GPR161 structure uncovers the redundant role of sterol-regulated ciliary cAMP signaling in the Hedgehog pathway.

The orphan G protein-coupled receptor (GPCR) GPR161 plays a central role in development by suppressing Hedgehog signaling. The fundamental basis of how GPR161 is activated remains unclear. Here, we determined a cryogenic-electron microscopy structure of active human GPR161 bound to heterotrimeric Gs. This structure revealed an extracellular loop 2 that occupies the canonical GPCR orthosteric ligand pocket. Furthermore, a sterol that binds adjacent to transmembrane helices 6 and 7 stabilizes a GPR161 conformation required for Gs coupling. Mutations that prevent sterol binding to GPR161 suppress Gs-mediated signaling. These mutants retain the ability to suppress GLI2 transcription factor accumulation in primary cilia, a key function of ciliary GPR161. By contrast, a protein kinase A-binding site in the GPR161 C terminus is critical in suppressing GLI2 ciliary accumulation. Our work highlights how structural features of GPR161 interface with the Hedgehog pathway and sets a foundation to understand the role of GPR161 function in other signaling pathways.

Gut microbiome-derived butyrate inhibits the immunosuppressive factors PD-L1 and IL-10 in tumor-associated macrophages in gastric cancer.

Early detection and surgical treatment are essential to achieve a good outcome in gastric cancer (GC). Stage IV and recurrent GC have a poor prognosis. Therefore, new treatments for GC are needed. We investigated the intestinal microbiome of GC patients and attempted to reverse the immunosuppression of the immune and cancer cells of GC patients through the modulation of microbiome metabolites. We evaluated the levels of programmed death-ligand 1 (PD-L1) and interleukin (IL)-10 in the peripheral blood immunocytes of GC patients. Cancer tissues were obtained from patients who underwent surgical resection of GC, and stained sections of cancer tissues were visualized via confocal microscopy. The intestinal microbiome was analyzed using stool samples of healthy individuals and GC patients. Patient-derived avatar model was developed by injecting peripheral blood mononuclear cells (PBMCs) from advanced GC (AGC) patients into NSG mice, followed by injection of AGS cells. PD-L1 and IL-10 had higher expression levels in immune cells of GC patients than in those of healthy controls. The levels of immunosuppressive factors were increased in the immune and tumor cells of tumor tissues of GC patients. The abundances of Faecalibacterium and Bifidobacterium in the intestinal flora were lower in GC patients than in healthy individuals. Butyrate, a representative microbiome metabolite, suppressed the expression levels of PD-L1 and IL-10 in immune cells. In addition, the PBMCs of AGC patients showed increased levels of immunosuppressive factors in the avatar mouse model. Butyrate inhibited tumor growth in mice. Restoration of the intestinal microbiome and its metabolic functions inhibit tumor growth and reverse the immunosuppression due to increased PD-L1 and IL-10 levels in PBMCs and tumor cells of GC patients.

Effect of IL-10-producing B cells in peripheral blood and tumor tissue on gastric cancer.

BACKGROUND: Interleukin (IL)-10-producing B (B10) cells are generated in response to signals from the tumor microenvironment and promote tumor growth by interacting with B10 cells. We investigated the distributions of immune cells in peripheral blood and tumor tissue samples from patients with gastric cancer (GC). METHODS: Patients with GC who underwent radical gastrectomy in Seoul St. Mary's Hospital between August 2020 and May 2021 were enrolled in this study. Forty-two samples of peripheral blood were collected, and a pair of gastric mucosal samples (normal and cancerous mucosa; did not influence tumor diagnosis or staging) was collected from each patient after surgery. B10 cells in peripheral blood and cancer mucosa samples were investigated by flow cytometry and immunofluorescence. AGS cells, gastric cancer cell line, were cultured with IL-10 and measured cell death and cytokine secretion. Also, AGS cells were co-cultured with CD19 + B cells and measured cytokine secretion. RESULTS: The population of B10 cells was significantly larger in the blood of patients with GC compared with controls. In confocal images of gastric mucosal tissues, cancerous mucosa contained more B10 cells than normal mucosa. The population of B10 cells in cancerous mucosa increased with cancer stage. When AGS cells were cultured under cell-death conditions, cellular necrosis was significantly decreased, and proliferation was increased, for 1 day after IL-10 stimulation. Tumor necrosis factor (TNF)-α, IL-8, IL-1ß, and vascular endothelial growth factor secretion by cancer cells was significantly increased by coculture of AGS cells with GC-derived CD19+ B cells. CONCLUSIONS: B cells may be one of the populations that promote carcinogenesis by inducing the production of inflammatory mediators, such as IL-10, in GC. Targeting B10 cells activity could improve the outcomes of antitumor immunotherapy. Video Abstract.

Context-dependent ciliary regulation of hedgehog pathway repression in tissue morphogenesis.

A fundamental problem in tissue morphogenesis is identifying how subcellular signaling regulates mesoscale organization of tissues. The primary cilium is a paradigmatic organelle for compartmentalized subcellular signaling. How signaling emanating from cilia orchestrates tissue organization-especially, the role of cilia-generated effectors in mediating diverse morpho-phenotypic outcomes-is not well understood. In the hedgehog pathway, bifunctional GLI transcription factors generate both GLI-activators (GLI-A) and GLI-repressors (GLI-R). The formation of GLI-A/GLI-R requires cilia. However, how these counterregulatory effectors coordinate cilia-regulated morphogenetic pathways is unclear. Here we determined GLI-A/GLI-R requirements in phenotypes arising from lack of hedgehog pathway repression (derepression) during mouse neural tube and skeletal development. We studied hedgehog pathway repression by the GPCR GPR161, and the ankyrin repeat protein ANKMY2 that direct cAMP/protein kinase-A signaling by cilia in GLI-R generation. We performed genetic epistasis between Gpr161 or Ankmy2 mutants, and Gli2/Gli3 knockouts, Gli3R knock-in and knockout of Smoothened, the hedgehog pathway transducer. We also tested the role of cilia-generated signaling using a Gpr161 ciliary localization knock-in mutant that is cAMP signaling competent. We found that the cilia-dependent derepression phenotypes arose in three modes: lack of GLI-R only, excess GLI-A formation only, or dual regulation of either lack of GLI-R or excess GLI-A formation. These modes were mostly independent of Smoothened. The cAMP signaling-competent non-ciliary Gpr161 knock-in recapitulated Gpr161 loss-of-function tissue phenotypes solely from lack of GLI-R only. Our results show complex tissue-specific GLI-effector requirements in morphogenesis and point to tissue-specific GLI-R thresholds generated by cilia in hedgehog pathway repression. Broadly, our study sets up a conceptual framework for rationalization of different modes of signaling generated by the primary cilium in mediating morphogenesis in diverse tissues.

GPR161 structure uncovers the redundant role of sterol-regulated ciliary cAMP signaling in the Hedgehog pathway.

The orphan G protein-coupled receptor (GPCR) GPR161 is enriched in primary cilia, where it plays a central role in suppressing Hedgehog signaling1. GPR161 mutations lead to developmental defects and cancers2,3,4. The fundamental basis of how GPR161 is activated, including potential endogenous activators and pathway-relevant signal transducers, remains unclear. To elucidate GPR161 function, we determined a cryogenic-electron microscopy structure of active GPR161 bound to the heterotrimeric G protein complex Gs. This structure revealed an extracellular loop 2 that occupies the canonical GPCR orthosteric ligand pocket. Furthermore, we identify a sterol that binds to a conserved extrahelical site adjacent to transmembrane helices 6 and 7 and stabilizes a GPR161 conformation required for Gs coupling. Mutations that prevent sterol binding to GPR161 suppress cAMP pathway activation. Surprisingly, these mutants retain the ability to suppress GLI2 transcription factor accumulation in cilia, a key function of ciliary GPR161 in Hedgehog pathway suppression. By contrast, a protein kinase A-binding site in the GPR161 C-terminus is critical in suppressing GLI2 ciliary accumulation. Our work highlights how unique structural features of GPR161 interface with the Hedgehog pathway and sets a foundation to understand the broader role of GPR161 function in other signaling pathways.

Lactobacillus acidophilus and propionate attenuate Sjögren's syndrome by modulating the STIM1-STING signaling pathway.

BACKGROUND: Sjögren's syndrome (SS) is an autoimmune disease characterized by inflammation of the exocrine gland. An imbalance of gut microbiota has been linked to SS. However, the molecular mechanism is unclear. We investigated the effects of Lactobacillus acidophilus (L. acidophilus) and propionate on the development and progression of SS in mouse model. METHODS: We compared the gut microbiomes of young and old mice. We administered L. acidophilus and propionate up to 24 weeks. The saliva flow rate and the histopathology of the salivary glands were investigated, and the effects of propionate on the STIM1-STING signaling pathway were evaluated in vitro. RESULTS: Lactobacillaceae and Lactobacillus were decreased in aged mice. SS symptoms were ameliorated by L. acidophilus. The abundance of propionate-producing bacterial was increased by L. acidophilus. Propionate ameliorated the development and progression of SS by inhibiting the STIM1-STING signaling pathway. CONCLUSIONS: The findings suggest that Lactobacillus acidophilus and propionate have therapeutic potential for SS. Video Abstract.

Human IFT-A complex structures provide molecular insights into ciliary transport.

Intraflagellar transport (IFT) complexes, IFT-A and IFT-B, form bidirectional trains that move along the axonemal microtubules and are essential for assembling and maintaining cilia. Mutations in IFT subunits lead to numerous ciliopathies involving multiple tissues. However, how IFT complexes assemble and mediate cargo transport lacks mechanistic understanding due to missing high-resolution structural information of the holo-complexes. Here we report cryo-EM structures of human IFT-A complexes in the presence and absence of TULP3 at overall resolutions of 3.0-3.9 Å. IFT-A adopts a "lariat" shape with interconnected core and peripheral subunits linked by structurally vital zinc-binding domains. TULP3, the cargo adapter, interacts with IFT-A through its N-terminal region, and interface mutations disrupt cargo transport. We also determine the molecular impacts of disease mutations on complex formation and ciliary transport. Our work reveals IFT-A architecture, sheds light on ciliary transport and IFT train formation, and enables the rationalization of disease mutations in ciliopathies.

Interactions between TULP3 tubby domain and ARL13B amphipathic helix promote lipidated protein transport to cilia.

The primary cilium is a nexus for cell signaling and relies on specific protein trafficking for function. The tubby family protein TULP3 transports integral membrane proteins into cilia through interactions with the intraflagellar transport complex-A (IFT-A) and phosphoinositides. It was previously shown that short motifs called ciliary localization sequences (CLSs) are necessary and sufficient for TULP3-dependent ciliary trafficking of transmembrane cargoes. However, the mechanisms by which TULP3 regulates ciliary compartmentalization of nonintegral, membrane-associated proteins and whether such trafficking requires TULP3-dependent CLSs is unknown. Here we show that TULP3 is required for ciliary transport of the Joubert syndrome-linked palmitoylated GTPase ARL13B through a CLS. An N-terminal amphipathic helix, preceding the GTPase domain of ARL13B, couples with the TULP3 tubby domain for ciliary trafficking, irrespective of palmitoylation. ARL13B transport requires TULP3 binding to IFT-A but not to phosphoinositides, indicating strong membrane-proximate interactions, unlike transmembrane cargo transport requiring both properties of TULP3. TULP3-mediated trafficking of ARL13B also regulates ciliary enrichment of farnesylated and myristoylated downstream effectors of ARL13B. The lipidated cargoes show distinctive depletion kinetics from kidney epithelial cilia with relation to Tulp3 deletion-induced renal cystogenesis. Overall, these findings indicate an expanded role of the tubby domain in capturing analogous helical secondary structural motifs from diverse cargoes.

EC-18 prevents autoimmune arthritis by suppressing inflammatory cytokines and osteoclastogenesis.

BACKGROUND: EC-18, a synthetic monoacetyldiaglyceride, exhibits protective effects against lung inflammation, allergic asthma, and abdominal sepsis. However, there have been no investigations to determine whether EC-18 has preventive potential in autoimmune diseases, especially rheumatoid arthritis (RA). METHODS: To investigate the efficacy of EC-18 on the development of RA, EC-18 was administered in a collagen-induced arthritis (CIA) murine model and disease severity and the level of inflammatory cytokines in the joint were investigated. The effect of EC-18 on the inflammation-related factors was investigated by flow cytometry, ELISA, western blot, and real-time PCR in splenocytes from mice and in peripheral blood mononuclear cells from healthy and patients with RA. The effect of EC-18 on osteoclastogenesis was investigated. RESULTS: EC-18 effectively reduced the clinical and histological severity of arthritis, similar to Janus kinase inhibitors include tofacitinib and baricitinib, in CIA. Furthermore, EC-18 exhibited a synergistic effect with methotrexate in preventing CIA. Treatment with EC-18 effectively reduced the production of inflammatory cytokines in immune cells and osteoclast differentiation in mice and patients with RA. CONCLUSION: These results suggest that EC-18 may be an effective strategy for RA.

Cilia-Localized Counterregulatory Signals as Drivers of Renal Cystogenesis.

Primary cilia play counterregulatory roles in cystogenesis-they inhibit cyst formation in the normal renal tubule but promote cyst growth when the function of polycystins is impaired. Key upstream cilia-specific signals and components involved in driving cystogenesis have remained elusive. Recent studies of the tubby family protein, Tubby-like protein 3 (TULP3), have provided new insights into the cilia-localized mechanisms that determine cyst growth. TULP3 is a key adapter of the intraflagellar transport complex A (IFT-A) in the trafficking of multiple proteins specifically into the ciliary membrane. Loss of TULP3 results in the selective exclusion of its cargoes from cilia without affecting their extraciliary pools and without disrupting cilia or IFT-A complex integrity. Epistasis analyses have indicated that TULP3 inhibits cystogenesis independently of the polycystins during kidney development but promotes cystogenesis in adults when polycystins are lacking. In this review, we discuss the current model of the cilia-dependent cyst activation (CDCA) mechanism in autosomal dominant polycystic kidney disease (ADPKD) and consider the possible roles of ciliary and extraciliary polycystins in regulating CDCA. We then describe the limitations of this model in not fully accounting for how cilia single knockouts cause significant cystic changes either in the presence or absence of polycystins. Based on available data from TULP3/IFT-A-mediated differential regulation of cystogenesis in kidneys with deletion of polycystins either during development or in adulthood, we hypothesize the existence of cilia-localized components of CDCA (cCDCA) and cilia-localized cyst inhibition (CLCI) signals. We develop the criteria for cCDCA/CLCI signals and discuss potential TULP3 cargoes as possible cilia-localized components that determine cystogenesis in kidneys during development and in adult mice.

B Cell-Specific Deletion of CR6-Interacting Factor 1 Drives Lupus-like Autoimmunity by Activation of Interleukin-17, Interleukin-6, and Pathogenic Follicular Helper T Cells in a Mouse Model.

OBJECTIVE: CR6-interacting factor 1 (CRIF1) is a nuclear transcriptional regulator and a mitochondrial inner membrane protein; however, its functions in B lymphocytes have been poorly defined. This study was undertaken to investigate the effects of CRIF1 on B cell metabolic regulation, cell function, and autoimmune diseases. METHODS: Using mice with B cell-specific deletion of CRIF1 (Crif1ΔCD19 mice), we assessed the relevance of CRIF1 function for lupus disease parameters, including anti-double-stranded DNA (anti-dsDNA), cytokines, and kidney pathology. RNA sequencing was performed on B cells from Crif1ΔCD19 mice. The phenotypic and metabolic changes in immune cells were evaluated in Crif1ΔCD19 mice. Roquinsan/+ mice crossed with Crif1ΔCD19 mice were monitored to assess the functionality of CRIF1-deficient B cells in lupus development. RESULTS: Crif1ΔCD19 mice showed an autoimmune lupus-like phenotype, including high levels of autoantibodies to dsDNA and severe lupus nephritis with increased mesangial hypercellularity. While loss of CRIF1 in B cells showed impaired mitochondrial oxidative function, CRIF1-deficient B cells promoted the production of interleukin-17 (IL-17) and IL-6 and was more potent in helping T cells develop into follicular helper T cells. In a mouse model of autoimmune lupus, depletion of CRIF1 in B cells exacerbated lupus severity, and CRIF1 overexpression prevented lupus development in roquinsan/san mice. CONCLUSION: These results demonstrated that CRIF1 negatively correlates with disease severity and that overexpression of CRIF1 ameliorates disease development. Our findings suggest that CRIF1 is essential for preventing lupus development by maintaining B cell self tolerance.

IL-17 and CCR9+α4ß7- Th17 Cells Promote Salivary Gland Inflammation, Dysfunction, and Cell Death in Sjögren's Syndrome.

Previous studies have evaluated the roles of T and B cells in the pathogenesis of Sjögren's syndrome (SS); however, their relationships with age-dependent and metabolic abnormalities remain unclear. We examined the impacts of changes associated with aging or metabolic abnormalities on populations of T and B cells and SS disease severity. We detected increased populations of IL-17-producing T and B cells, which regulate inflammation, in the salivary glands of NOD/ShiLtJ mice. Inflammation-induced human submandibular gland cell death, determined based on p-MLKL and RIPK3 expression levels, was significantly increased by IL-17 treatment. Among IL-17-expressing cells in the salivary gland, peripheral blood, and spleen, the α4ß7 (gut-homing integrin)-negative population was significantly increased in aged NOD/ShiLtJ mice. The α4ß7-positive population markedly increased in the intestines of aged NOD/ShiLtJ mice following retinoic acid (RA) treatment. A significant increase in α4ß7-negative IL-17-expressing cells in salivary glands may be involved in the onset and progression of SS. These results suggest the potential therapeutic utility of RA in SS treatment.

Ciliary and extraciliary Gpr161 pools repress hedgehog signaling in a tissue-specific manner.

The role of compartmentalized signaling in primary cilia during tissue morphogenesis is not well understood. The cilia localized G protein-coupled receptor, Gpr161, represses hedgehog pathway via cAMP signaling. We engineered a knock-in at the Gpr161 locus in mice to generate a variant (Gpr161mut1), which was ciliary localization defective but cAMP signaling competent. Tissue phenotypes from hedgehog signaling depend on downstream bifunctional Gli transcriptional factors functioning as activators or repressors. Compared to knockout (ko), Gpr161mut1/ko had delayed embryonic lethality, moderately increased hedgehog targets, and partially down-regulated Gli3 repressor. Unlike ko, the Gpr161mut1/ko neural tube did not show Gli2 activator-dependent expansion of ventral-most progenitors. Instead, the intermediate neural tube showed progenitor expansion that depends on loss of Gli3 repressor. Increased extraciliary receptor levels in Gpr161mut1/mut1 prevented ventralization. Morphogenesis in limb buds and midface requires Gli repressor; these tissues in Gpr161mut1/mut1 manifested hedgehog hyperactivation phenotypes-polydactyly and midfacial widening. Thus, ciliary and extraciliary Gpr161 pools likely establish tissue-specific Gli repressor thresholds in determining morpho-phenotypic outcomes.

Metformin-Inducible Small Heterodimer Partner Interacting Leucine Zipper Protein Ameliorates Intestinal Inflammation.

Small heterodimer partner interacting leucine zipper protein (SMILE) is an orphan nuclear receptor and a member of the bZIP family of proteins. We investigated the mechanism by which SMILE suppressed the development of inflammatory bowel disease (IBD) using a DSS-induced colitis mouse model and peripheral blood mononuclear cells (PBMCs) from patients with ulcerative colitis (UC). Metformin, an antidiabetic drug and an inducer of AMPK, upregulated the level of SMILE in human intestinal epithelial cells and the number of SMILE-expressing cells in colon tissues from DSS-induced colitis mice compared to control mice. Overexpression of SMILE using a DNA vector reduced the severity of DSS-induced colitis and colitis-associated intestinal fibrosis compared to mock vector. Furthermore, SMILE transgenic mice showed ameliorated DSS-induced colitis compared with wild-type mice. The mRNA levels of SMILE and Foxp3 were downregulated and SMILE expression was positively correlated with Foxp3 in PBMCs from patients with UC and an inflamed mucosa. Metformin increased the levels of SMILE, AMPK, and Foxp3 but decreased the number of interleukin (IL)-17-producing T cells among PBMCs from patients with UC. These data suggest that SMILE exerts a therapeutic effect on IBD by modulating IL-17 production.

Gait parameters as tools for analyzing phenotypic alterations of a mouse model of Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT), a genetically heterogeneous group of diseases in the peripheral nervous system, is characterized by progressive and symmetrical distal weakness resulting in gait abnormality. The necessity of the diagnostic and prognostic biomarkers has been raised for both basic research and clinical practice in CMT. Since biomarkers for animal study of CMT are limited, we evaluated the feasibility of gait parameters as tool for measuring disease phenotype of CMT mouse model. Using a Trembler-J (Tr-J) mouse, a CMT type 1 (CMT1) mouse model, we analyzed kinematic parameters such as angles of hip, knee and ankle (sagittal plane), and spatial parameters including step width and stride length (transverse plane). Regarding of kinematic parameters, Tr-J mice exhibited less plantarflexed ankle during the swing phase and more dorsiflexed ankle at the terminal stance compared to control mice. The range of motion in ankle angle of Tr-J mice was significantly greater than that of control mice. In spatial parameter, Tr-J mice exhibited wider step width compared to control mice. These results are similar to previously reported gait patterns of CMT1 patients. In comparison with other markers such as nerve conduction study and rotarod test, gait parameters dynamically reflected the disease progression of CMT1 mice. Therefore, these data imply that gait parameters can be used as useful tools to analyzed the disease phenotype and progression during preclinical study of peripheral neuropathy such as CMT.

Ankmy2 Prevents Smoothened-Independent Hyperactivation of the Hedgehog Pathway via Cilia-Regulated Adenylyl Cyclase Signaling.

The mechanisms underlying subcellular targeting of cAMP-generating adenylyl cyclases and processes regulated by their compartmentalization are poorly understood. Here, we identify Ankmy2 as a repressor of the Hedgehog pathway via adenylyl cyclase targeting. Ankmy2 binds to multiple adenylyl cyclases, determining their maturation and trafficking to primary cilia. Mice lacking Ankmy2 are mid-embryonic lethal. Knockout embryos have increased Hedgehog signaling and completely open neural tubes showing co-expansion of all ventral neuroprogenitor markers, comparable to the loss of the Hedgehog receptor Patched1. Ventralization in Ankmy2 knockout is completely independent of the Hedgehog pathway transducer Smoothened. Instead, ventralization results from the reduced formation of Gli2 and Gli3 repressors and early depletion of adenylyl cyclase III in neuroepithelial cilia, implicating deficient pathway repression. Ventralization in Ankmy2 knockout requires both cilia and Gli2 activation. These findings indicate that cilia-dependent adenylyl cyclase signaling represses the Hedgehog pathway and promotes morphogenetic patterning.

Metabolic abnormalities exacerbate Sjögren's syndrome by and is associated with increased the population of interleukin-17-producing cells in NOD/ShiLtJ mice.

BACKGROUND: Sjögren's syndrome (SS) is an autoimmune disease mediated by lymphocytic infiltration into exocrine glands, resulting in progressive lacrimal and salivary destruction and dysfunctional glandular secretion. Metabolic syndrome influences the immune system. To investigate its relationship with metabolic abnormalities, we evaluated the pathogenesis of SS and the immune cell populations in non-obese diabetic NOD/ShiLtJ mice with type 1 diabetes (T1D). METHODS: To induce metabolic abnormalities, streptozotocin (STZ)-a glucosamine-nitrosourea compound that destroys pancreatic ß cells, resulting in T1D-was injected into NOD/ShiLtJ mice. The blood glucose level was measured to evaluate induction of T1D. The severity of SS was assessed by determining the body weight, salivary flow rate, and histologic parameters. The expression levels of proinflammatory factors in the salivary glands, lacrimal gland, and spleen were quantified by real-time PCR. The populations of various T- and B-cell subtypes in the peripheral blood, spleen, and salivary glands were assessed by flow cytometry. RESULTS: Induction of T1D in NOD/ShiLtJ mice increased both the severity of SS and the levels of proinflammatory cytokines in the salivary glands compared to the controls. Furthermore, the number of interleukin-17-producing immune cells in the peripheral blood, spleen, and salivary glands was increased in STZ- compared to vehicle-treated NOD/ShiLtJ mice. CONCLUSIONS: Metabolic abnormalities play an important role in the development of SS.

Effect of Peat Intervention on Pain and Gait in Patients with Knee Osteoarthritis: A Prospective, Double-Blind, Randomized, Controlled Study.

METHODS: Knee osteoarthritis patients with Visual Analog Scale (VAS) of 3 or more and Kellgren-Lawrence osteoarthritis grades 1 to 3 were included. Patients with history of intraarticular injection treatment were excluded. Forty-one participants were randomly allocated to the peat intervention group (n = 22) or the hot-pack-only control group (n = 19). Peat and hot pack were applied to both knees of each group of patients. Each intervention session lasted 20 minutes, and eight sessions were completed over five days. VAS, serum cartilage oligomeric matrix protein (COMP), and gait parameters were evaluated before and after the whole interventions. RESULTS: VAS in the peat group decreased from 6.000 to 3.409 after intervention (p < 0.001) and also decreased in the control group from 5.737 to 4.421 (p < 0.001). VAS score reduction between two periods was greater in the peat group than that in the control group (p < 0.001). There was no significant difference in the serum COMP level in either intergroup or intragroup analysis. In gait analysis, the gait velocity of the peat group increased from 0.781 m/s to 0.873 m/s after intervention (p=0.002), while it decreased in the control group. The knee varus/valgus range of motion during gaits was reduced from 11.455° to 8.439° after intervention in the peat group (p=0.006). CONCLUSIONS: This study showed that peat can be considered as a therapeutic option for pain relief of knee osteoarthritis patients. The reduction in knee joint varus/valgus range of motion and the increase in gait velocity after peat intervention were also identified through this research, which is the first to analyze the effects of peat on gait.

A bispecific soluble receptor fusion protein that targets TNF-α and IL-21 for synergistic therapy in inflammatory arthritis.

This study was aimed at investigating the therapeutic effects of BITRAP, a bispecific fusion protein targeting TNF-α and IL-21, on the development of autoimmune arthritis in humans and mice. To verify the effects of BITRAP in human, peripheral blood mononuclear cells were cultured with BITRAP under IL-17-producing T (Th17) cell-polarizing conditions or osteoclast differentiation conditions. BITRAP treatment inhibited the production of IL-17 and vascular endothelial growth factor but increased the production of IL-10 in CD4+ T cells, as well as directly suppressed osteoclastogenesis. Collagen-induced arthritis (CIA) and IL-1R antagonist (IL-1Ra) knockout mice were treated with BITRAP. Following injection in CIA mice, BITRAP rapidly migrated into the inflamed joints and remained there for 72 hours. Application of BITRAP attenuated the severity of autoimmune arthritis in CIA and IL-1Ra knockout mice by reducing the numbers of inflammatory cytokine-expressing cells and Th17 cells and antibody secretion. Finally, BITRAP suppressed STAT3 phosphorylation, as well as production of IL-17 and TNF-α, in murine splenic CD4+ T cells. These findings suggest that BITRAP, a bispecific fusion protein targeting TNF-α and IL-21, may be an effective treatment to overcome the limitations of anti-TNF therapy for patients with rheumatoid arthritis.

Interleukin-21-mediated suppression of the Pax3-Id3 pathway exacerbates the development of Sjögren's syndrome via follicular helper T cells.

Sjögren's syndrome (SS) is a systemic autoimmune disease with severe dysfunction of glandular secretory function mediated by T and B lymphocyte infiltration into the exocrine glands, including the salivary and lacrimal glands. Follicular helper T (Tfh) cells exacerbate the disease by causing B cell hyperactivity. Inhibitor of DNA binding 3 (Id3) deficiency causes activation of Tfh cells and is known to be a clinical manifestation of human SS disease. In this study, we investigated the mechanism of action of Pax3, which is reduced in SS and can interact with Id3, in NOD/ShiLtJ mice as an animal model of SS. Treatment with interleukin (IL)-21, a major cytokine secreted from Tfh cells, suppressed Pax3 and Id3 expression via STAT3 in splenic T cells in vitro. Administration of pCMV14-3xFlag PAX3 vector improved the severity of SS by reducing the number of Tfh cells in NOD/ShiLtJ mice. Application of IL-21R-Fc increased the number of Pax3- and Id3-positive cells in the salivary glands, while reducing the proportion of Tfh cells and IL-17-producing T cells in NOD/ShiLtJ mice. The salivary glands from SS patients showed decreased levels of Pax3 or Id3 expression compared with healthy controls. Our findings regarding reinforcement of the Pax3-Id3 signal pathway may facilitate the development of novel therapeutic strategies for SS.