Identifying a Ubiquitinated Adaptor Protein by a Viral E3 Ligase Through Co-immunoprecipitation.

Co-immunoprecipitation is a technique widely utilized to isolate protein complexes and study protein-protein interactions. Ubiquitinated proteins could be identified by combining co-immunoprecipitation with SDS-PAGE followed by immunoblotting. In this chapter, we use Herpes Simplex Virus 1 immediate-early protein ICP0-mediated polyubiquitination of p50 as an example to describe the method to identify a ubiquitinated adaptor protein by a viral E3 ligase by co-immunoprecipitation.

Cholesterol promotes IFNG mRNA expression in CD4+ effector/memory cells by SGK1 activation.

IFNγ-secreting T cells are central for the maintenance of immune surveillance within the central nervous system (CNS). It was previously reported in healthy donors that the T-cell environment in the CNS induces distinct signatures related to cytotoxic capacity, CNS trafficking, tissue adaptation, and lipid homeostasis. These findings suggested that the CNS milieu consisting predominantly of lipids mediated the metabolic conditions leading to IFNγ-secreting brain CD4 T cells. Here, we demonstrate that the supplementation of CD4+CD45RO+CXCR3+ cells with cholesterol modulates their function and increases IFNG expression. The heightened IFNG expression was mediated by the activation of the serum/glucocorticoid-regulated kinase (SGK1). Inhibition of SGK1 by a specific enzymatic inhibitor significantly reduces the expression of IFNG Our results confirm the crucial role of lipids in maintaining T-cell homeostasis and demonstrate a putative role of environmental factors to induce effector responses in CD4+ effector/memory cells. These findings offer potential avenues for further research targeting lipid pathways to modulate inflammatory conditions.

Upregulation of keratin 15 is required for varicella-zoster virus replication in keratinocytes and is attenuated in the live attenuated vOka vaccine strain.

Varicella-zoster virus (VZV) is the etiological agent of chickenpox and shingles, diseases characterised by epidermal virus replication in skin and mucosa and the formation of blisters. We have previously shown that VZV infection has a profound effect on keratinocyte differentiation, altering the normal pattern of epidermal gene expression. In particular, VZV infection reduces expression of suprabasal keratins 1 and 10 and desmosomal proteins, disrupting epidermal structure to promote expression of a blistering phenotype. Here, we extend these findings to show that VZV infection upregulates the expression of keratin 15 (KRT15), a marker expressed by basal epidermal keratinocytes and hair follicles stem cells. We demonstrate that KRT15 is essential for VZV replication in the skin, since downregulation of KRT15 inhibits VZV replication in keratinocytes, while KRT15 exogenous overexpression supports viral replication. Importantly, our data show that VZV upregulation of KRT15 depends on the expression of the VZV immediate early gene ORF62. ORF62 is the only regulatory gene that is mutated in the live attenuated VZV vaccine and contains four of the five fixed mutations present in the VZV Oka vaccine. Our data indicate that the mutated vaccine ORF62 is not capable of upregulating KRT15, suggesting that this may contribute to the vaccine attenuation in skin. Taken together our data present a novel association between VZV and KRT15, which may open a new therapeutic window for a topical targeting of VZV replication in the skin via modulation of KRT15.

The Pentamer glycoprotein complex inhibits viral Immediate Early transcription during Human Cytomegalovirus infections.

The Pentamer complex of Human Cytomegalovirus (HCMV) consists of the viral glycoproteins gH, gL, UL128, UL130, and UL131 and is incorporated into infectious virions. HCMV strains propagated extensively in vitro in fibroblasts carry UL128, UL130, or UL131 alleles that do not make a functional complex and thus lack Pentamer function. Adding functional Pentamer to such strains decreases virus growth in fibroblasts. Here, we show that the Pentamer inhibits productive HCMV replication in fibroblasts by repressing viral Immediate Early (IE) transcription. We show that ectopic expression of the viral IE1 protein, a target of Pentamer-mediated transcriptional repression, complements the growth defect of a Pentamer-positive virus. Furthermore, we show that the Pentamer also represses viral IE transcription in cell types where HCMV in vitro latency is studied. Finally, we identify UL130 as a functional subunit of the Pentamer for IE transcriptional repression and demonstrate that cyclic AMP Response Element (CRE) and NFkB sites within the Major Immediate Early Promoter that drives IE1 transcription contribute to this repression. We conclude that the HCMV Pentamer represses viral IE transcription.

G-quadruplex as an essential structural element in cytomegalovirus replication origin.

G-quadruplex (G4) structures are found in eukaryotic cell replication origins, but their role in origin function remains unclear. In this study G4 motifs are found in the lytic DNA replication origin (oriLyt) of human cytomegalovirus (HCMV) and recombinant viruses show that a G4 motif in oriLyt essential region I (ER-I) is necessary for viral growth. Replication assays of oriLyt-containing plasmids and biochemical/biophysical analyses show that G4 formation in ER-I is crucial for viral DNA replication. G4 pull-down analysis identifies viral DNA replication factors, such as IE2, UL84, and UL44, as G4-binding proteins. In enzyme-linked immunosorbent assays, specific G4-binding ligands inhibit G4 binding by the viral proteins. The Epstein-Barr virus oriLyt core element also forms a stable G4 that could substitute for the oriLyt ER-I G4 in HCMV. These results demonstrate that viral G4s in replication origins represent an essential structural element in recruiting replication factors and might be a therapeutic target against viral infections.

Inhibitory effects of Eplerenone on angiogenesis via modulating SGK1/TGF-ß pathway in contralateral kidney of CKD pregnancy rats.

BACKGROUND: Eplerenone is a selective aldosterone receptor blocker that is effective in preventing the progression of chroinic kidney disease (CKD). However, its mechanism and role in CKD pregnancy still remain uncertain. The aim of this study was to evaluate whether eplerenone could attenuated the fibrosis of unilateral ureteral obstruction (UUO) pregnant rats' contralateral kidney, improved pregnancy outcome and explore its therapeutic mechanisms. METHODS: A pregnancy rat model of UUO established, female Wistar rats were randomly assigned into sham-operated group (Sham group),sham-operated combined pregnancy group (SP group), unilateral ureteral obstruction combined pregnancy group (UUO + Pregnancy group), unilateral ureteral obstruction combined pregnancy, administered eplerenone (UUO + Pregnancy+Eplerenone group). On the 18th day of pregnancy, the rats were placed in a metabolic cage, 24 h urine was collected and stored at -80 °C. Next day, all animals were euthanized, and serum was collected by centrifugation and stored at -20 °C. Then the right kidney was extracted, a part of the kidney was placed in 4% paraformaldehyde for morphology, immunohistochemical staining, and immunofluorescence staining, and the other part was placed in a - 80 °C refrigerator for RNA and protein extraction. In vitro, HUVECs was treated with aldosterone, progesterone and estradiol, VEGFA and its receptor blocker bevacizumab. The ability of proliferation, migration and tubularization of HUVECs was detected by CCK-8, scratch wound assay and endothelial tube formation assay. And the co-expression of CD34 and α-SMA of HUVECs was detected by Flow cytometry. RESULTS: Immunofluorescence results showed that the co-expression of CD34 and α-SMA increased in the UUO + Pregnancy group was significantly increased. The expression of SGK-1, TGFß-1, Smad2, Smad3, VEGF-A, VEGFR2, CD34, α-SMA and Collagen I was significantly higher in the kidneys of the UUO + Pregnancy group compared to the Sham group and SP group. Eplerenone inhibited the expression of those results. In vitro, the ability of proliferation, migration and tubularization was increased after treated with aldosterone, aldosterone with progesterone and estradiol or VEGFA. Similarly, the expression of α-SMA on the surface of HUVECs treated with aldosterone, aldosterone with progesterone and estradiol were increased, while eplerenone supressed its expression. CONCLUSION: Eplerenone inhibits renal angiogenesis by blocking the SGK-1/TGFß signal transduction pathway, thereby inhibiting the phenotypic transformation of endothelial cells, slowing down renal fibrosis, and reducing kidney damage caused by pregnancy.

Serum and glucocorticoid-regulated kinase 1 (SGK1) as an emerging therapeutic target for cardiac diseases.

Cardiac diseases encompass a wide range of conditions that affect the structure and function of the heart. These conditions are a leading cause of morbidity and mortality worldwide. The serum- and glucocorticoid-inducible kinase 1 (SGK1) is a serine/threonine kinase that plays a significant role in various cellular processes, including cell survival and stress response. Alterations in SGK1 activity can have significant impacts on health and disease. Multiple research findings have indicated that SGK1 is associated with heart disease due to its involvement in cardiac hypertrophy and fibrosis. This article reviews different signaling pathways associated with SGK1 activity in various heart conditions, including the SGK1/NF-κB and PI3K/SGK1 pathways.

Placental growth factor mediates pathological uterine angiogenesis by activating the NFAT5-SGK1 signaling axis in the endometrium: implications for preeclampsia development.

After menstruation the uterine spiral arteries are repaired through angiogenesis. This process is tightly regulated by the paracrine communication between endometrial stromal cells (EnSCs) and endothelial cells. Any molecular aberration in these processes can lead to complications in pregnancy including miscarriage or preeclampsia (PE). Placental growth factor (PlGF) is a known contributing factor for pathological angiogenesis but the mechanisms remain poorly understood. In this study, we investigated whether PlGF contributes to pathological uterine angiogenesis by disrupting EnSCs and endothelial paracrine communication. We observed that PlGF mediates a tonicity-independent activation of nuclear factor of activated T cells 5 (NFAT5) in EnSCs. NFAT5 activated downstream targets including SGK1, HIF-1α and VEGF-A. In depth characterization of PlGF - conditioned medium (CM) from EnSCs using mass spectrometry and ELISA methods revealed low VEGF-A and an abundance of extracellular matrix organization associated proteins. Secreted factors in PlGF-CM impeded normal angiogenic cues in endothelial cells (HUVECs) by downregulating Notch-VEGF signaling. Interestingly, PlGF-CM failed to support human placental (BeWo) cell invasion through HUVEC monolayer. Inhibition of SGK1 in EnSCs improved angiogenic effects in HUVECs and promoted BeWo invasion, revealing SGK1 as a key intermediate player modulating PlGF mediated anti-angiogenic signaling. Taken together, perturbed PlGF-NFAT5-SGK1 signaling in the endometrium can contribute to pathological uterine angiogenesis by negatively regulating EnSCs-endothelial crosstalk resulting in poor quality vessels in the uterine microenvironment. Taken together the signaling may impact on normal trophoblast invasion and thus placentation and, may be associated with an increased risk of complications such as PE.

The role of BTG2/PI3K/AKT pathway-mediated microglial activation in T-2 toxin-induced neurotoxicity.

T-2 toxin is one of the mycotoxins widely distributed in human food and animal feed. Our recent work has shown that microglial activation may contribute to T-2 toxin-induced neurotoxicity. However, the molecular mechanisms involved need to be further clarified. To address this, we employed high-throughput transcriptome sequencing and found altered B cell translocation gene 2 (BTG2) expression levels in microglia following T-2 toxin treatment. It has been shown that altered BTG2 expression is involved in a range of neurological pathologies, but whether it's involved in the regulation of microglial activation is unclear. The aim of this study was to investigate the role of BTG2 in T-2 toxin-induced microglial activation. The results of animal experiments showed that T-2 toxin caused neurobehavioral disorders and promoted the expression of microglial BTG2 and pro-inflammatory activation of microglia in hippocampus and cortical, while microglial inhibitor minocycline inhibited these changes. The results of in vitro experiments showed that T-2 toxin enhanced BTG2 expression and pro-inflammatory microglial activation, and inhibited BTG2 expression weakened T-2 toxin-induced microglial activation. Moreover, T-2 toxin activated PI3K/AKT and its downstream NF-κB signaling pathway, which could be reversed after knock-down of BTG2 expression. Meanwhile, the PI3K inhibitor LY294002 also blocked this process. Therefore, BTG2 may be involved in T-2 toxin's ability to cause microglial activation through PI3K/AKT/NF-κB pathway.

Linear ubiquitination regulates the KSHV replication and transcription activator protein to control infection.

Like many other viruses, KSHV has two life cycle modes: the latent phase and the lytic phase. The RTA protein from KSHV is essential for lytic reactivation, but how this protein's activity is regulated is not fully understood. Here, we report that linear ubiquitination regulates the activity of RTA during KSHV lytic reactivation and de novo infection. Overexpressing OTULIN inhibits KSHV lytic reactivation, whereas knocking down OTULIN or overexpressing HOIP enhances it. Intriguingly, we found that RTA is linearly polyubiquitinated by HOIP at K516 and K518, and these modifications control the RTA's nuclear localization. OTULIN removes linear polyubiquitin chains from cytoplasmic RTA, preventing its nuclear import. The RTA orthologs encoded by the EB and MHV68 viruses are also linearly polyubiquitinated and regulated by OTULIN. Our study establishes that linear polyubiquitination plays a critically regulatory role in herpesvirus infection, adding virus infection to the list of biological processes known to be controlled by linear polyubiquitination.

MicroRNA-223-3p Targeting SGK1 Regulates Apoptosis and Inflammation in Sepsis-Associated Acute Kidney Injury.

INTRODUCTION: Sepsis and septic shock are significant contributors to the development of acute kidney injury (AKI) in critically ill patients. This study aimed to elucidate the role and mechanism of microRNA-223-3p in sepsis-associated AKI (SA-AKI). METHODS: Bioinformatics methods were used to analyze the expression of microRNA-223-3p in sepsis patients, its correlation with inflammatory cytokines, and to predict the binding site of microRNA-223-3p with SGK1. The binding relationship between microRNA-223-3p and SGK1 was validated using a dual-luciferase reporter gene assay. The expression of microRNA-223-3p was assayed using qPCR in patient serum or lipopolysaccharide (LPS)-treated HK-2 cells. Cell apoptosis; expression of Bax, Bcl-2, cleaved caspase-3; and levels of TNF-α, IL-1ß, and IL-6 were measured using TUNEL assay, Western blot (WB), and ELISA, respectively. SGK1 expression of HK-2 cells with different treatments was detected using qPCR and WB. RESULTS: The expression of microRNA-223-3p was found to be upregulated in sepsis patients and HK-2 cells treated with LPS. Furthermore, microRNA-223-3p promoted apoptosis and inflammation in LPS-induced HK-2 cells. This promotion was mediated by the negative regulation of SGK1 by microRNA-223-3p. CONCLUSION: The microRNA-223-3p was found to regulate SGK1 and promote apoptosis and inflammation in LPS-induced HK-2 cells. Our study has elucidated the mechanism of microRNA-223-3p in SA-AKI, providing a potential target for sepsis treatment.

SGK1 promotes the lipid accumulation via regulating the transcriptional activity of FOXO1 in bovine.

OBJECTIVES: Serum/glucocorticoid-inducible kinase 1 (SGK1) gene encodes a serine/threonine protein kinase that plays an essential role in cellular stress response and regulation of multiple metabolic processes. However, its role in bovine adipogenesis remains unknown. In this study, we aimed to clarify the role of SGK1 in bovine lipid accumulation and improvement of meat quality. METHODS: Preadipocytes were induced to differentiation to detect the temporal expression pattern of SGK1. Heart, liver, lung, spleen, kidney, muscle and fat tissues were collected to detect its tissue expression profile. Recombinant adenovirus and the lentivirus were packaged for overexpression and knockdown. Oil Red O staining, quantitative real-time PCR, Western blot analysis, Yeast two-hybrid assay, luciferase assay and RNA-seq were performed to study the regulatory mechanism of SGK1. RESULTS: SGK1 showed significantly higher expression in adipose and significantly induced expression in differentiated adipocytes. Furthermore, overexpression of SGK1 greatly promoted adipogenesis and inhibited proliferation, which could be shown by the remarkable increasement of lipid droplet, and the expression levels of adipogenic marker genes and cell cycle-related genes. Inversely, its knockdown inhibited adipogenesis and facilitated proliferation. Mechanistically, SGK1 regulates the phosphorylation and expression of two critical proteins of FoxO family, FOXO1/FOXO3. Importantly, SGK1 attenuates the transcriptional repression role of FOXO1 for PPARγ via phosphorylating the site S256, then promoting the bovine fat deposition. CONCLUSIONS: SGK1 is a required epigenetic regulatory factor for bovine preadipocyte proliferation and differentiation, which contributes to a better understanding of fat deposition and meat quality improvement in cattle.

Impact of the interaction between herpes simplex virus 1 ICP22 and FACT on viral gene expression and pathogenesis.

Facilitates chromatin transcription (FACT) interacts with nucleosomes to promote gene transcription by regulating the dissociation and reassembly of nucleosomes downstream and upstream of RNA polymerase II (Pol II). A previous study reported that herpes simplex virus 1 (HSV-1) regulatory protein ICP22 interacted with FACT and was required for its recruitment to the viral DNA genome in HSV-1-infected cells. However, the biological importance of interactions between ICP22 and FACT in relation to HSV-1 infection is unclear. Here, we mapped the minimal domain of ICP22 required for its efficient interaction with FACT to a cluster of five basic amino acids in ICP22. A recombinant virus harboring alanine substitutions in this identified cluster led to the decreased accumulation of viral mRNAs from UL54, UL38, and UL44 genes, reduced Pol II occupancy of these genes in MRC-5 cells, and impaired HSV-1 virulence in mice following ocular or intracranial infection. Furthermore, the treatment of mice infected with wild-type HSV-1 with CBL0137, a FACT inhibitor currently being investigated in clinical trials, significantly improved the survival rate of mice. These results suggested that the interaction between ICP22 and FACT was required for efficient HSV-1 gene expression and pathogenicity. Therefore, FACT might be a potential therapeutic target for HSV-1 infection.IMPORTANCEICP22 is a well-known regulatory factor of HSV-1 gene expression, but its mechanism(s) are poorly understood. Although the interaction of FACT with ICP22 was reported previously, its significance in HSV-1 infection is unknown. Given that FACT is involved in gene transcription, it is of interest to investigate this interaction as it relates to HSV-1 gene expression. To determine a direct link between the interaction and HSV-1 infection, we mapped a minimal domain of ICP22 required for its efficient interaction with FACT and generated a recombinant virus carrying mutations in the identified domain. Using the recombinant virus, we obtained evidence suggesting that the interaction between ICP22 and FACT promoted Pol II transcription from HSV-1 genes and viral virulence in mice. In addition, CBL0137, an inhibitor of FACT, effectively protected mice from lethal HSV-1 infection, suggesting FACT might be a potential target for the development of novel anti-HSV drugs.

The cellular Notch1 protein promotes KSHV reactivation in an Rta-dependent manner.

The cellular Notch signal transduction pathway is intimately associated with infections by Kaposi's sarcoma-associated herpesvirus (KSHV) and other gamma-herpesviruses. RBP-Jk, the cellular DNA binding component of the canonical Notch pathway, is the key Notch downstream effector protein in virus-infected and uninfected animal cells. Reactivation of KSHV from latency requires the viral lytic switch protein, Rta, to form complexes with RBP-Jk on numerous sites within the viral DNA. Constitutive Notch activity is essential for KSHV pathophysiology in models of Kaposi's sarcoma (KS) and Primary Effusion Lymphoma (PEL), and we demonstrate that Notch1 is also constitutively active in infected Vero cells. Although the KSHV genome contains >100 RBP-Jk DNA motifs, we show that none of the four isoforms of activated Notch can productively reactivate the virus from latency in a highly quantitative trans-complementing reporter virus system. Nevertheless, Notch contributed positively to reactivation because broad inhibition of Notch1-4 with gamma-secretase inhibitor (GSI) or expression of dominant negative mastermind-like1 (dnMAML1) coactivators severely reduced production of infectious KSHV from Vero cells. Reduction of KSHV production is associated with gene-specific reduction of viral transcription in both Vero and PEL cells. Specific inhibition of Notch1 by siRNA partially reduces the production of infectious KSHV, and NICD1 forms promoter-specific complexes with viral DNA during reactivation. We conclude that constitutive Notch activity is required for the robust production of infectious KSHV, and our results implicate activated Notch1 as a pro-viral member of a MAML1/RBP-Jk/DNA complex during viral reactivation. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) manipulates the host cell oncogenic Notch signaling pathway for viral reactivation from latency and cell pathogenesis. KSHV reactivation requires that the viral protein Rta functionally interacts with RBP-Jk, the DNA-binding component of the Notch pathway, and with promoter DNA to drive transcription of productive cycle genes. We show that the Notch pathway is constitutively active during KSHV reactivation and is essential for robust production of infectious virus progeny. Inhibiting Notch during reactivation reduces the expression of specific viral genes yet does not affect the growth of the host cells. Although Notch cannot reactivate KSHV alone, the requisite expression of Rta reveals a previously unappreciated role for Notch in reactivation. We propose that activated Notch cooperates with Rta in a promoter-specific manner that is partially programmed by Rta's ability to redistribute RBP-Jk DNA binding to the virus during reactivation.

Kv1.3-induced hyperpolarization is required for efficient Kaposi's sarcoma-associated herpesvirus lytic replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus that is linked directly to the development of Kaposi's sarcoma. KSHV establishes a latent infection in B cells, which can be reactivated to initiate lytic replication, producing infectious virions. Using pharmacological and genetic silencing approaches, we showed that the voltage-gated K+ channel Kv1.3 in B cells enhanced KSHV lytic replication. The KSHV replication and transcription activator (RTA) protein increased the abundance of Kv1.3 and led to enhanced K+ channel activity and hyperpolarization of the B cell membrane. Enhanced Kv1.3 activity promoted intracellular Ca2+ influx, leading to the Ca2+-driven nuclear localization of KSHV RTA and host nuclear factor of activated T cells (NFAT) proteins and subsequently increased the expression of NFAT1 target genes. KSHV lytic replication and infectious virion production were inhibited by Kv1.3 blockers or silencing. These findings highlight Kv1.3 as a druggable host factor that is key to the successful completion of KSHV lytic replication.

Matrix stiffness influences response to chemo and targeted therapy in brain metastatic breast cancer cells.

Breast cancer is the most common malignancy accounting for 12.5% of all newly diagnosed cancer cases across the globe. Breast cancer cells are known to metastasize to distant organs (i.e., brain), wherein they can exhibit a dormant phenotype for extended time periods. These dormant cancer cells exhibit reduced proliferation and therapeutic resistance. However, the mechanisms by which dormant cancer cells exhibit resistance to therapy, in the context of brain metastatic breast cancer (BMBC), is not well understood. Herein, we utilized hyaluronic acid (HA) hydrogels with varying stiffnesses to study drug responsiveness in dormant vs. proliferative BMBC cells. It was found that cells cultured on soft HA hydrogels (∼0.4 kPa) that showed a non-proliferative (dormant) phenotype exhibited resistance to Paclitaxel or Lapatinib. In contrast, cells cultured on stiff HA hydrogels (∼4.5 kPa) that showed a proliferative phenotype exhibited responsiveness to Paclitaxel or Lapatinib. Moreover, dormancy-associated resistance was found to be due to upregulation of the serum/glucocorticoid regulated kinase 1 (SGK1) gene which was mediated, in part, by the p38 signaling pathway. Accordingly, SGK1 inhibition resulted in a dormant-to-proliferative switch and response to therapy. Overall, our study demonstrates that matrix stiffness influences dormancy-associated therapy response mediated, in part, via the p38/SGK1 axis.

A viral E3 ubiquitin ligase produced by herpes simplex virus 1 inhibits the NLRP1 inflammasome.

Guard proteins initiate defense mechanisms upon sensing pathogen-encoded virulence factors. Successful viral pathogens likely inhibit guard protein activity, but these interactions have been largely undefined. Here, we demonstrate that the human pathogen herpes simplex virus 1 (HSV-1) stimulates and inhibits an antiviral pathway initiated by NLRP1, a guard protein that induces inflammasome formation and pyroptotic cell death when activated. Notably, HSV-1 infection of human keratinocytes promotes posttranslational modifications to NLRP1, consistent with MAPK-dependent NLRP1 activation, but does not result in downstream inflammasome formation. We identify infected cell protein 0 (ICP0) as the critical HSV-1 protein that is necessary and sufficient for inhibition of the NLRP1 pathway. Mechanistically, ICP0's cytoplasmic localization and function as an E3 ubiquitin ligase prevents proteasomal degradation of the auto-inhibitory NT-NLRP1 fragment, thereby preventing inflammasome formation. Further, we demonstrate that inhibiting this inflammasome is important for promoting HSV-1 replication. Thus, we have established a mechanism by which HSV-1 overcomes a guard-mediated antiviral defense strategy in humans.

Sodium chloride promotes macrophage pyroptosis and aggravates rheumatoid arthritis by activating SGK1 through GABA receptors Slc6a12.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovial inflammation and the production of autoantibodies. Previous studies have indicated an association between high-salt diets (HSD) and an increased risk of RA, yet the underlying mechanisms remain unclear. Macrophage pyroptosis, a pro-inflammatory form of cell death, plays a pivotal role in RA. In this study, we demonstrate that HSD exacerbates the severity of arthritis in collagen-induced arthritis (CIA) mice, correlating with macrophage infiltration and inflammatory lesions. Given the significant alterations observed in macrophages from CIA mice subjected to HSD, we specifically investigate the impact of HSD on macrophage responses in the inflammatory milieu of RA. In our in vitro experiments, pretreatment with NaCl enhances LPS-induced pyroptosis in RAW.264.7 and THP-1 cells through the p38 MAPK/NF-κB signaling pathway. Subsequent experiments reveal that Slc6a12 inhibitors and SGK1 silencing inhibit sodium-induced activation of macrophage pyroptosis and the p38 MAPK/NF-κB signaling pathway, whereas overexpression of the SGK1 gene counteracts the effect of sodium on macrophages. In conclusion, our findings verified that high salt intake promotes the progression of RA and provided a detailed elucidation of the activation of macrophage pyroptosis induced by sodium transportation through the Slc6a12 channel.

Effect of IL-9 neutralising antibody on pyroptosis via SGK1/NF-κB/NLRP3/GSDMD in allergic rhinitis mice.

Allergic rhinitis is a common non-infectious inflammatory disease that affects approximately 15 % of people worldwide and has a complex and unclear aetiology. In recent years, pyroptosis has been found to play a role in the development of allergic rhinitis. IL-9, pyroptosis, serum and glucocorticoid-induced protein kinase 1 (SGK1), NOD-like receptor 3 (NLRP3), and nuclear factor kappa B (NF-κB) have been shown to influence each other. Herein, we aimed to explore the role of IL-9 neutralising antibody in pyroptosis involving IL-9, SGK1, NF-κB, and NLRP3 in allergic rhinitis. We observed a decrease in cytokines involved in pyroptosis and gasdermin D (GSDMD) compared with those in mice with allergic rhinitis. Further, phosphorylation of NF-κB/p65 decreased compared with that in mice with allergic rhinitis; NLRP3 and ASC also decreased, although the levels were higher than those in controls. SGK1 levels decreased compared with that in mice with allergic rhinitis and increased after using IL-9 neutralising antibodies, thus demonstrating its negative regulatory effects. The IL-9 neutralising antibody reduced the inflammatory and pyroptosis responses via SGK1 and NF-κB/NLRP3/GSDMD pathway. Our research results indicate that IL-9 regulates allergic rhinitis via the influence of SGK1 and NF-κB/NLRP3/GSDMD signalling pathway, providing new insights for developing novel drugs to treat allergic rhinitis.

Deciphering the molecular nexus of BTG2 in periodontitis and diabetic kidney disease.

OBJECTIVE: To investigate the role of BTG2 in periodontitis and diabetic kidney disease (DKD) and its potential underlying mechanism. METHODS: Gene expression data for periodontitis and DKD were acquired from the Gene Expression Omnibus (GEO) database. Differential expression analysis identified co-expressed genes between these conditions. The Nephroseq V5 online nephropathy database validated the role of these genes in DKD. Pearson correlation analysis identified genes associated with our target gene. We employed Gene Set Enrichment Analysis (GSEA) and Protein-Protein Interaction (PPI) networks to elucidate potential mechanisms. Expression levels of BTG2 mRNA were examined using quantitative polymerase Chain Reaction (qPCR) and immunofluorescence assays. Western blotting quantified proteins involved in epithelial-to-mesenchymal transition (EMT), apoptosis, mTORC1 signaling, and autophagy. Additionally, wound healing and flow cytometric apoptosis assays evaluated podocyte migration and apoptosis, respectively. RESULTS: Analysis of GEO database data revealed BTG2 as a commonly differentially expressed gene in both DKD and periodontitis. BTG2 expression was reduced in DKD compared to normal conditions and correlated with proteinuria. GSEA indicated enrichment of BTG2 in the EMT and mTORC1 signaling pathways. The PPI network highlighted BTG2's relevance to S100A9, S100A12, and FPR1. Immunofluorescence assays demonstrated significantly lower BTG2 expression in podocytes under high glucose (HG) conditions. Reduced BTG2 expression in HG-treated podocytes led to increased levels of EMT markers (α-SMA, vimentin) and the apoptotic protein Bim, alongside a decrease in nephrin. Lower BTG2 levels were associated with increased podocyte mobility and apoptosis, as well as elevated RPS6KB1 and mTOR levels, but reduced autophagy marker LC3. CONCLUSION: Our findings suggest that BTG2 is a crucial intermediary gene linking DKD and periodontitis. Modulating autophagy via inhibition of the mTORC1 signaling pathway, and consequently suppressing EMT, may be pivotal in the interplay between periodontitis and DKD.