Immunomodulatory Effects of Yu-Ping-Feng Formula on Primary Sjögren's Syndrome: Interrogating the T Cell Response.

Ethnopharmacological treatments have shown beneficial effects in the clinical practice of autoimmune disorders. However, the underlying mechanism of immunomodulatory effects remains challenging, given the complicate composition of herbal medicines. Here, we developed an immunological approach to interrogate the T helper cell response. Through data mining we hypothesized that Chinese medicine formula, Yu-Ping-Feng (YPF) might be a promising candidate for treating primary Sjögren's syndrome (pSS), a common autoimmune disease manifested by exocrine gland dysfunction. We took advantage of a mouse model of experimental Sjögren's syndrome (ESS) that we previously established for YPF formula treatment. YPF therapy ameliorated the ESS pathology in mice with active disease, showing improved salivary function and decreased serum levels of autoantibodies. Phenotypic analysis suggested that both effector T and B cells were significantly suppressed. Using co-culture assay and adoptive transfer models, we demonstrated that YPF formula directly restrained effector/memory T cell expansion and differentiation into Th17 and T follicular helper (Tfh) cells, the key subsets in ESS pathogenesis. Importantly, we recruited 20 pSS patients and conducted a pilot study of 8-week therapy of YPF formula. YPF treatment effectively improved fatigue symptoms, exocrine gland functions and reduced serum IgG/IgA levels, while effector T and B cell subsets were significantly decreased. There was a trend of reduction on disease activity, but not statistically significant. Together, our findings suggested a novel approach to assess the immunomodulatory effects of YPF formula, which may be favorable for patients with autoimmune disorders.

Peroxynitrite reduces Treg cell expansion and function by mediating IL-2R nitration and aggravates multiple sclerosis pathogenesis.

T-helper 17 cells and regulatory T cells (Treg) are critical regulators in the pathogenesis of multiple sclerosis (MS) but the factors affecting Treg/Th17 balance remains largely unknown. Redox balance is crucial to maintaining immune homeostasis and reducing the severity of MS but the underlying mechanisms are unclear yet. Herein, we tested the hypothesis that peroxynitrite, a representative molecule of reactive nitrogen species (RNS), could inhibit peripheral Treg cells, disrupt Treg/Th17 balance and aggravate MS pathology by inducing nitration of interleukin-2 receptor (IL-2R) and down-regulating RAS/JNK-AP-1 signalling pathway. Experimental autoimmune encephalomyelitis (EAE) mouse model and serum samples of MS patients were used in the study. We found that the increases of 3-nitrotyrosine and IL-2R nitration in Treg cells were coincided with disease severity in the active EAE mice. Mechanistically, peroxynitrite-induced IL-2R nitration down-regulated RAS/JNK signalling pathway, subsequently impairing peripheral Treg expansion and function, increasing Teff infiltration into the central nerve system (CNS), aggravating demyelination and neurological deficits in the EAE mice. Those changes were abolished by peroxynitrite decomposition catalyst (PDC) treatment. Furthermore, transplantation of the PDC-treated-autologous Treg cells from donor EAE mice significantly decreased Th17 cells in both axillary lymph nodes and lumbar spinal cord, and ameliorated the neuropathology of the recipient EAE mice. Those results suggest that peroxynitrite could disrupt peripheral Treg/Th17 balance, and aggravate neuroinflammation and neurological deficit in active EAE/MS pathogenesis. The underlying mechanisms are related to induce the nitration of IL-2R and inhibit the RAS/JNK-AP-1 signalling pathway in Treg cells. The study highlights that targeting peroxynitrite-mediated peripheral IL-2R nitration in Treg cells could be a novel therapeutic strategy to restore Treg/Th17 balance and ameliorate MS/EAE pathogenesis. The study provides valuable insights into potential role of peripheral redox balance in maintaining CNS immune homeostasis.

The Future of Targeted Treatment of Primary Sjögren's Syndrome: A Focus on Extra-Glandular Pathology.

Primary Sjögren's syndrome (pSS) is a chronic, systemic autoimmune disease defined by exocrine gland hypofunction resulting in dry eyes and dry mouth. Despite increasing interest in biological therapies for pSS, achieving FDA-approval has been challenging due to numerous complications in the trials. The current literature lacks insight into a molecular-target-based approach to the development of biological therapies. This review focuses on novel research in newly defined drug targets and the latest clinical trials for pSS treatment. A literature search was conducted on ClinicalTrials.gov using the search term "Primary Sjögren's syndrome". Articles published in English between 2000 and 2021 were included. Our findings revealed potential targets for pSS treatment in clinical trials and the most recent advances in understanding the molecular mechanisms underlying the pathogenesis of pSS. A prominent gap in current trials is in overlooking the treatment of extraglandular symptoms such as fatigue, depression, and anxiety, which are present in most patients with pSS. Based on dryness and these symptom-directed therapies, emerging biological agents targeting inflammatory cytokines, signal pathways, and immune reaction have been studied and their efficacy and safety have been proven. Novel therapies may complement existing non-pharmacological methods of alleviating symptoms of pSS. Better grading systems that add extraglandular symptoms to gauge disease activity and severity should be created. The future of pSS therapies may lie in gene, stem-cell, and tissue-engineering therapies.

Aryl hydrocarbon receptor activation drives polymorphonuclear myeloid-derived suppressor cell response and efficiently attenuates experimental Sjögren's syndrome.

Myeloid-derived suppressor cells (MDSCs) comprise heterogeneous myeloid cell populations with immunosuppressive capacity that contribute to immune regulation and tolerance induction. We previously reported impaired MDSC function in patients with primary Sjögren's syndrome (pSS) and mice with experimental SS (ESS). However, the molecular mechanisms underlying MDSC dysfunction remain largely unclear. In this study, we first found that aryl hydrocarbon receptor (AhR) was highly expressed by human and murine polymorphonuclear MDSCs (PMN-MDSCs). Indole-3-propionic acid (IPA), a natural AhR ligand produced from dietary tryptophan, significantly promoted PMN-MDSC differentiation and suppressive function on CD4+ T cells. In contrast, feeding a tryptophan-free diet resulted in a decreased PMN-MDSC response, a phenotype that could be reversed by IPA supplementation. The functional importance of PMN-MDSCs was demonstrated in ESS mice by using a cell-depletion approach. Notably, AhR expression was reduced in PMN-MDSCs during ESS development, while AhR antagonism resulted in exacerbated ESS pathology and dysregulated T effector cells, which could be phenocopied by a tryptophan-free diet. Interferon regulatory factor 4 (IRF4), a repressive transcription factor, was upregulated in PMN-MDSCs during ESS progression. Chromatin immunoprecipitation analysis revealed that IRF4 could bind to the promoter region of AhR, while IRF4 deficiency markedly enhanced AhR-mediated PMN-MDSC responses. Furthermore, dietary supplementation with IPA markedly ameliorated salivary glandular pathology in ESS mice with restored MDSC immunosuppressive function. Together, our results identify a novel function of AhR in modulating the PMN-MDSC response and demonstrate the therapeutic potential of targeting AhR for the treatment of pSS.

Mitochondrial regulation of acute extrafollicular B-cell responses to COVID-19 severity.

BACKGROUND: Patients with COVID-19 display a broad spectrum of manifestations from asymptomatic to life-threatening disease with dysregulated immune responses. Mechanisms underlying the detrimental immune responses and disease severity remain elusive. METHODS: We investigated a total of 137 APs infected with SARS-CoV-2. Patients were divided into mild and severe patient groups based on their requirement of oxygen supplementation. All blood samples from APs were collected within three weeks after symptom onset. Freshly isolated PBMCs were investigated for B cell subsets, their homing potential, activation state, mitochondrial functionality and proliferative response. Plasma samples were tested for cytokine concentration, and titer of Nabs, RBD-, S1-, SSA/Ro- and dsDNA-specific IgG. RESULTS: While critically ill patients displayed predominantly extrafollicular B cell activation with elevated inflammation, mild patients counteracted the disease through the timely induction of mitochondrial dysfunction in B cells within the first week post symptom onset. Rapidly increased mitochondrial dysfunction, which was caused by infection-induced excessive intracellular calcium accumulation, suppressed excessive extrafollicular responses, leading to increased neutralizing potency index and decreased inflammatory cytokine production. Patients who received prior COVID-19 vaccines before infection displayed significantly decreased extrafollicular B cell responses and mild disease. CONCLUSION: Our results reveal an immune mechanism that controls SARS-CoV-2-induced detrimental B cell responses and COVID-19 severity, which may have implications for viral pathogenesis, therapeutic interventions and vaccine development.

Acteoside promotes B cell-derived IL-10 production and ameliorates autoimmunity.

IL-10-producing regulatory B (Breg) cells are well recognized for maintaining immune tolerance. The impaired Breg cell function with decreased IL-10-producing capacity has been found in autoimmune diseases, such as rheumatoid arthritis, lupus, and primary Sjogren's syndrome (pSS). However, seldom therapeutic agents targeting Breg cells are available to treat those autoimmune diseases. Here, we showed that acteoside (AC), a caffeoyl phenylethanoid glycoside from a medicinal herb Radix Rehmanniae, could promote IL-10 production from both human and murine B cells via critically regulating the TLR4/PI3K axis. Moreover, TLR4 was found increased in Breg cells from mice with experimental SS (ESS), a mouse model that recapitulates human pSS. Thus, B cells from the ESS mice were susceptible to AC treatment, showing higher IL-10-producing capacity than those from naïve controls. In addition, AC treatment also promoted the production of IL-10 from TLR4+ CXCR4+ plasma cells of ESS mice. Notably, we found that AC was able to enter lymphoid organs upon oral administration. AC treatment effectively increased IL-10+ B cells in ESS mice and ameliorated disease pathology accompanied by reduced T effector cells, including Th17 and T follicular helper cells in the ESS mice. In conclusion, AC could promote Breg cell function and attenuate ESS pathology in vivo, which may be a promising drug candidate for treating pSS and other autoimmune diseases.

Detection of T Follicular Helper Cells and T Follicular Regulatory Cells in Experimental Sjögren's Syndrome.

With the compiling studies on the autoimmune pathogenesis in the developing of Sjögren's syndrome, the functional importance of T follicular helper cells (Tfh) and T follicular regulatory cells (Tfr) was investigated, including our recent findings, among which various techniques for detecting Tfh and Tfr cells in Sjögren's syndrome have been reported. In this chapter, we describe detailed methods for the effective detection of Tfh and Tfr cells in mice with experimental Sjögren's syndrome (ESS), a mouse model with evident salivary hypofunction, increased serum levels of autoantibodies, and histopathological changes in the salivary glands. We provide representative detections of surface markers, cytokines, and transcription factors of Tfh and Tfr cells by flow cytometry and ELISpot assay. Moreover, a detailed protocol for detecting Tfh and Tfr cells in the draining cervical lymph nodes (CLNs) in ESS mice by immunofluorescence microscopy is also described. Together, these techniques of Tfh and Tfr cell detection in ESS mice may facilitate the investigation of autoimmune pathogenesis and enhance the understanding of Tfh and Tfr cell functions in the development of Sjögren's syndrome.

Tetrahydroberberrubine attenuates lipopolysaccharide-induced acute lung injury by down-regulating MAPK, AKT, and NF-κB signaling pathways.

Acute lung injury (ALI) is a life-threatening syndrome that is characterized by overwhelming lung inflammation and increased microvascular permeability, which causes a high mortality worldwide. Here, we studied the protective effect of tetrahydroberberrubine (THBru), a berberine derivative, on a mouse model of lipopolysaccharide (LPS)-induced acute lung injury that was established in our previous studies. The results showed that a single oral administration of THBru significantly decreased the lung wet to dry weight (W/D) ratio at doses of 2, 10 and 50mg/kg administered 1h prior to LPS challenge (30mg/kg, intravenous injection). Histopathological changes, such as pulmonary edema, infiltration of inflammatory cells and coagulation, were also attenuated by THBru. In addition, THBru markedly decreased the total cell counts, total protein and nitrate/nitrite content in bronchoalveolar lavage fluid (BALF), significantly decreased tumor necrosis factor-α (TNF-α) and nitrate/nitrite content in the plasma, and reduced the myeloperoxidase (MPO) activity in the lung tissues. Additionally, THBru (10µM) significantly decreased the content of TNF-α and nitric oxide (NO) in LPS-induced THP-1 cells in vitro. Moreover, THBru significantly suppressed the activation of the MAPKs JNK and p38, AKT, and the NF-κB subunit p65 in LPS-induced THP-1 cells. These findings confirm that THBru attenuates LPS-induced acute lung injury by inhibiting the release of inflammatory cytokines and suppressing the activation of MAPKs, AKT, and NF-κB signaling pathways, which implicates it as a potential therapeutic agent for ALI or sepsis.

Molecular mechanisms in lipopolysaccharide-induced pulmonary endothelial barrier dysfunction.

The confluent pulmonary endothelium plays an important role as a semi-permeable barrier between the vascular space of blood vessels and the underlying tissues, and it contributes to the maintenance of circulatory fluid homeostasis. Pulmonary endothelial barrier dysfunction is a pivotal early step in the development of a variety of high mortality diseases, such as acute lung injury (ALI). Endothelium barrier dysfunction in response to inflammatory or infectious mediators, including lipopolysaccharide (LPS), is accompanied by invertible cell deformation and interendothelial gap formation. However, specific pharmacological therapies aiming at ameliorating pulmonary endothelial barrier function in patients are still lacking. A full understanding of the fundamental mechanisms that are involved in the regulation of pulmonary endothelial permeability is essential for the development of barrier protective therapeutic strategies. Therefore, this review summarizes several important molecular mechanisms involved in LPS-induced changes in pulmonary endothelial barrier function. As for barrier-disruption, the activation of myosin light chain kinase (MLCK), RhoA and tyrosine kinases; increase of calcium influx; and apoptosis of the endothelium lead to an elevation of lung endothelial permeability. Additionally, the activation of Rac1, Cdc42, protease activated receptor 1 (PAR1) and adenosine receptors (ARs), as well as the increase of cyclic AMP and sphingosine-1-phosphate (S1P) content, protect against LPS-induced lung endothelial barrier dysfunction. Furthermore, current regulatory factors and strategies against the development of LPS-induced lung endothelial hyper-permeability are discussed.

Advances in the pharmacological activities and mechanisms of diosgenin.

Diosgenin, a well-known steroid sapogenin derived from plants, has been used as a starting material for production of steroidal hormones. The present review will summarize published literature concerning pharmacological potential of diosgenin, and the underlying mechanisms of actions. Diosgenin has shown a vast range of pharmacological activities in preclinical studies. It exhibits anticancer, cardiovascular protective, anti-diabetes, neuroprotective, immunomodulatory, estrogenic, and skin protective effects, mainly by inducing apoptosis, suppressing malignant transformation, decreasing oxidative stress, preventing inflammatory events, promoting cellular differentiation/proliferation, and regulating T-cell immune response, etc. It interferes with cell death pathways and their regulators to induce apoptosis. Diosgenin antagonizes tumor metastasis by modulating epithelial-mesenchymal transition and actin cytoskeleton to change cellular motility, suppressing degradation of matrix barrier, and inhibiting angiogenesis. Additionally, diosgenin improves antioxidant status and inhibits lipid peroxidation. Its anti-inflammatory activity is through inhibiting production of pro-inflammatory cytokines, enzymes and adhesion molecules. Furthermore, diosgenin drives cellular growth/differentiation through the estrogen receptor (ER) cascade and transcriptional factor PPARγ. In summary, these mechanistic studies provide a basis for further development of this compound for pharmacotherapy of various diseases.