A lncRNA Dleu2-encoded peptide relieves autoimmunity by facilitating Smad3-mediated Treg induction.

Micropeptides encoded by short open reading frames (sORFs) within long noncoding RNAs (lncRNAs) are beginning to be discovered and characterized as regulators of biological and pathological processes. Here, we find that lncRNA Dleu2 encodes a 17-amino-acid micropeptide, which we name Dleu2-17aa, that is abundantly expressed in T cells. Dleu2-17aa promotes inducible regulatory T (iTreg) cell generation by interacting with SMAD Family Member 3 (Smad3) and enhancing its binding to the Foxp3 conserved non-coding DNA sequence 1 (CNS1) region. Importantly, the genetic deletion of Dleu2-17aa in mice by start codon mutation impairs iTreg generation and worsens experimental autoimmune encephalomyelitis (EAE). Conversely, the exogenous supplementation of Dleu2-17aa relieves EAE. Our findings demonstrate an indispensable role of Dleu2-17aa in maintaining immune homeostasis and suggest therapeutic applications for this peptide in treating autoimmune diseases.

Single-Cell RNA Sequencing Identifies WARS1+ Mesenchymal Stem Cells with Enhanced Immunomodulatory Capacity and Improved Therapeutic Efficacy.

Psoriasis is a common inflammatory skin disorder with no cure. Mesenchymal stem cells (MSCs) have immunomodulatory properties for psoriasis, but the therapeutic efficacies varied, and the molecular mechanisms were unknown. In this study, we improved the efficacy by enhancing the immunomodulatory effects of umbilical cord-derived MSCs (UC-MSCs). UC-MSCs stimulated by TNF-α and IFN-γ exhibited a better therapeutic effect in a mouse model of psoriasis. Single-cell RNA sequencing revealed that the stimulated UC-MSCs overrepresented a subpopulation expressing high tryptophanyl-tRNA synthetase 1 (WARS1). WARS1-overexpressed UC-MSCs treat psoriasis-like skin inflammation more efficiently than control UC-MSCs by restraining the proinflammatory macrophages. Mechanistically, WARS1 maintained a RhoA-Akt axis and governed the immunomodulatory properties of UC-MSCs. Together, we identify WARS1 as a master regulator of UC-MSCs with enhanced immunomodulatory capacities, which paves the way for the directed modification of UC-MSCs for escalated therapeutic efficacy.

The distinct initiation sites and processing activities of TTLL4 and TTLL7 in glutamylation of brain tubulin.

Mammalian brain tubulins undergo a reversible posttranslational modification-polyglutamylation-which attaches a secondary polyglutamate chain to the primary sequence of proteins. Loss of its erasers can disrupt polyglutamylation homeostasis and cause neurodegeneration. Tubulin tyrosine ligase like 4 (TTLL4) and TTLL7 were known to modify tubulins, both with preference for the ß-isoform, but differently contribute to neurodegeneration. However, differences in their biochemical properties and functions remain largely unknown. Here, using an antibody-based method, we characterized the properties of a purified recombinant TTLL4 and confirmed its sole role as an initiator, unlike TTLL7, which both initiates and elongates the side chains. Unexpectedly, TTLL4 produced stronger glutamylation immunosignals for α-isoform than ß-isoform in brain tubulins. Contrarily, the recombinant TTLL7 raised comparable glutamylation immunoreactivity for two isoforms. Given the site selectivity of the glutamylation antibody, we analyzed modification sites of two enzymes. Tandem mass spectrometry analysis revealed their incompatible site selectivity on synthetic peptides mimicking carboxyl termini of α1- and ß2-tubulins and a recombinant tubulin. Particularly, in the recombinant α1A-tubulin, a novel region was found glutamylated by TTLL4 and TTLL7, that again at distinct sites. These results pinpoint different site specificities between two enzymes. Moreover, TTLL7 exhibits less efficiency to elongate microtubules premodified by TTLL4, suggesting possible regulation of TTLL7 elongation activity by TTLL4-initiated sites. Finally, we showed that kinesin behaves differentially on microtubules modified by two enzymes. This study underpins the different reactivity, site selectivity, and function of TTLL4 and TTLL7 on brain tubulins and sheds light on their distinct role in vivo.

miPEP31 alleviates Ang II-induced hypertension in mice by occupying Cebpα binding sites in the pri-miR-31 promoter.

BACKGROUND: Previous studies have shown that peptides encoded by noncoding RNAs (ncRNAs) can be used as peptide drugs to alleviate diseases. We found that microRNA-31 (miR-31) is involved in the regulation of hypertension and that the peptide miPEP31, which is encoded by the primary transcript of miR-31 (pri-miR-31), can inhibit miR-31 expression. However, the role and mechanism of miPEP31 in hypertension have not been elucidated. METHODS: miPEP31 expression was determined by western blot analysis. miPEP31-deficient mice (miPEP31-/-) were used, and synthetic miPEP31 was injected into Ang II-induced hypertensive mice. Blood pressure was monitored through the tail-cuff method. Histological staining was used to evaluate renal damage. Regulatory T (Treg) cells were assessed by flow cytometry. Differentially expressed genes were analysed through RNA sequencing. The transcription factors were predicted by JASPAR. Luciferase reporter and electrophoretic mobility shift assays (EMSAs) were used to determine the effect of pri-miR-31 on the promoter activity of miPEP31. Images were taken to track the entry of miPEP31 into the cell. RESULTS: miPEP31 is endogenously expressed in target organs and cells related to hypertension. miPEP31 deficiency exacerbated but exogenous miPEP31 administration mitigated the Ang II-induced systolic blood pressure (SBP) elevation, renal impairment and Treg cell decreases in the kidney. Moreover, miPEP31 deletion increased the expression of genes related to Ang II-induced renal fibrosis. miPEP31 inhibited the transcription of miR-31 and promoted Treg differentiation by occupying the Cebpα binding site. The minimal functional domain of miPEP31 was identified and shown to regulate miR-31. CONCLUSION: miPEP31 was identified as a potential therapeutic peptide for treating hypertension by promoting Treg cell differentiation in vivo. Mechanistically, we found that miPEP31 acted as a transcriptional repressor to specifically inhibit miR-31 transcription by competitively occupying the Cebpα binding site in the pri-miR-31 promoter. Our study highlights the significant therapeutic effect of miPEP31 on hypertension and provides novel insight into the role and mechanism of miPEPs.

Transplantation of skin mesenchymal stem cells attenuated AngII-induced hypertension and vascular injury.

Skin mesenchymal stem cells (S-MSCs) revealed an important immunomodulatory activity to markedly suppress the formation of the atherosclerosis (AS) plaque by modulating macrophages, and also inhibit the development of experimental autoimmune encephalomyelitis (EAE) by regulating T helper 17 (Th17) cell differentiation. Macrophages and Th17 cells play important roles in hypertension. However, it remains unclear whether S-MSCs are capable of improving angiotensin (AngII)-induced hypertension by acting on inflammatory cells. Therefore, we studied a direct effect of S-MSC treatment on an AngII-induced hypertensive mouse model. Twenty-seven C57BL/6 (WT) mice were divided into three groups: Control group (WT-NC), AngII-infused group (WT-AngII), and S-MSC treatment group (WT-AngII + S-MSCs). In contrast to WT-AngII group, systolic blood pressure (SBP) and vascular damage were strikingly attenuated after tail-vein injection of S-MSCs. Numbers of Th17 cells in mouse peripheral blood of S-MSC treated group were significantly decreased, and IL-17 mRNA and protein levels were also reduced in the aorta and serum compared with WT-AngII group. Furthermore, macrophages in S-MSC treated group were switched to a regulatory profile characterized by a low ability to produce pro-inflammatory cytokine TNF-α and a high ability to produce anti-inflammatory cytokines Arg1 and IL-10. Mechanistically, we found that S-MSCs inhibited Th17 cell differentiation and induced M2 polarization. Moreover, we found proliferation and migration of S-MSCs were elevated, and expression of CXCR4, the receptor for Stromal derivated factor -1(SDF-1), was markedly increased in lipopolysaccharide (LPS)- stimulated S-MSCs. Given that SDF-1 expression was increased in the serum and aorta in AngII- induced hypertensive mice, the immunomodulatory effects exerted by S-MSCs involved the CXCR4/SDF-1 signaling. Collectively, our data demonstrated that S-MSCs attenuated AngII-induced hypertension by inhibiting Th17 cell differentiation and by modulating macrophage M2 polarization, suggesting that S-MSCs potentially have a role in stem cell based therapy for hypertension.

Tenascin C+ papillary fibroblasts facilitate neuro-immune interaction in a mouse model of psoriasis.

Dermal fibroblasts and cutaneous nerves are important players in skin diseases, while their reciprocal roles during skin inflammation have not been characterized. Here we identify an inflammation-induced subset of papillary fibroblasts that promotes aberrant neurite outgrowth and psoriasiform skin inflammation by secreting the extracellular matrix protein tenascin-C (TNC). Single-cell analysis of fibroblast lineages reveals a Tnc+ papillary fibroblast subset with pro-axonogenesis and neuro-regulation transcriptomic hallmarks. TNC overexpression in fibroblasts boosts neurite outgrowth in co-cultured neurons, while fibroblast-specific TNC ablation suppresses hyperinnervation and alleviates skin inflammation in male mice modeling psoriasis. Dermal γδT cells, the main producers of type 17 pathogenic cytokines, frequently contact nerve fibers in mouse psoriasiform lesions and are likely modulated by postsynaptic signals. Overall, our results highlight the role of an inflammation-responsive fibroblast subset in facilitating neuro-immune synapse formation and suggest potential avenues for future therapeutic research.

Protein phosphatase 6 (Pp6) is crucial for regulatory T cell function and stability in autoimmunity.

Regulatory T (Treg) cells constitute a dynamic population that is critical in autoimmunity. Treg cell therapies for autoimmune diseases are mainly focused on enhancing their suppressive activities. However, recent studies demonstrated that certain inflammatory conditions induce Treg cell instability with diminished FoxP3 expression and convert them into pathogenic effector cells. Therefore, the identification of novel targets crucial to both Treg cell function and plasticity is of vital importance to the development of therapeutic approaches in autoimmunity. In this study, we found that conditional Pp6 knockout (cKO) in Treg cells led to spontaneous autoinflammation, immune cell activation, and diminished levels of FoxP3 in CD4+ T cells in mice. Loss of Pp6 in Treg cells exacerbated two classical mouse models of Treg-related autoinflammation. Mechanistically, Pp6 deficiency increased CpG motif methylation of the FoxP3 locus by dephosphorylating Dnmt1 and enhancing Akt phosphorylation at Ser473/Thr308, leading to impaired FoxP3 expression in Treg cells. In summary, our study proposes Pp6 as a critical positive regulator of FoxP3 that acts by decreasing DNA methylation of the FoxP3 gene enhancer and inhibiting Akt signaling, thus maintaining Treg cell stability and preventing autoimmune diseases.

Mitochondrial activity regulates the differentiation of skin-derived mesenchymal stem cells into brown adipocytes to contribute to hypertension.

BACKGROUND: Brown adipocytes (BAs) are major components of brown adipose tissue (BAT), which is involved in blood pressure regulation. BAs are derived from multiple progenitors, including PDGFRα+ adipose-derived stem cells (ASCs). Skin-derived mesenchymal stem cells (S-MSCs) have the capacity to differentiate into adipocytes; however, their ability to differentiate into BAs remains unexplored. We aim to study the ability and regulatory mechanism of the differentiation of S-MSCs into BAs and the direct role of BAT in blood pressure regulation. METHODS: Protein expression was measured by flow cytometry or Western blotting, and gene mRNA levels were quantified by real-time quantitative PCR (RT-PCR). To induce the differentiation of S-MSCs into BAs, S-MSCs were stimulated with a brown adipogenic cocktail comprising insulin, IBMX, dexamethasone, triiodothyronine (T3), and rosiglitazone for the indicated periods. The oxygen consumption rate (OCR) was measured with an XF24 Extracellular Flux Analyzer. Mitochondrial mass was determined by flow cytometry and fluorescence staining. Hypertension was induced in WT mice by infusion of angiotensin II (Ang II), and systolic blood pressure (SBP) was measured using a tail cuff. Interscapular brown adipose tissue (iBAT)-deficient mice were generated by surgical removal of the iBAT depot, after which the animals were allowed to recover for 6 days. Aortic, iBAT, and heart tissue sections were analyzed by hematoxylin and eosin (HE) staining. RESULTS: We found that in vitro, S-MSCs isolated from the mouse dermis expressed the stem cell markers CD90/105 and PDGFRα and readily differentiated into BAs. Mitochondrial biogenesis and oxygen consumption were markedly increased during differentiation of S-MSCs into BAs. In vivo, iBAT was converted to white adipose tissue (WAT) in Ang II-induced hypertensive mice. We assessed the direct role of BAT in blood pressure (BP) regulation by using iBAT-deficient mice (generated by surgical removal of iBAT) and C57BL/6 (wild-type (WT)) mice and found that Ang II-induced BP elevation and vascular damage were markedly aggravated in iBAT-deficient mice compared with WT mice. CONCLUSIONS: This study demonstrates that PDGFRα+ S-MSCs are able to differentiate into BAs and that this differentiation is regulated by mitochondrial activity. We also show that BAT plays a direct role in ameliorating Ang II-induced hypertension. The therapeutic potential of BAT for the prevention of hypertension-induced organ remodeling thus warrants further investigation.

HIF-1α-BNIP3-mediated mitophagy in tubular cells protects against renal ischemia/reperfusion injury.

In the present study, we hypothesized that hypoxia-inducible factor 1α (HIF-1α)-mediated mitophagy plays a protective role in ischemia/reperfusion (I/R)-induced acute kidney injury (AKI). Mitophagy was evaluated by measuring the changes of mitophagy flux, mitochondria DNA copy number, and the changes of mitophagy-related proteins including translocase of outer mitochondrial membrane 20 (TOMM20), cytochrome c oxidase IV (COX IV), microtubule-associated protein 1 light chain 3B (LC3B), and mitochondria adaptor nucleoporin p62 in HK2 cells, a human tubular cell line. Results show that HIF-1α knockout significantly attenuated hypoxia/reoxygenation (H/R)-induced mitophagy, aggravated H/R-induced apoptosis, and increased the production of reactive oxygen species (ROS). Similarly, H/R induced significantly increase in Bcl-2 19-kDa interacting protein 3 (BNIP3), a downstream regulator of HIF-1α. Notably, BNIP3 overexpression reversed the inhibitory effect of HIF-1α knockout on H/R-induced mitophagy, and prevented the enhancing effect of HIF-1α knockout on H/R-induced apoptosis and ROS production. For in vivo study, we established HIF-1αflox/flox; cadherin-16-cre mice in which tubular HIF-1α was specifically knockout. It was found that tubular HIF-1α knockout significantly inhibited I/R-induced mitophagy, and aggravated I/R-induced tubular apoptosis and kidney damage. In contrast, adenovirus-mediated BNIP3 overexpression significantly reversed the decreased mitophagy, and prevented enhanced kidney damage in tubular HIF-1α knockout mice with I/R injury. In summary, our study demonstrated that HIF-1α-BNIP3-mediated mitophagy in tubular cells plays a protective role through inhibition of apoptosis and ROS production in acute kidney damage.

MicroRNA-31 Regulates Immunosuppression in Ang II (Angiotensin II)-induced Hypertension by Targeting Ppp6C (Protein Phosphatase 6c).

Regulatory T cells (Treg cells) play important roles in hypertension and organ damages. MicroRNA-31 (miR-31) is a critical regulator for Treg cell generation. However, the role of miR-31 in hypertension has not been elucidated. We aim to study the functionality of miR-31 and the detailed mechanism in Ang II (Angiotensin II)-induced hypertensive mouse model. We found: In vitro, miR-31 expression was higher in T helper 17 cells and lower in Treg cells than that of naïve T cells. The genetic deficiency of miR-31 promoted Treg cell differentiation, whereas no impact on T helper 17 cells differentiation. Ang II-induced hypertension resulted in increased expression of miR-31 in the aorta, splenic CD4+ T cells, and kidney leukocytes. MiR-31 deficiency strikingly decreased systolic blood pressure and diastolic blood pressure and attenuated renal and vascular damage. MiR-31 deletion altered the accumulation of Treg cells and macrophages and expression of inflammatory cytokines in kidneys in Ang II-induced hypertensive mice. Ang II treatment reduced the levels of anti-inflammatory cytokines and increased proinflammatory cytokines in plasma that were blunted by the miR-31 deletion. Ppp6C (protein phosphatase 6c; a direct target of miR-31) specific deletion in Treg cells led to marked impairment of Treg cell induction, increased Ang II-induced blood pressure elevation, and organ damage in mice. In conclusion, we provided novel evidence of miR-31 as an emerging key posttranscriptional regulator of hypertension-associated immunosuppression through targeting ppp6C which is a critical regulator in the differentiation of Treg cells. This study offers new perspectives on miRNA-based therapeutic approaches.

Catalyst and additive-free regioselective oxidative C-H thio/selenocyanation of arenes and heteroarenes with elemental sulfur/selenium and TMSCN.

A regioselective oxidative C-H thio/selenocyanation of arenes and heteroarenes with TMSCN and elemental sulfur/selenium was demonstrated under catalyst-free and additive-free conditions. Dimethyl sulfoxide (DMSO) was employed as the mild oxidant as well as the solvent. The reaction is operationally simple and scalable with a broad substrate scope.