated upon mouse sacrifice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Consistently, WT recipients reconstructed with Cav-1-/- bone marrows showed higher autoantibody levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH) and histopathological changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI-J), which was comparable with those mice of global Cav-1 deletion. Together, these results suggest the importance of Cav-1 in regulating immune responses during ESS development.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCaveolin-1 constrains ICOS expression and follicular migration of Tfh cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious studies, including our findings, identified a central role of Th17 cells in both EAE and ESS pathogenesis \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. However, phenotypic analyses showed that Cav-1 deficiency did not increase Th17 cell frequencies and counts upon ESS development (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This was consistent with previous findings in Cav-1-/- EAE mice\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Instead, we detected significantly increased Tfh cells in Cav-1-/- ESS mice, which was associated with their follicular displacement in the germinal centers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). This may explain the expanded GC areas and GC B cells (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-B). Thus, we sought to determine Cav-1 expression levels in CD4\u0026thinsp;+\u0026thinsp;T cells during ESS development. Upon ESS induction, we observed a transient increase of Cav-1 in CD4\u0026thinsp;+\u0026thinsp;T cells at disease onset, followed by persistent downregulation during ESS progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This was negatively correlated with increased Tfh cell responses \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Since Cav-1-/- B cells could respond to T cell-dependent antigens \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, we next investigated whether Cav-1 deficiency in CD4\u0026thinsp;+\u0026thinsp;T cells would be sufficient to mount humoral dysregulation. WT or Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T cells were co-transferred with WT B cells into SG-antigen immunized immunodeficient NOD-\u003cem\u003escid\u003c/em\u003e IL2Rg\u003csup\u003enull\u003c/sup\u003e (NSG) mice. Notably, Cav-1 deficiency in CD4\u0026thinsp;+\u0026thinsp;T cells markedly promoted autoreactive B cell response (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D), resulting in elevated autoantibodies production and IgG deposition on the salivary epithelium (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). These data suggest that Cav-1 deficiency in CD4\u0026thinsp;+\u0026thinsp;T cells might promote Tfh cell response \u003cem\u003ein vivo\u003c/em\u003e. To validate this finding, WT or Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T cells were purified and cultured for Tfh polarization. Indeed, Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T cells exhibited higher capacity of Tfh cell differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). A previous structural biology study implicates a central role of scaffolding domain of Cav-1 (CSD) as a docking site in signaling transduction \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Cavtratin, a cell-permeable peptide of CSD, binds to caveolin-binding motifs \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Herein, it dose-dependently restrained Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T cell response to Tfh polarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Collectively, these data indicate the role of endogenous Cav-1 in regulating Tfh cell differentiation. We next determined the featured molecules in Cav-1-/- Tfh cells, in which PD-1 and ICOS, but not CXCR5 expressions were found markedly increased (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD). However, we did not observe the elevation of CD40 ligand and representative cytokine productions, in particular IL-17 and IL-21 in Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T cells under the polarization conditions (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE). Moreover, Cav-1 deficiency-mediated Tfh cell response was also found stable in long term, as revealed by flow cytometric analysis of BrdU-incorporated WT or Cav-1-/- donor CD4\u0026thinsp;+\u0026thinsp;T cells in the recipient ESS mice over 10 weeks post adoptive transfer (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eICOS-ICOSL plays an indispensable role of in Tfh cell motility \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Notably, flow cytometric analysis showed comparable ICOSL levels between WT and Cav-1-/- B cells from ESS mice (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eG). Thus, we hypothesized that increased ICOS expression in Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T cells might largely strengthen their follicular migration. Consistent with the previous finding \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, anti-ICOS activating antibody rapidly induced WT Tfh cell polarization, as revealed by pseudopod protrusion and persistent movement (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-H). Quantitatively, Cav-1-/- Tfh cells showed higher shape index with augmented pseudopod extension (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG) upon ICOS ligation, and thus resulted in increased centroid speed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI) and cell displacement in random directions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eH). This was validated in ICOSL-mediated transwell assay in response to CXCL13 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eI). Consistently, ICOS expression was significantly increased in CD4\u0026thinsp;+\u0026thinsp;T cells obtained from draining cervical lymph nodes of Cav-1-/- ESS mice, accompanied with enhanced ICOS\u0026thinsp;+\u0026thinsp;CD4\u0026thinsp;+\u0026thinsp;T cells present in the lymphocytic foci of SG (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eJ-K). We further testified Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T cell motility \u003cem\u003ein vivo\u003c/em\u003e, in particular those at T-B borders, by two-photon intravital imaging analysis. In this adoptive transfer model, CD4\u0026thinsp;+\u0026thinsp;T cell zone could be clearly distinguished in the spleen at 72h post transfer (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). Consistent with the findings \u003cem\u003ein vitro\u003c/em\u003e, Cav-1-/-CD4\u0026thinsp;+\u0026thinsp;T cells exhibited more polarized status, as reflected by less sphericity in morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). By analyzing the cell tracks, we observed markedly increased motility of Cav-1 deficient CD4\u0026thinsp;+\u0026thinsp;T cells \u003cem\u003ein situ\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK), as reflected by enhanced cell displacement (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eL) and velocity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM) by quantification, in particular those at the T-B borders (Supplementary Video 1\u0026ndash;2). Since the follicular homing of CD4\u0026thinsp;+\u0026thinsp;T cells, as the consequence of Tfh cell motility, was defined as the fundamental feature to mount B-cell response \u003cem\u003ein vivo\u003c/em\u003e, we next evaluated their follicular homing capacity in the draining cervical lymph nodes under the identical conditions. CFSE-labelled WT and Qtracker-labeled Cav-1-/- na\u0026iuml;ve CD4\u0026thinsp;+\u0026thinsp;T cells were co-transferred into WT ESS mice with active disease, while the draining cervical lymph nodes were analyzed after 72h. As expected, Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T cells exhibited overt follicular displacement when compared with those WT counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eN). Together, these results demonstrated that Cav-1 critically restrained Tfh cell migratory capacity toward B cell follicles. To further assess the B-cell help functions other than T cell motility, we sorting-purified WT or Cav-1-/- Tfh cells from ESS mice, and co-cultured with cognate WT B cells for 72h. Interestingly, Cav-1 deficiency did not obviously affect the effector molecules of T-B interactions, as revealed by comparable plasmacytic differentiation and autoantibody productions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eO). This was in line with the findings of CD40 ligand and cytokines above mentioned. Together, our data suggest that increased ICOS expression in Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T cells were responsible for enhanced humoral autoimmunity and ESS pathology. To validate this notion, we performed anti-ICOS blocking antibodies treatment in Cav-1-/- mice upon ESS induction. The efficacy was validated by reduced intrafollicular ICOS\u0026thinsp;+\u0026thinsp;CD4\u0026thinsp;+\u0026thinsp;T cell counts, which was associated with similar Tfh cell numbers and follicular homing coefficient to those in WT ESS mice (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA-C). Consequently, the reduced Tfh cell response upon ICOS blockage led to decreased plasma cell numbers and anti-SSA IgG levels in Cav-1-/- ESS mice (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eD-E). Therefore, these data indicate a critical role of Cav-1 in ICOS expression and Tfh cell response.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImpaired PPAR\u003c/b\u003eα \u003cb\u003eexpression in CD4\u0026thinsp;+\u0026thinsp;T cells contributes to caveolin-1-mediated Tfh cell response.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCav-1 is mainly distributed at the plasma membrane and cytoplasm, but not in the nucleus in endothelial cells and B cells \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, Similarly, this was also observed in CD4\u0026thinsp;+\u0026thinsp;T cells (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA-B). Thus, we reasonably addressed the question that enhanced \u003cem\u003eIcos\u003c/em\u003e transcription in CD4\u0026thinsp;+\u0026thinsp;T cells could be indirectly regulated by Cav-1. We first performed RNA-seq analysis of WT and Cav-1-/- Tfh cells for transcriptome comparison. Notably, peroxisome proliferator-activated receptors (PPARs) signaling pathway was significantly affected by the absence of Cav-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). PPARs are a family of transcription factors including PPARα, PPARβ/δ and PPARγ \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Real time-PCR analysis revealed that Cav-1 deficiency mainly affected the transcription of PPARα, while PPARγ and PPARδ were comparable with WT Tfh cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Indeed, significantly reduced protein levels of PPARα were also found in Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). This was similar to the recent findings in hepatocytes \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. To investigate whether impaired PPARα expression contributes to Tfh cell response, we first performed genetic ablation of PPARα in CD4\u0026thinsp;+\u0026thinsp;T cells by CRISPR/Cas9. Interestingly, PPARα-/- CD4\u0026thinsp;+\u0026thinsp;T cells phenocopied Cav-1 deficiency and showed significantly augmented Tfh cell differentiation, regardless of cavtratin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), suggesting that PPARα served as the downstream of Cav-1 in CD4\u0026thinsp;+\u0026thinsp;T cells. This could be also achieved by the treatment of GW6471, a selective PPARα antagonist, which markedly promoted Tfh cell differentiation in dose dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Functional studies using transwell assay further validated enhanced ICOS-mediated T cell motility upon PPARα antagonism (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Given an intense search for PPARs ligands in the past decades, selective agonists were reported to activate differential PPAR members \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Thus, based on the binding affinity, 8-hydroxyeicosapentaenoic (8-HEPE), 15-deoxy-D\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e-prostaglandin J2 (15-Deoxy) and GW0742\u003csup\u003e32\u003c/sup\u003e were used to differentially activate PPARα, PPARβ/δ and PPARγ respectively. In contrast to PPARα deficiency, PPARα agonist effectively suppressed Tfh cell differentiation, while no difference was observed in the presence of 15-Deoxy and GW0742 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). To avoid the possible off-target effect, we adopted fenofibrate, the pharmaceutical agonist of both human and murine PPARα \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, which dose dependently suppressed both WT and Cav-1-/- Tfh differentiation in culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Thus, these data suggest that PPARα would be responsible for Cav-1 downstream signal and serves as negative regulator of Tfh cell response. In the context of ESS development, similar to Cav-1 expression in CD4\u0026thinsp;+\u0026thinsp;T cells, we also observed a transient increase of PPARα, but progressively decreased during disease development (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-J), in particular at disease chronic stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK). However, PPARγ was not correlated with Cav-1 and PPARα expressions in CD4\u0026thinsp;+\u0026thinsp;T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC-D). Consequently, treatment with PPARα antagonist GW6471 accelerated ESS development and Tfh cell responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL-M). Together, these results demonstrated the functional importance of Cav-1/PPARα axis in restraining Tfh cell response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePPAR\u003c/b\u003eα \u003cb\u003erepresses ICOS transcription in Tfh cells.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA previous study suggested downregulation of lipid metabolic processes as a major consequence of Cav-1 deficiency, among which PPARα was responsible \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Indeed, we observed significantly lower levels of lipid droplets in Cav-1 deficient CD4\u0026thinsp;+\u0026thinsp;T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), in particular in the effector population (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA). Thus, we sought to investigate whether lipid metabolism would be involved in Cav-1/PPARα axis-mediated ICOS expression in Tfh cells. We first monitored the transcriptional regulation of \u003cem\u003eIcos\u003c/em\u003e during Tfh polarization. Notably, the mRNA levels of ICOS were significantly increased as early as 16h in Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T cells, while protein levels at 48h upon Tfh differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Since lipid metabolism-mediated energy generation consisted of several rate-limiting steps \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, in which fatty acid β-oxidation (FAO) produced acetyl-CoA and entered mitochondrial tricarboxylic acid (TCA) cycle, we next determined whether \u003cem\u003eIcos\u003c/em\u003e transcription was altered during this process. We first measured the mitochondrial respiration from fatty acids by using palmitic acid (16:0, PA) as sole extracellular substrate \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. After 48h Tfh polarization, as expected, Cav-1 deficiency significantly decreased the basal and maximal respiratory capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB). Accordingly, ACADM protein levels, the representative enzyme of FAO initiation, were found significantly decreased in the absence of Cav-1. Moreover, succinyl-CoA synthetase (SDH), and aconitase (ACO2), the key regulatory enzymes of TCA cycle, were also significantly decreased at 48h in Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). This was associated with decreased cellular fatty acid content, as reflected by free fatty acid assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). However, this was not seen at the early stages upon Tfh polarization. FAO and cellular fatty acid content were minimal or undetectable in both WT and Cav-/- CD4\u0026thinsp;+\u0026thinsp;T cells at 16h in culture, while there was no obvious difference of enzyme expression levels. Together, these results validated that Cav-1 deficiency indeed impaired FAO process. However, the rapid \u003cem\u003eIcos\u003c/em\u003e transcription in Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T cells, prior to energy status transition, suggests its transcriptional regulation in relatively direct manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo validate this notion, we next looked into the target genes of PPARα in CD4\u0026thinsp;+\u0026thinsp;T cells, which were previously identified for fatty acid transportation and β-oxidation through carnitine palmitoyltransferase (CPT) system \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Cpt1a, a highly conserved PPARα target gene and metabolic regulator, catalyzes the long chain fatty acids from acyl-CoA to carnitine for translocation across the mitochondrial membranes \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Indeed, PPARα agonist significantly increased Cpt1a expression \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Thus, we first determined the expression levels of Cpt1a during Tfh differentiation. Consistent with the findings of the lipid metabolism above, Cpt1a expression was elevated in WT CD4\u0026thinsp;+\u0026thinsp;T cells at 48h under Tfh differentiation, which was found much lower in the Cav-1-/- counterparts. However, there was no obvious difference of Cpt1a level at early phase upon Tfh polarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). We next performed Cpt1a overexpression in CD4\u0026thinsp;+\u0026thinsp;T cells to restore, at least in part, the PPARα deficiency-mediated lipid metabolism. Surprisingly, overexpression of Cpt1a did not restrain, but rather promoted Tfh cell differentiation in PPARα-/- CD4\u0026thinsp;+\u0026thinsp;T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Similar findings were also observed following irreversible Cpt1a antagonism by etomoxir, as the etomoxir treatment did not phenocopy PPARα or Cav-1 deficiency, but rather suppressed Tfh cell development (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH), which may attribute to globally constrained energy generation and requirement. This notion was further supported by fatty acid-free culture conditions, in which Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T cell retained higher capacity of ICOS expression and Tfh differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI, Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eC). In this context, nutrient addition by BSA further gave rise to both WT and Cav-1-/- Tfh cell differentiation. Thus, these results demonstrate that impaired lipid metabolism in Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T cells would not affect the increased \u003cem\u003eIcos\u003c/em\u003e transcription under Tfh polarized conditions.\u003c/p\u003e \u003cp\u003eAdditionally, we also determined the glucose oxidation of WT and Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T cells under Tfh polarization conditions. Using glucose as a substrate, we detected minimal oxygen consumption rates (OCR) at 16h, but markedly increased at 48h in both WT and Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eD). Among the glucose oxidation cascade, the phospho-PFK2 to PFK2 ratio represents the glycolytic rate \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, in which a higher ratio leads to increased gluconeogenesis \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Consistent with the OCR findings, using glucose as the sole extracellular source of carbon, the ratio of p-PFK2:PFK2 was found comparable in WT and Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T cells during Tfh differentiation (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eE), as well as the expressions of SDH and ACO2. These data suggest that glucose oxidation might not be involved in Cav-1-mediated Tfh response.\u003c/p\u003e \u003cp\u003ePPARα is also recognized to limit inflammatory responses \u003cem\u003evia\u003c/em\u003e transcriptional repression\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. This is achieved by a conserved mechanism that PPARs could form heterodimer with 9-cis-retinoic acid receptor (RXR), which binds to peroxisome proliferator response element (PPRE) at the promoter region of target genes \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Indeed, in the transcriptomic screening analysis, both PPAR and RXR binding activities were found significantly reduced in Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). Thus, we next assessed the binding capacity of PPARα to \u003cem\u003eIcos\u003c/em\u003e promoter. ChIP-PCR analysis indicated that PPARα rapidly bound to \u003cem\u003eIcos\u003c/em\u003e promoter region 12h upon Tfh polarization, an effect could be largely abrogated by selective antagonist (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK). This was further supported by interfering the intranuclear translocation. Time series imaging showed that intranuclear translocation of PPARα initiated at 30 min and persisted for hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL). Early studies have suggested a role of COX-1 in the nuclear translocation of PPARs \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. We also validated this finding in CD4\u0026thinsp;+\u0026thinsp;T cells, as SC-560, a selective COX-1 inhibitor effectively retained PPARα in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM). Importantly, PPARα agonist-mediated \u003cem\u003eIcos\u003c/em\u003e trans-repression was abolished by SC-560 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eN). Conversely, selective RXR antagonist HX531 augmented, while RXR agonist LG100754 \u003csup\u003e43\u003c/sup\u003e inhibited ICOS and Tfh cell differentiation \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eO, Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eF). Thus, these data suggest that PPARα could serve as a repressor of \u003cem\u003eIcos\u003c/em\u003e transcription.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCav-1/PPARα axis critically regulates human Tfh cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe next sought to investigate whether Cav-1/PPARα axis also operates in human subjects. Purified CD4\u0026thinsp;+\u0026thinsp;T cells from healthy donors were transduced with GFP-incorporated plasmids for Cav-1 deletion. Similar to the murine system, Cav-1-/- hCD4\u0026thinsp;+\u0026thinsp;T cells also exhibited strong capacity towards Tfh differentiation, as well as increased ICOS expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). In addition, PPARα expression levels were also significantly decreased upon Cav-1 deficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Thus, we evaluated the effects of fenofibrate in suppressing Tfh cell responses in dose dependent manner, which yielded an IC50 of 7.024 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Upon RNA-seq analysis, fenofibrate significantly reduced \u003cem\u003eIcos\u003c/em\u003e transcript copies at 16h post stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), which strongly supported our findings in mice. Consistently, human PPARα also rapidly bound to \u003cem\u003eIcos\u003c/em\u003e promoter region under Tfh polarization, revealed by ChIP-PCR analysis, which was largely abolished upon PPARα antagonism by GW6471 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Consequently, blockage of intranuclear entrance of PPARα by SC-560 prevented the transcriptional repression of \u003cem\u003eIcos\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Functional assay further verified the ICOS-mediated migration could be inhibited by fenofibrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). To validate this phenotype under disease conditions, we analyzed the circulating Tfh (cTfh) cells from pSS patients. By measuring the protein expression of each patient, we first detected strongly positive correlation between PPARα and Cav-1 in CD4\u0026thinsp;+\u0026thinsp;T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). Interestingly, patients with higher frequencies of cTfh cells exhibited relatively lower levels of Cav-1 and PPARα in CD4\u0026thinsp;+\u0026thinsp;T cells, rendering a negative correlation between Cav-1 with ICOS expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ-K). Thus, these results demonstrate Cav-1/PPARα axis as a negative regulator in human Tfh cells, while targeting PPARα may be a promising approach in treating Tfh cell dysregulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003ePharmaceutical activation of PPARα ameliorates ESS development\u003c/h3\u003e\n\u003cp\u003eFenofibrate has a similar affinity for both murine and human PPARα with high efficacies \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. We found that fenofibrate had a half maximal inhibitory concentration of 8.39 \u0026micro;M in suppressing murine Tfh cell differentiation (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). This prompted us to explore the therapeutic potential of this pharmaceutical PPARα agonist on mice with established ESS. Previous studies reported that oral administration of 100 mg/kg body weight fenofibrate was sufficient to induce global PPARα activation and downstream signaling pathways \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Thus, we first treated ESS mice with fenofibrate at disease onset (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), as diagnosed by reduced saliva secretion and elevated autoantibodies. Fenofibrate effectively ameliorated salivary hypofunction and decreased serum levels of anti-SSA IgG, although anti-SSA IgM levels were not altered (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-C). We next sought to determine the therapeutic potential of fenofibrate treatment at disease chronic stages. As revealed by mild lymphocytic infiltrations in the SG, ESS mice immunized for 20wk were treated with fenofibrate for 10wk. Notably, histopathological findings showed significantly reduced tissue damages and inflammation in SG of the fenofibrate-treated ESS mice, as evidenced by diminished lymphocytic infiltration and few apoptotic epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Phenotypic analyses further showed that fenofibrate treatment significantly suppressed Tfh cell responses, in particular GC-Tfh cell counts in the lymphoid tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-G). This was consequently associated with restrained GC area, GC B cell and plasma cell counts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-H). A recent study reported a humanized mouse model by transplanting PBMCs in NSG mice \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Thus, we adopted this method by dividing PBMCs from each pSS patient into two groups for paired analysis, followed by PBS or fenofibrate treatments. Although NSG mice xenografted with PBMCs from pSS patients exhibited higher frequencies of Tfh cells than those from healthy donors, fenofibrate administration effectively suppressed human Tfh cells in matched control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). Together, these results provide strong evidence to support the notion that targeting PPARα could be a promising therapeutic approach in pSS and related autoimmune disorders with dysregulated Tfh cell responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTfh cells play a central role in the pathogenesis of humoral autoimmunity \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. However, the mechanism underlying the Tfh cell-intrinsic tolerance was not fully understood. In this study, we for the first time showed that Cav-1/PPARα axis negatively regulated Tfh cell migration capacity, in which PPARα rapidly bound to the promoter region of \u003cem\u003eIcos\u003c/em\u003e upon Tfh polarization. This was further exemplified by the Cav-1 deletion and PPARα activation in human CD4\u0026thinsp;+\u0026thinsp;T cells. In the context of autoimmunity, negative correlation between Cav-1 and ICOS in CD4\u0026thinsp;+\u0026thinsp;T cells was observed in pSS patients. Importantly, pharmaceutical activation of PPARα effectively ameliorated the disease pathology of both ESS mice at acute and chronic stages. This study provided a previously unrecognized role of Cav-1/PPARα axis in Tfh cell response, and suggested the therapeutic potential of targeting PPARα in treating autoimmune patient cohorts with Tfh cell dysregulation.\u003c/p\u003e \u003cp\u003eCurrently, the investigation regarding how Cav-1 modulates Th cell response is limited. Early study reported that Cav-1 was dispensable in T cell development \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Although Cav-1 could optimize TCR-induced membrane raft polarity in CD8\u0026thinsp;+\u0026thinsp;T cells, as supported by impaired IFN-gamma and NFAT-dependent transcription in Cav-1-/- CD8\u0026thinsp;+\u0026thinsp;T cells, the TCR-mediated expansion and cytokine production was comparable between WT and Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T cells \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, suggesting the differential function of Cav-1 in T cell subsets. Cav-1 was reported to modulate regulatory T (Treg) cell response under differential conditions. At steady state, WT and Cav-1-/- mice exhibited comparable Treg cells. However, in an adoptive transfer model of murine graft-versus-host disease (GVHD), Cav-1-/- donor CD4\u0026thinsp;+\u0026thinsp;T cells elicited superior suppressive function than those WT compartments \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, suggesting that endogenous deficiency of Cav-1 may sensitize the T cells to acquire Treg phenotype in the microenviroment shaped by GVHD. In the present study, we did not observe statistical differences of Treg cell frequencies between WT and Cav-1-/- upon ESS induction. In contrast to Treg cells, recent studies, including our findings reported that Th1 and Th17 cell counts were comparable between WT and Cav-1-/- mice with active EAE \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, while the remission of EAE mainly owed to the altered tight junction remodeling in the blood-brain barrier, and thus decreased immune cell transmigration towards spinal cord. Notably, early studies demonstrated that transfer of myelin-specific Tfh cells did not induce EAE development \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, which may explain the differential outcomes between EAE and ESS mice upon Cav-1 deficiency. Moreover, although NFAT was reported to orchestrate CD4\u0026thinsp;+\u0026thinsp;T cell responses \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, in our transcriptome screening, we did not detect obvious change of NFAT activity in Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T cells, which was consistent with the previous study in Cav-1 deficient T cells \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePrevious studies, including our findings, reported a variety of signaling molecules affected by Cav-1 \u003csup\u003e50\u003c/sup\u003e, including lipid metabolism, nitric oxide \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and vascular endothelial growth factor pathways \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In several studies, altered PPARs and FAO pathways from Gene Ontology were highlighted in both immune cells (i.e. macrophages\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e) and tissue cells (i.e. hepatocytes \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e). However, few RNA-seq analysis has been done in CD4\u0026thinsp;+\u0026thinsp;T cells. In this study, we also observed differential expressed genes related to PPARs pathway and nitrogen metabolism between WT and Cav-1-/- Tfh cells. Extensive studies have reported PPAR isoforms as lipid sensors and regulators of lipid metabolism. Indeed, in the absence of Cav-1, we detected significantly decreased lipid droplet in effector T cells, impaired FAO cascade and reduced PPARα target gene \u003cem\u003eCpt1a\u003c/em\u003e was detected in CD4\u0026thinsp;+\u0026thinsp;T cells. However, the lipid metabolism was a rate-limiting process during the relative long-term culture of T cell differentiation, and thus did not explain the rapid \u003cem\u003eIcos\u003c/em\u003e transcription in Cav-1-/- CD4\u0026thinsp;+\u0026thinsp;T cells at the initiation stage of Tfh polarization. This was further validated under lipid-free culture conditions, regardless of exogenous lipids supplementation. Another pivotal function of PPARs is the inhibition of inflammatory gene expression. Notably, the expression levels of PPARα and PPARγ were diverse in na\u0026iuml;ve and activated, among which PPARγ was mostly studied \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Ligand-mediated PPARγ activation could induce T cell apoptosis, inhibit T cell activation, and suppress Th1 cytokines \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. By contrast, PPARα activation did not affect T cell fate, but selectively regulate the Th1 differentiation in indirect manner, showing higher IFN-γ but lower IL-2 transcription in CD4\u0026thinsp;+\u0026thinsp;T cells deficient for PPARα \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. In this study, we for the first time showed that PPARα could bind to \u003cem\u003eIcos\u003c/em\u003e promoter for repression. Since the nuclear localization signal within the PPARα domain enables the cytoplasmic and nuclear shuttling \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, we also showed that blockade of nuclear transcription of PPARα could effectively prevent ICOS repression. Together, our results suggested that Cav-1/PPARα axis served as a checkpoint in Tfh cell tolerance, rather than a metabolic regulator.\u003c/p\u003e \u003cp\u003eAlthough altered Cav-1 expression levels have been reported in various diseases, including metabolic disorders, cerebrovascular disease, and cancer, however, investigation of the Cav-1 in autoimmune pathogenesis is scarce. Reduced Cav-1 expression has been reported in the skin tissues and dermal fibroblasts isolated from patients with systemic sclerosis \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. In this study, we observed decreased Cav-1 expression in CD4\u0026thinsp;+\u0026thinsp;T cells from both ESS mice and pSS patients, which was positively correlated with PPARα, but negatively correlated with ICOS expression. Although the relationship between Cav-1 and PPARα has been reported in other cell types, including cancer cells and hepatocytes, the underlying mechanism of how Cav-1 regulates PPARα was not clear. Recent finding suggested that lower bile acid levels in Cav-1-/- mice may account for insufficient FXRα activation, a critical regulator of PPARα in hepatocytes \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. This may be coincided with the comorbidity of liver involvement in pSS, as biliary cirrhosis was commonly seen in pSS, together with reduced bile acid levels \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Nonetheless, how Cav-1 regulates PPARα in CD4\u0026thinsp;+\u0026thinsp;T cells requires further investigation.\u003c/p\u003e \u003cp\u003eIn contrast to Cav-1, emerging evidence has reported the role of PPARs in autoimmune disorders. Agonists of PPARγ have been reported to interfere pro-inflammatory cytokines produced by various cell types, showing beneficial effects on mouse models of autoimmune thyroid diseases, multiple sclerosis and systemic lupus erythematosus \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. PPARα is well-conserved between humans and mice, as pharmaceutical agonists could selectively activate both human and murine PPARα \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, which potentiates the clinical application from drug screening using mouse models including experimental cholestatic liver disease and autoimmune dry eyes \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Fenofibrate has been clinically prescribed for decades, showing good tolerance and safety in long-term usage in humans \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Here, we demonstrated that fenofibrate effectively ameliorated established ESS mice, in particular under the chronic inflammatory conditions. Together, our findings suggest that targeting Cav-1 or PPARα may serve as a promising strategy for autoimmune disorders with Tfh cell dysregulation, while the expression levels of Cav-1 or PPARα may be considered as factors in patients partition and cohort stratification.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by grants through National Key Research and Development Program of China (2023YFE0203100), General Research Fund, Hong Kong Research Grants Council (17116521 and 27111820), Health and Medical Research Fund (19201121 and 20212601), Hong Kong Research Grants Council Area of Excellence Scheme 2016/2017 (No. AoE/P-705/16). We thanked the staff of Faculty Core Facility, Li Ka Shing Faculty of Medicine, the University of Hong Kong for their kind support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXL and JS conceptualized and supervised this study. XL designed the experiments. XL and JS provided research agents and animals. XL, JS and YF received the research fund. SY, MW and XL conducted the animal experiments and human samples from healthy donors. YC and JX assisted the animal experiments. YF and XL oversaw the conduct of patient recruitment and clinical investigations. Patient recruitment and screening were done by PHL, WZ and YF. Patient samples were analyzed by WF, WZ and PHL. XL constructed and JS revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no conflict of interest exists.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRothberg, K.G.\u003cem\u003e, et al.\u003c/em\u003e Caveolin, a protein component of caveolae membrane coats. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e68\u003c/strong\u003e, 673-682 (1992).\u003c/li\u003e\n\u003cli\u003eRazani, B.\u003cem\u003e, et al.\u003c/em\u003e Caveolin-1-deficient mice are lean, resistant to diet-induced obesity, and show hypertriglyceridemia with adipocyte abnormalities. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e277\u003c/strong\u003e, 8635-8647 (2002).\u003c/li\u003e\n\u003cli\u003eHaddad, D., Al Madhoun, A., Nizam, R. \u0026amp; Al-Mulla, F. 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A Review of Currently Available Fenofibrate and Fenofibric Acid Formulations. \u003cem\u003eCardiol Res\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 47-55 (2013).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3230861/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3230861/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eT follicular helper (Tfh) cells play a central role in humoral autoimmunity, including primary Sjögren’s syndrome (pSS). However, targeting Tfh cells is challenging in clinical management. Previous studies suggested inducible T­cell co­stimulator (ICOS) directed Tfh cell motility in engaging bystander B cells. Here, we identified a novel function of caveolin-1 (Cav-1) in restraining Tfh cell motility, in which \u003cem\u003eIcos\u003c/em\u003e transcription was repressed by peroxisome proliferator-activated receptor alpha (PPARα), unexpectedly, independence of lipid metabolism. In the context of autoimmunity, Cav-1 and PPARα expressions were decreased in CD4+ T cells from pSS patients and mice with experimental SS (ESS), while Cav-1 deficiency significantly exacerbated Tfh cell response and ESS pathology. Importantly, pharmaceutical activation of PPARα with fenofibrate effectively ameliorated ESS in mice with acute or chronic inflammation. These results revealed an unrecognized role of Cav-1/PPARα axis in Tfh cell tolerance, suggesting PPARα as a promising target in the treatment of humoral autoimmunity.\u003c/p\u003e","manuscriptTitle":"Caveolin-1 restrains pathogenic T follicular helper cell response in primary Sjögren’s syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-09-18 16:16:36","doi":"10.21203/rs.3.rs-3230861/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e091c573-d9bd-4610-a906-852c754e014d","owner":[],"postedDate":"September 18th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":24726839,"name":"Health sciences/Rheumatology/Rheumatic diseases/Connective tissue diseases/Sj\u0026#x00F6;gren's disease"},{"id":24726840,"name":"Biological sciences/Immunology/Autoimmunity"}],"tags":[],"updatedAt":"2023-12-08T15:05:54+00:00","versionOfRecord":[],"versionCreatedAt":"2023-09-18 16:16:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3230861","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3230861","identity":"rs-3230861","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}