Endoscopic assessment of the J pouch in ulcerative colitis: A narrative review.

Patients with ulcerative colitis sometimes need a total colectomy with ileal pouch-anal anastomosis due to medically refractory disease or colitis-associated neoplasia. Up to 50% of patients with ulcerative colitis postoperatively develop pouchitis and the rate of chronic inflammatory pouch conditions requiring pouch excision or diverting ileostomy is reported to be 10%. In order to diagnose and monitor pouchitis, pouchoscopy is essential to assess endoscopic inflammatory findings of the J pouch and to survey neoplasia development, particularly in the remnant distal rectum. However, endoscopic protocols for the evaluation of the pouch may not be standardized worldwide and the reliability of existing disease activity indices for pouchitis has been questioned due to the lack of validation. Recently, reliable endoscopic scoring systems based on an observation of the anatomical location of the J pouch were reported and a significant association between the distribution pattern of endoscopic inflammation (i.e., endoscopic phenotype) and pouch outcomes was also uncovered. In this review, we discuss how to survey the J pouch using pouchoscopy, endoscopic indices for pouchitis disease activity, endoscopic phenotypes and classification, and the pathological mechanisms of pouchitis phenotype in patients with ulcerative colitis.

Protective effect of trans-nerolidol on vascular endothelial cell injury induced by lipopolysaccharides / Efectos protectores del trans-nerolidol en lesiones inducidas por lipopolisacáridos en células endoteliales vasculares

This article aimed to discuss the protection of trans - nerolidol on vascular endothelial cells (ECs) injured by lipopolysac charides. ECs were divided into four groups: normal, model, low and high dose trans - nerolidol treatment groups. The cell survival rate and the contents of NO in the cell culture supernatant were determined. The protein expression and transcript level of pe roxisome proliferator - activated receptor - γ (PPARγ), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS) were determined by western blotting and RT - PCR respectively. Compared with the normal group, cell livability, protein e xpression and mRNA transcript level of PPARγ and eNOS decreased, NO contents, protein expression and mRNA transcript tlevel of iNOS increased in model group significantly. Compared with model group, all the changes recovered in different degree in treatmen t groups. Hence, it was concluded that trans - nerolidol can alleviate the ECs injuryby the regulation of iNOS/eNOS through activating PPARγ in a dose - dependent manner

Este artículo tiene como objetivo discutir la protección del trans - nerolidol en las células endoteliales vasculares (CE) dañadas por lipopolisacáridos. Las CE se di vidieron en cuatro grupos: normal, modelo, grupos de tratamiento con trans - nerolidol de baja y alta dosis. Se determinó la tasa de supervivencia de las células y los contenidos de óxido nítrico (NO) en el sobrenadante del cultivo celular. La expresión de p roteínas y el nivel de transcripción del receptor activado por proliferadores de peroxisomas - γ (PPARγ), el óxido nítrico sint et asa endotelial (eNOS) y el óxido nítrico sint et asa inducible (iNOS) se determinaron mediante western blot y RT - PCR, respectivamen te. En comparación con el grupo normal, la viabilidad celular, la expresión de proteínas y el nivel de transcripción de PPARγ y eNOS disminuyeron, los contenidos de NO, la expresión de proteínas y el nivel de transcripción de iNOS aumentaron significativam ente en el grupo modelo. En comparación con el grupo modelo, todos los cambios se recuperaron en diferentes grados en los grupos de tratamiento. Por lo tanto, se concluyó que el trans - nerolidol puede aliviar el daño en las CE regulando iNOS/eNOS a través d e la activación de PPARγ de manera dependiente de la dosis.

Lactic acid: a narrative review of a promoter of the liver cancer microenvironment.

Background and Objective: Lactic acid is a metabolite of glycolysis produced in the body, and its production is thought to be a mechanism by which cancer cells evade immune surveillance. Immune evasion and metabolic changes are well established as basic hallmarks of cancer. Although lactate has long been considered a waste product, it is now generally recognized to be a versatile small-molecule chemical that plays an important part in the tumor microenvironment (TME), with increased lactate production linked to the development of human malignancies. Metabolism in liver cancer is redirected toward glycolysis, which enhances the production of metabolic compounds used by tumor cells to produce proteins, lipids, and nucleotides, enabling them to maintain high proliferation rates and to establish the TME. Dysregulation of metabolic activity in liver cancer may impair antitumor responses owing to the immunosuppressive activity of the lactate produced by anaerobic glycolytic rates in tumor cells. This review primarily explores the link connection between lactic acid and the TME; evaluates the role of lactic acid in the occurrence, metastasis, prognosis, and treatment of liver cancer. Additionally, it investigates the associated pathways as potential targets for liver cancer treatment. Methods: Literature searches were conducted in PubMed, Web of Science, and Google Scholar, with the publication date of the most recent article included being January 2024. After eliminating duplicate articles and less relevant articles through titles and abstracts, we selected 113 articles for this review. We categorized references into two categories. One is to classify the content into lactate-related, liver cancer-related and tumor metabolism-related. The other is to classify the article types, which are divided into reviews, research articles and clinical trials. Additionally, we consulted the reference lists of the relevant articles to ensure coverage was comprehensive and unbiased. Key Content and Findings: The connection between lactic acid and the TME has recently become an area of intense research interest, and many related articles have been published in this field. The main finding of this review is to summarize the proven link between lactate and the TME and its possible impact on the TME of liver cancer. And analyzed the potential of lactate in liver cancer treatment and prognosis prediction. Conclusions: Lactate may be key to developing novel approaches in the future treatment of liver cancer. Related research on the combination of classic therapies and molecular targeted drugs may provide innovative medicines that more selectively regulate immune cell activity.

Promotion of perineural invasion of cholangiocarcinoma by Schwann cells via nerve growth factor.

Background: Cholangiocarcinoma (CCA), a highly lethal tumor of the hepatobiliary system originating from bile duct epithelium, can be divided into the intrahepatic, hilar, and extrahepatic types. Due to its insidious onset and atypical early clinical symptoms, the overall prognosis is poor. One of the important factors contributing to the poor prognosis of CCA is the occurrence of perineural invasion (PNI), but the specific mechanisms regarding how it contributes to the occurrence of PNI are still unclear. The main purpose of this study is to explore the molecular mechanism leading to the occurrence of PNI and provide new ideas for clinical treatment. Methods: CCA cell lines and Schwann cells (SCs) were stimulated to observe the changes in cell behavior. SCs cocultured with tumor supernatant and SCs cultured in normal medium were subjected to transcriptome sequencing to screen the significantly upregulated genes. Following this, the two types of tumor cells were cultured with SC supernatant, and the changes in behavior of the tumor cells were observed. Nonobese diabetic-severe combined immunodeficiency disease (NOD-SCID) mice were injected with cell suspension supplemented with nerve growth factor (NGF) via the sciatic nerve. Four weeks later, the mice were euthanized and the tumor sections were removed and stained. Results: Nerve invasion by tumor cells was common in CCA tissues. SCs were observed in tumor tissues, and the number of SCs in tumor tissues and the degree of PNI were much higher than were those in normal tissues or tissues without PNI. The overall survival time was shorter in patients with CCA with PNI than in patients without PNI. SCs were enriched in CCA tissues, indicating the presence of PNI and associated with poor prognosis in CCA patients. CCA was found to promote NGF secretion from SCs in vitro. After the addition of exogenous NGF in CCA cell culture medium, the proliferation activity and migration ability of CCA cells were significantly increased, suggesting that SCs can promote the proliferation and migration of CCA through the secretion of NGF. NGF, in turn, was observed to promote epithelial-mesenchymal transition in CCA through tropomyosin receptor kinase A (TrkA), thus promoting its progression. Tumor growth in mice shows that NGF can promote PNI in CCA. Conclusions: In CCA, tumor cells can promote the secretion of NGF by SCs, which promotes the progression of CCA and PNI by binding to its high-affinity receptor TrkA, leading to poor prognosis.

Evaluating the ability of cultivated odontoblasts to form dentin-like tissue in vitro using fibroblast growth factor and insulin-like growth factor.

Aim: The aim of the study was to evaluate the ability of cultivated odontoblast to form dentin-like tissue using fibroblast growth factor (FGF) and insulin-like growth factor (IGF). Materials and Methods: Dental pulp stem cells (DPSCs) were extracted from 10 human teeth. They were isolated and cultivated in vitro with the use of stem cell markers. The human DPSCs were characterized for trilineage differentiation. They were then differentiated into odontoblasts. The ability of cultivated odontoblasts to form dentin-like tissue was evaluated using FGF and IGF. Results: IGF showed superior ability to form dentin-like tissue as compared to FGF. The addition of FGF showed no significant difference in the formation of dentin-like tissue. A combination of FGF and IGF in odontoblast showed an enhanced ability to form dentin-like tissue. Conclusion: The use of growth factors IGF and FGF with dental stem cells showed a greater potential to form dentin-like tissue. This can profoundly alter the paradigms of conservative vital pulp therapy, which may eventually make it possible to treat dental diseases by regeneration of lost dentine.

Decoding Endothelial MPL and JAK2V617F Mutation: Insight Into Cardiovascular Dysfunction in Myeloproliferative Neoplasms.

BACKGROUND: Patients with JAK2V617F-positive myeloproliferative neoplasms (MPNs) and clonal hematopoiesis of indeterminate potential face a significantly elevated risk of cardiovascular diseases. Endothelial cells carrying the JAK2V617F mutation have been detected in many patients with MPN. In this study, we investigated the molecular basis for the high incidence of cardiovascular complications in patients with MPN. METHODS: We investigated the impact of endothelial JAK2V617F mutation on cardiovascular disease development using both transgenic murine models and MPN patient-derived induced pluripotent stem cell lines. RESULTS: Our investigations revealed that JAK2V617F mutant endothelial cells promote cardiovascular diseases under stress, which is associated with endothelial-to-mesenchymal transition and endothelial dysfunction. Importantly, we discovered that inhibiting the endothelial TPO (thrombopoietin) receptor MPL suppressed JAK2V617F-induced endothelial-to-mesenchymal transition and prevented cardiovascular dysfunction caused by mutant endothelial cells. Notably, the endothelial MPL receptor is not essential for the normal physiological regulation of blood cell counts and cardiac function. CONCLUSIONS: JAK2V617F mutant endothelial cells play a critical role in the development of cardiovascular diseases in JAK2V617F-positive MPNs, and endothelial MPL could be a promising therapeutic target for preventing or ameliorating cardiovascular complications in these patients.

FOXC1 and FOXC2 Ablation Causes Abnormal Valvular Endothelial Cell Junctions and Lymphatic Vessel Formation in Myxomatous Mitral Valve Degeneration.

BACKGROUND: Mitral valve (MV) disease including myxomatous degeneration is the most common form of valvular heart disease with an age-dependent frequency. Genetic evidence indicates that mutations of the transcription factor FOXC1 are associated with MV defects, including MV regurgitation. In this study, we sought to determine whether murine Foxc1 and its closely related factor, Foxc2, are required in valvular endothelial cells (VECs) for the maintenance of MV leaflets, including VEC junctions and the stratified trilaminar ECM (extracellular matrix). METHODS: Adult mice carrying tamoxifen-inducible, vascular endothelial cell (EC), and lymphatic EC-specific, compound Foxc1;Foxc2 mutations (ie, EC-Foxc-DKO and lymphatic EC-Foxc-DKO mice, respectively) were used to study the function of Foxc1 and Foxc2 in the maintenance of MVs. The EC and lymphatic EC mutations of Foxc1/c2 were induced at 7 to 8 weeks of age by tamoxifen treatment, and abnormalities in the MVs of these mutant mice were assessed via whole-mount immunostaining, immunohistochemistry/RNAscope, Movat pentachrome/Masson Trichrome staining, and Evans blue injection. RESULTS: EC deletions of Foxc1 and Foxc2 in mice resulted in abnormally extended and thicker MVs by causing defects in the regulation of ECM organization with increased proteoglycan and decreased collagen. Notably, reticular adherens junctions were found in VECs of control MV leaflets, and these reticular structures were severely disrupted in EC-Foxc-DKO mice. PROX1 (prospero homeobox protein 1), a key regulator in a subset of VECs on the fibrosa side of MVs, was downregulated in EC-Foxc1/c2 mutant VECs. Furthermore, we determined the precise location of lymphatic vessels in murine MVs, and these lymphatic vessels were aberrantly expanded and dysfunctional in EC-Foxc1/c2 mutant MVs. Lymphatic EC deletion of Foxc1/c2 also resulted in similar structural/ECM abnormalities as seen in EC-Foxc1/c2 mutant MVs. CONCLUSIONS: Our results indicate that Foxc1 and Foxc2 are required for maintaining the integrity of the MV, including VEC junctions, ECM organization, and lymphatic vessel formation/function to prevent myxomatous MV degeneration.

Synergy Between NK Cells and Monocytes in Potentiating Cardiovascular Disease Risk in Severe COVID-19.

BACKGROUND: Evidence suggests that COVID-19 predisposes to cardiovascular diseases (CVDs). While monocytes/macrophages play a central role in the immunopathogenesis of atherosclerosis, less is known about their immunopathogenic mechanisms that lead to CVDs during COVID-19. Natural killer (NK) cells, which play an intermediary role during pathologies like atherosclerosis, are dysregulated during COVID-19. Here, we sought to investigate altered immune cells and their associations with CVD risk during severe COVID-19. METHODS: We measured plasma biomarkers of CVDs and determined phenotypes of circulating immune subsets using spectral flow cytometry. We compared these between patients with severe COVID-19 (severe, n=31), those who recovered from severe COVID-19 (recovered, n=29), and SARS-CoV-2-uninfected controls (controls, n=17). In vivo observations were supported using in vitro assays to highlight possible mechanistic links between dysregulated immune subsets and biomarkers during and after COVID-19. We performed multidimensional analyses of published single-cell transcriptome data of monocytes and NK cells during severe COVID-19 to substantiate in vivo findings. RESULTS: During severe COVID-19, we observed alterations in cardiometabolic biomarkers including oxidized-low-density lipoprotein, which showed decreased levels in severe and recovered groups. Severe patients exhibited dysregulated monocyte subsets, including increased frequencies of proinflammatory intermediate monocytes (also observed in the recovered) and decreased nonclassical monocytes. All identified NK-cell subsets in the severe COVID-19 group displayed increased expression of activation and tissue-resident markers, such as CD69. We observed significant correlations between altered immune subsets and plasma oxidized-low-density lipoprotein levels. In vitro assays revealed increased uptake of oxidized-low-density lipoprotein into monocyte-derived macrophages in the presence of NK cells activated by plasma of patients with severe COVID-19. Transcriptome analyses confirmed enriched proinflammatory responses and lipid dysregulation associated with epigenetic modifications in monocytes and NK cells during severe COVID-19. CONCLUSIONS: Our study provides new insights into the involvement of monocytes and NK cells in the increased CVD risk observed during and after COVID-19.

Amniotic Membrane-Derived Multichannel Hydrogels for Neural Tissue Repair.

In the pursuit of advancing neural tissue regeneration, biomaterial scaffolds have emerged as promising candidates, offering potential solutions for nerve disruptions. Among these scaffolds, multichannel hydrogels, characterized by meticulously designed micrometer-scale channels, stand out as instrumental tools for guiding axonal growth and facilitating cellular interactions. This study explores the innovative application of human amniotic membranes modified with methacryloyl domains (AMMA) in neural stem cell (NSC) culture. AMMA hydrogels, possessing a tailored softness resembling the physiological environment, are prepared in the format of multichannel scaffolds to simulate native-like microarchitecture of nerve tracts. Preliminary experiments on AMMA hydrogel films showcase their potential for neural applications, demonstrating robust adhesion, proliferation, and differentiation of NSCs without the need for additional coatings. Transitioning into the 3D realm, the multichannel architecture fosters intricate neuronal networks guiding neurite extension longitudinally. Furthermore, the presence of synaptic vesicles within the cellular arrays suggests the establishment of functional synaptic connections, underscoring the physiological relevance of the developed neuronal networks. This work contributes to the ongoing efforts to find ethical, clinically translatable, and functionally relevant approaches for regenerative neuroscience.

Tissue-Engineered Implant for Hemilaryngectomy Reconstruction with Recurrent Laryngeal Nerve Injury.

OBJECTIVE: Laryngeal cancer resections often require excision of portions of the larynx along with sacrifice of the ipsilateral recurrent laryngeal nerve (RLN). In such cases, there are no reconstructive options that reliably restore laryngeal function, rendering patients with severe functional impairment. To address this unmet clinical need, we extend our evaluation of a 3-implant mucosal, muscle, cartilage reconstruction approach aimed at promoting functional laryngeal restoration in a porcine hemilaryngectomy model with ipsilateral RLN transection. METHODS: Six Yucatan mini-pigs underwent full-thickness hemilaryngectomies with RLN transection followed by transmural reconstruction using fabricated collagen polymeric mucosal, muscle, and cartilage replacements. To determine the effect of adding therapeutic cell populations, subsets of animals received collagen muscle implants containing motor-endplate-expressing muscle progenitor cells (MEEs) and/or collagen cartilage implants containing adipose stem cell (ASC)-derived chondrocyte-like cells. Acoustic vocalization and laryngeal electromyography (L-EMG) provided functional assessments and histopathological analysis with immunostaining was used to characterize the tissue response. RESULTS: Five of six animals survived the 4-week postoperative period with weight gain, airway maintenance, and audible phonation. No tracheostomy or feeding tube was required. Gross and histological assessments of all animals revealed implant integration and regenerative remodeling of airway mucosa epithelium, muscle, and cartilage in the absence of a material-mediated foreign body reaction or biodegradation. Early voice and L-EMG data were suggestive of positive functional outcomes. CONCLUSION: Laryngeal reconstruction with collagen polymeric mucosa, muscle, and cartilage replacements may provide effective restoration of function after hemilaryngectomy with RLN transection. Future preclinical studies should focus on long-term functional outcomes. LEVEL OF EVIDENCE: NA Laryngoscope, 2024.

Allergen-specific B cell responses in oral immunotherapy-induced desensitization, remission, and natural outgrowth in cow's milk allergy.

BACKGROUND: Antigen-specific memory B cells play a key role in the induction of desensitization and remission to food allergens in oral immunotherapy and in the development of natural tolerance (NT). Here, we characterized milk allergen Bos d 9-specific B cells in oral allergen-specific immunotherapy (OIT) and in children spontaneously outgrowing cow's milk allergy (CMA) due to NT. METHODS: Samples from children with CMA who received oral OIT (before, during, and after), children who naturally outgrew CMA (NT), and healthy individuals were received from Stanford biobank. Bos d 9-specific B cells were isolated by flow cytometry and RNA-sequencing was performed. Protein profile of Bos d 9-specific B cells was analyzed by proximity extension assay. RESULTS: Increased frequencies of circulating milk allergen Bos d 9-specific B cells were observed after OIT and NT. Milk-desensitized subjects showed the partial acquisition of phenotypic features of remission, suggesting that desensitization is an earlier stage of remission. Within these most significantly expressed genes, IL10RA and TGFB3 were highly expressed in desensitized OIT patients. In both the remission and desensitized groups, B cell activation-, Breg cells-, BCR-signaling-, and differentiation-related genes were upregulated. In NT, pathways associated with innate immunity characteristics, development of marginal zone B cells, and a more established suppressor function of B cells prevail that may play a role in long-term tolerance. The analyses of immunoglobulin heavy chain genes in specific B cells demonstrated that IgG2 in desensitization, IgG1, IgA1, IgA2, IgG4, and IgD in remission, and IgD in NT were predominating. Secreted proteins from allergen-specific B cells revealed higher levels of regulatory cytokines, IL-10, and TGF-ß after OIT and NT. CONCLUSION: Allergen-specific B cells are essential elements in regulating food allergy towards remission in OIT-received and naturally resolved individuals.

Quantification of cell cycle re-entry during dedifferentiation of primary adipocytes in vitro.

Dedifferentiated adipose tissue (DFAT) has been proposed as a promising source of patient-specific multipotent progenitor cells (MPPs). During induced dedifferentiation, adipocytes exhibit profound gene expression and cell morphology changes. However, dedifferentiation of post-mitotic cells is expected to enable proliferation, which is critical if enough MPPs are to be obtained. Here, lineage tracing was employed to quantify cell proliferation in mouse adipocytes subjected to a dedifferentiation-inducing protocol commonly used to obtain DFAT cells. No evidence of cell proliferation in adipocyte-derived cells was observed, in contrast to the robust proliferation of non-adipocyte cells present in adipose tissue. We conclude that proliferative MPPs derived using the ceiling culture method most likely arise from non-adipocyte cells in adipose tissue.

Unveiling Key Interface Characteristics of Ni/Yttria-Stabilized Zirconia Solid Oxide Cell Electrodes in H2O Electroreduction Using Operando X-ray Photoelectron Spectroscopy.

Nickel/yttria-stabilized zirconia (YSZ) composites are the most commonly used fuel electrodes for solid oxide cells. While microstructural changes of Ni/YSZ during operational conditions have been thoroughly investigated, there is limited knowledge regarding Ni/YSZ surface chemistry under working conditions. In this study, we examine the interaction between Ni/YSZ electrodes and water vapor under open circuit and polarization conditions, utilizing near ambient pressure soft and hard X-ray photoelectron spectroscopies. Miniature cells with conventional porous Ni/YSZ composite cermet cathodes were modified to facilitate the direct spectroscopic observation of the functional electrode's areas close to the interface with the YSZ electrolyte. The results highlight dynamic changes in the oxidation state and composition of Ni/YSZ under H2 and H2O atmospheres. We also quantify the accumulation of impurities on the electrode surface. Through adjustments in the pretreatment of the cell, the correlation between the nickel surface oxidation state and the cell's electrochemical performance during H2O electroreduction is established. It is unequivocally shown that nickel surface oxidation in H2O electrolysis favors NiO over Ni(OH)x, providing critical insights into the mechanism of Ni-phase redistribution within the electrode during long-term operation. Depth-dependent photoemission measurements, combined with theoretical quantitative simulations, reveal that NiO and Ni phases are uniformly mixed on the surface during H2O electrolysis. This differs from the conventional expectation of a NiO-shell/Ni-core configuration in gas phase oxidation. These findings provide crucial insights into the surface chemistry of Ni/YSZ electrodes under conditions relevant to H2O electrolysis, elucidating their impact on the electrochemical performance of the cell.

Metabolic Responses to Redox Stress in Vascular Cells.

Significance: Redox stress underlies numerous vascular disease mechanisms. Metabolic adaptability is essential for vascular cells to preserve energy and redox homeostasis. Recent Advances: Single-cell technologies and multiomic studies demonstrate significant metabolic heterogeneity among vascular cells in health and disease. Increasing evidence shows that reductive or oxidative stress can induce metabolic reprogramming of vascular cells. A recent example is intracellular L-2-hydroxyglutarate accumulation in response to hypoxic reductive stress, which attenuates the glucose flux through glycolysis and mitochondrial respiration in pulmonary vascular cells and provides protection against further reductive stress. Critical Issues: Regulation of cellular redox homeostasis is highly compartmentalized and complex. Vascular cells rely on multiple metabolic pathways, but the precise connectivity among these pathways and their regulatory mechanisms is only partially defined. There is also a critical need to understand better the cross-regulatory mechanisms between the redox system and metabolic pathways as perturbations in either systems or their cross talk can be detrimental. Future Directions: Future studies are needed to define further how multiple metabolic pathways are wired in vascular cells individually and as a network of closely intertwined processes given that a perturbation in one metabolic compartment often affects others. There also needs to be a comprehensive understanding of how different types of redox perturbations are sensed by and regulate different cellular metabolic pathways with specific attention to subcellular compartmentalization. Lastly, integration of dynamic changes occurring in multiple metabolic pathways and their cross talk with the redox system is an important goal in this multiomics era.

Mast cell secretory granule fusion with amphisomes coordinates their homotypic fusion and release of exosomes.

Secretory granule (SG) fusion is an intermediate step in SG biogenesis. However, the precise mechanism of this process is not completely understood. We show that Golgi-derived mast cell (MC) SGs enlarge through a mechanism that is dependent on phosphoinositide (PI) remodeling and fusion with LC3+ late endosomes (amphisomes), which serve as hubs for the fusion of multiple individual SGs. Amphisome formation is regulated by the tyrosine phosphatase PTPN9, while the subsequent SG fusion event is additionally regulated by the tetraspanin protein CD63 and by PI4K. We also demonstrate that fusion with amphisomes imparts to SGs their capacity of regulated release of exosomes. Finally, we show that conversion of PI(3,4,5)P3 to PI(4,5)P2 and the subsequent recruitment of dynamin stimulate SG fission. Our data unveil a key role for lipid-regulated interactions with the endocytic and autophagic systems in controlling the size and number of SGs and their capacity to release exosomes.

STING trafficking activates MAPK-CREB signaling to trigger regulatory T cell differentiation.

The Type-I interferon (IFN-I) response is the major outcome of stimulator of interferon genes (STING) activation in innate cells. STING is more abundantly expressed in adaptive T cells; nevertheless, its intrinsic function in T cells remains unclear. Intriguingly, we previously demonstrated that STING activation in T cells activates widespread IFN-independent activities, which stands in contrast to the well-known STING-mediated IFN response. Here, we have identified that STING activation induces regulatory T cells (Tregs) differentiation independently of IRF3 and IFN. Specifically, the translocation of STING from the endoplasmic reticulum to the Golgi activates mitogen-activated protein kinase (MAPK) activity, which subsequently triggers transcription factor cAMP response element-binding protein (CREB) activation. The activation of the STING-MAPK-CREB signaling pathway induces the expression of many cytokine genes, including interleukin-2 (IL-2) and transforming growth factor-beta 2 (TGF-ß2), to promote the Treg differentiation. Genetic knockdown of MAPK p38 or pharmacological inhibition of MAPK p38 or CREB markedly inhibits STING-mediated Treg differentiation. Administration of the STING agonist also promotes Treg differentiation in mice. In the Trex1-/- autoimmune disease mouse model, we demonstrate that intrinsic STING activation in CD4+ T cells can drive Treg differentiation, potentially counterbalancing the autoimmunity associated with Trex1 deficiency. Thus, STING-MAPK-CREB represents an IFN-independent signaling axis of STING that may have profound effects on T cell effector function and adaptive immunity.

IFIH1 (MDA5) is required for innate immune detection of intron-containing RNA expressed from the HIV-1 provirus.

Intron-containing RNA expressed from the HIV-1 provirus activates type 1 interferon in primary human blood cells, including CD4+ T cells, macrophages, and dendritic cells. To identify the innate immune receptor required for detection of intron-containing RNA expressed from the HIV-1 provirus, a loss-of-function screen was performed with short hairpin RNA-expressing lentivectors targeting twenty-one candidate genes in human monocyte-derived dendritic cells. Among the candidate genes tested, only knockdown of XPO1 (CRM1), IFIH1 (MDA5), or MAVS prevented activation of the interferon-stimulated gene ISG15. The importance of IFIH1 protein was demonstrated by rescue of the knockdown with nontargetable IFIH1 coding sequence. Inhibition of HIV-1-induced ISG15 by the IFIH1-specific Nipah virus V protein, and by IFIH1-transdominant 2-CARD domain-deletion or phosphomimetic point mutations, indicates that IFIH1 (MDA5) filament formation, dephosphorylation, and association with MAVS are all required for innate immune activation in response to HIV-1 transduction. Since both IFIH1 (MDA5) and DDX58 (RIG-I) signal via MAVS, the specificity of HIV-1 RNA detection by IFIH1 was demonstrated by the fact that DDX58 knockdown had no effect on activation. RNA-Seq showed that IFIH1 knockdown in dendritic cells globally disrupted the induction of IFN-stimulated genes by HIV-1. Finally, specific enrichment of unspliced HIV-1 RNA by IFIH1 (MDA5), over two orders of magnitude, was revealed by formaldehyde cross-linking immunoprecipitation (f-CLIP). These results demonstrate that IFIH1 is the innate immune receptor for intron-containing RNA from the HIV-1 provirus and that IFIH1 potentially contributes to chronic inflammation in people living with HIV-1, even in the presence of effective antiretroviral therapy.

The effectiveness of ruxolitinib and cyclophosphamide combination on T helper 17 and regulatory T cells in rat experimental membranous glomerulonephritis.

The progression and pathogenesis of membranous glomerulonephritis (MGN) are inextricably linked to chronic inflammation. Despite improving clinical remission rates due to the application of cyclophosphamide (CYC), treatment of MGN still requires further exploration. Ruxolitinib (Ruxo) negatively affects the signaling pathways participating in the production of pro-inflammatory cytokines. Hence, we investigated whether the combination of CYC and Ruxo can modulate inflammation through influencing T helper 17 (Th17) lineages and regulatory T cells (Tregs). Passive Heymann nephritis (PHN), an experimental model of MGN, was induced in a population of rats. Then, the animals were divided into five groups: PHN, CYC-receiving, Ruxo-receiving, CYC-Ruxo-receiving PHN rats, and healthy controls. After 28 days of treatment, biochemistry analysis was performed and splenocytes were isolated for flowcytometry investigation of Th17 cells and Tregs. The correlative transcription factors of the cells, alongside their downstream cytokine gene expressions, were also assessed using real-time PCR. Furthermore, serum cytokine signatures for the lymphocytes were determined through ELISA. The combination of CYC and Ruxo significantly reduced the serum values of urea in rats versus the PHN group (24.62 ± 7.970 vs. 40.60 ± 10.81 mg/dL). In contrast to Treg's activities, the functionality of Th17 cells noticeably increased not only in PHN rats but also in CYC or Ruxo-receiving PHN animals when compared with the control (10.60 ± 2.236, 8.800 ± 1.465, 8.680 ± 1.314 vs. 4.420 ± 1.551 %). However, in comparison to the PHN group, the incidence of Th17 cells notably fell in rats receiving CYC and Ruxo (10.60 ± 2.236 vs. 6.000 ± 1.373 %) in favor of the Treg's percentage (5.020 ± 1.761 vs. 8.980 ± 1.178 %), which was verified by the gene expressions and cytokine productions correlative to these lymphocytes. The combination of CYC and Ruxo was able to decline Th17 cells in favor of Tregs improvement in PHN rats, suggesting an innovative combination therapy in MGN treatment approaches.