Secretion Of Inflammatory Cytokines Research Articles

055 ANB032, an Investigational BTLA Agonist Antibody, Reduced T Cell Proliferation, Inflammatory Cytokine Secretion, and Prolonged Survival in a Mouse Model of Graph versus Host Disease (GvHD)

055 ANB032, an Investigational BTLA Agonist Antibody, Reduced T Cell Proliferation, Inflammatory Cytokine Secretion, and Prolonged Survival in a Mouse Model of Graph versus Host Disease (GvHD)

Clinically relevant cell culture model of inflammatory bowel diseases for identification of new therapeutic approaches.

Inflammatory Bowel Diseases (IDB) are chronic disorders characterized by gut inflammation, mucosal damage, increased epithelial permeability and altered mucus layer. No accurate in vitro model exists to simulate these characteristics. In this context, drug development for IBD or intestinal inflammation requires in vivo evaluations to verify treatments efficacy. A new model with altered mucus layer composition; altered epithelial permeability and pro-inflammatory crosstalk between immune and epithelial cells will be developed to enhance in vitro models for studying IBD treatments. The effects of dextran sulfate sodium and/or lipopolysaccharides on intestinal permeability, cytokines synthesis (IL-6, IL-8, TNF-α and IL-1β), mucins (MUC2, MUC5AC) and tight junction proteins expression (Claudin-1, ZO-1 and Occludin) were investigated in a tri-coculture model combining differentiated Caco-2/HT29-MTX cells and THP-1 cells. Two anti-inflammatory agents were evaluated to assess the model's therapeutic strategy applicability (corticoids and pro-resolving factors). Two in vitro models have been developed. The first model, characterized by increased permeability of the epithelial layer and subsequent secretion of inflammatory cytokines, can reproduce the different phases of inflammation, and enables the evaluation of preventive treatments. The second model simulates the acute phase of inflammation and allows for the assessment of curative treatments. Both models demonstrated reversibility when treated with betamethasone and pro-resolving factors. These in vitro models are valuable for selecting therapeutic agents prior to their application in in vivo models. They enable the assessment of agents' anti-inflammatory effects and their ability to permeate the inflamed epithelial layer and interact with immune cells.

Transcriptomic and proteomic analysis of the jejunum revealed the effects and mechanism of protocatechuic acid on alleviating Salmonella typhimurium infection in chickens

Salmonella typhimurium is a common cause of gastroenteritis, which infects animals and human. Protocatechuic acid has anti-inflammatory, immunoregulatory and anti-pathogenic effects, and is expected to be an effective choice for alleviating Salmonella infection and intestinal injury. A total of 180 1-d female, yellow-feathered chickens were randomly allocated into 3 treatment groups, the controls (Ctr), the Salmonella-challenged treatment (Sal) and the protocatechuic acid treatment (PA). Birds were fed a basal diet for 18 d, with birds in PA supplemented with 600 mg/kg protocatechuic acid. On 14 and 16 d, birds in Sal and PA were orally challenged with 109 CFU S. typhimurium, while birds in Ctr received an equal amount of PBS. The results showed that protocatechuic acid improved growth performance and jejunal structure in Salmonella-infected chickens (P < 0.05). In addition, protocatechuic acid suppressed (P < 0.05) the secretion of plasmal IgG, IL-1β, and IFN-β, and jejunal mucosal IL-6, IFN-β, complement protein 3 and 4. Mechanistically, the transcriptomic results showed that dietary protocatechuic acid inhibited acute inflammatory response and activated the signaling pathways of cytokine-cytokine receptor interaction, complement and coagulation cascades. Results of proteomic showed that protocatechuic acid inhibited the positive regulation of dendritic cell cytokine production and I-κB kinase/NF-κB signaling, as well as cytokine-cytokine receptor interaction and MAPK signaling pathways, and activated the mTOR signaling pathways. These results were critical to both suppressing the secretion of inflammatory cytokines and restoring the intestinal function. Dietary protocatechuic acid (600 mg/kg) was effective to alleviate Salmonella infection, as it suppressed the levels of cytokines and complement protein by inhibiting MAPK, I-κB kinase/NF-κB and enhancing the mTOR signaling pathway, thus offset the decline in weight loss and intestinal injury caused by infection.

Gut-on-a-Chip Reveals Enhanced Peristalsis Reduces Nanoplastic-Induced Inflammation.

Nanoplastics (NPs) pollution is a global issue posing potential threats to human health, particularly the digestive system. NPs may exacerbate intestinal inflammation, increasing the risk of inflammatory bowel disease. However, the impact of intestinal peristalsis on NP-induced inflammation remains unknown. Here, a biomimetic gut-on-a-chip (GOC) with integrated online sensing is presented to investigate NPs' impact on intestinal inflammation and propose enhanced peristalsis as a potential intervention. The GOC simulates intestinal peristalsis through periodic stretching and the optimized sensors dynamically detect inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) over 12 d with low detection limits (0.0095 and 0.0020 pg mL-1). Exposure to NPs led to vacuolization, apoptosis, and loss of tight junction proteins in intestinal cells, with IL-6 and TNF-α secretion peaking at 24 h (1341.55 ± 64.91 and 862.03 ± 66.45 pg mL-1). Notably, increasing periodic strain alleviates inflammatory cytokines secretion induced by NPs. With strain increased from 5% to 6.5%, IL-6 and TNF-α secretion decrease by 2.73-fold and 3.34-fold, respectively. This highlights the protective role of intestinal peristalsis in reducing NP-induced inflammation.

Therapeutic potential of Lacticaseibacillus rhamnosus grx10 and its derived postbiotic through gut microbiota and MAPK/MLCK/MLC pathway-mediated intestinal barrier repairment in ulcerative colitis.

Lacticaseibacillus rhamnosus grx10 (grx10) has shown promising potential in promoting intestinal health as predicted by genomic and metabolomic analyses. Given the increasing prevalence of ulcerative colitis (UC) and the limitations of existing treatments, exploring alternative therapeutic strategies is essential. This study explored the therapeutic effects and underlying mechanisms of grx10 and its derived postbiotic (P-grx10) in a mouse model of dextran sulfate sodium (DSS)-induced chronic UC. The intervention with grx10 and P-grx10 significantly alleviated clinical symptoms and improved biochemical markers in UC mice. These effects included reducing the disease activity index (DAI), improving colon length and histopathological damage, decreasing the secretion of inflammatory cytokines, and preventing the reduction of antioxidant enzymes. Additionally, grx10 and P-grx10 downregulated key proteins in the Mitogen-Activated Protein Kinase (MAPK)/myosin light chain kinase (MLCK)/myosin light chain (MLC) pathway, prevented the dissociation of tight junction (TJ) proteins and E-cadherin, reduced intestinal permeability, and restored the integrity of the intestinal barrier. Furthermore, both grx10 and P-grx10 modulated the composition and abundance of gut microbiota, helping to maintain intestinal microbiome homeostasis. In conclusion, this study provided evidence regarding the role of grx10 and P-grx10 in alleviating intestinal barrier dysfunction associated with UC and restoring gut microbiota balance. Notably, P-grx10 exhibited higher anti-inflammatory activity and better restoration of intestinal barrier function, whereas the live probiotic grx10 showed a stronger regulatory effect on the gut microbiota. These findings suggest that grx10 and P-grx10 could serve as promising nutritional adjunct therapies for UC, providing novel insights into the distinct roles of probiotic and its derived postbiotic in UC treatment.

Metrnl/IL-41 Alleviates Lipopolysaccharide-Induced Acute Lung Injury by Suppressing Inflammatory Responses through the AMPK/SIRT1 Signaling Pathway

Introduction: Interleukin-41 (IL-41), also known as Metrnl, is a multifunctional adipokine recognized for its neurotrophic and anti-inflammatory properties that play a significant role in diseases such as sepsis and chronic obstructive pulmonary disease. Despite its crucial biological functions, the mechanisms by which IL-41 mitigates lipopolysaccharide (LPS)-induced acute lung injury (ALI) are not well understood. This study aimed to elucidate the protective effects of IL-41 against LPS-induced inflammation and apoptosis in a murine model and in vitro using bronchial epithelial cells (Beas-2b). Methods: We administered recombinant IL-41 to mice subjected to LPS-induced ALI and observed changes in lung histopathology, inflammation, and apoptosis. Concurrently, Beas-2b cells were transfected with IL-41 constructs, and the role of the AMPK/SIRT1 pathway was investigated using specific inhibitors and agonists. Results: Our results demonstrated that LPS-induced ALI is characterized by increased inflammatory cell chemotaxis in lung lavage fluid, enhanced phosphorylation of NFκB p65, and elevated Bax protein expression, coupled with a decrease in IL-41 protein levels. Treatment with recombinant IL-41 effectively mitigated these pathological changes by upregulating AMPK phosphorylation and SIRT1 expression and inhibiting IκB/NFκB p65 phosphorylation. In cellular assays, overexpression of IL-41 reversed LPS-induced oxidative stress, apoptosis, and the secretion of inflammatory cytokines, whereas suppression of IL-41 or inhibition of AMPK reversed these protective effects. Conclusions: In conclusion, IL-41 exerts significant protective effects against ALI by activating the AMPK/SIRT1 signaling pathway and reducing excessive inflammation and apoptosis. These findings suggest that IL-41 holds promise as a therapeutic target for ALI, potentially allowing for personalized treatments based on serum IL-41 levels.

SENP1 Promotes Caspase-11 Inflammasome Activation and Aggravates Inflammatory Response in Murine Acute Lung Injury Induced by Lipopolysaccharide.

Infection is the leading cause of acute lung injury (ALI). Macrophages, which are pivotal innate immune cells, play a critical role in mediating inflammatory processes. Intracellular lipopolysaccharide (LPS) from invasive Gram-negative bacteria can activate the caspase-11 inflammasome, leading to the induction of pyroptosis in macrophages. This process subsequently triggers the release of inflammatory cytokines and damage-associated molecular patterns from pyroptotic macrophages, thereby exacerbating inflammatory progression in ALI. However, the precise regulatory mechanisms governing caspase-11 activation is still unclear. Sentrin-specific proteases (SENPs) have been identified as notable targets for their anti-inflammatory properties. Nevertheless, the specific role of SENPs in macrophage pyroptosis during the pathogenesis of ALI remains unknown. We used LPS as an endotoxin to induce ALI. We analyzed the expression and location of sentrin-specific protease 1 (SENP1), pulmonary impairment, macrophage infiltration, caspase-11 inflammasome expression and activation, caspase-11 SUMOylation, and inflammatory cytokine secretion. Upregulated expression of SENP1 in lung tissue and macrophages was observed following LPS stimulation. SENP1 mediates de-SUMOylation and activation of caspase-11 inflammasome in macrophages. Moreover, pharmacological inhibition or genetic deficiency of SENP1 in macrophages significantly improved ALI-related histological damage by reducing the secretion of inflammatory cytokines and suppressing caspase-11-dependent pyroptosis. Collectively, our findings highlight the involvement of SENP1 in caspase-11 activation and inflammatory progression in macrophages, thereby establishing a scientific foundation for the exploration of novel therapeutic strategies aimed at treating ALI.

PTX3 impairs granulosa cell function by promoting the secretion of inflammatory cytokines in M1 macrophages via the JAK pathway

Polycystic Ovary Syndrome (PCOS) is an endocrine disorder syndrome among women in their reproductive years and is often linked to chronic inflammation. Pentraxin 3 (PTX3), a member of the pentraxin protein family, plays a key role in inflammation. In our study, we explored whether PTX3 influences granulosa cell function via its involvement in inflammation. Our analysis revealed elevated PTX3 concentrations in the follicular fluid and granulosa cells of patients with PCOS. Overexpression of PTX3 promoted apoptosis in the cultured murine granulosa cell line KK1 and inhibited the proliferation of these cells. Additionally, it suppressed the expression of luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR), as well as those of key enzymes involved in steroid hormone synthesis, CYP19A1, and HSD3β, leading to reduced secretion of estradiol and progesterone. Moreover, both recombinant PTX3 protein and PTX3 secreted by granulosa cells (GCs) promoted the secretion of inflammatory cytokines IL-1β, IL-6, and TNF-α by M1 macrophages via the JAK pathway, which impaired the function of granulosa cells. This study may advance the understanding of cell–cell interactions in follicles and the inflammatory factors that contribute to PCOS pathophysiology.

Fe-capsaicin nanozyme attenuates sepsis-induced acute lung injury by regulating the functions of macrophages.

In sepsis, the lung is one of the worst affected organs, often leading to acute lung injury (ALI). More and more evidence suggests that macrophages are also involved in the pathogenesis of ALI. In our previous study, we successfully synthesized Iron-capsaicin-based nanoparticles (Fe-CAP NPs) and found that it could inhibit the secretion of inflammatory cytokines to alleviate ALI. Here, we further explore the anti-inflammatory mechanism of Fe-CAP NPs. Bone marrow-derived macrophages (BMDM) and C57 mice were divided into four groups: control group, lipopolysaccharide (LPS) group, CAP + LPS group and Fe-CAP + LPS group. Western blot and Immunofluorescence were used to detect the expression of macrophage phenotypic markers CD86 and CD206 in BMDM and lung tissue. Fluorescence microbeads, Transwell and ROS kit were used to detect the phagocytosis, migration and ROS clearing capability of BMDM. Western blot was used to detect the expression of JAK2/STAT3 pathway and apoptosis proteins in BMDM. TUNEL kit and H&E staining were used to evaluate apoptosis and pathological changes in lung tissue. In vitro, CD86 expression was increased in LPS, but decreased after Fe-CAP pretreatment. CD206 expression was the opposite. Fe-CAP reduced phagocytosis, migration and scavenged ROS in LPS-treated BMDM. Fe-CAP inhibited P-JAK2 and P-STAT3 expression and reduced apoptosis. In vivo, Fe-CAP improved lung histopathology and reduced apoptosis in lung tissue of LPS group. CD86 expression was increased in lung tissue of LPS group, but decreased in Fe-CAP preconditioning, and CD206 expression was reversed. Fe-CAP NPs could alleviate sepsis-induced ALI by regulating the polarization and function of macrophages, reducing ROS level and apoptosis.

Human Tendon-on-a-Chip for Modeling the Myofibroblast Microenvironment in Peritendinous Fibrosis.

Understanding the myofibroblast microenvironment is critical to developing therapies for fibrotic diseases. Here the development of a novel human tendon-on-a-chip (hToC) is reported to model this crosstalk in peritendinous adhesions, which currently lacks biological therapies. The hToC facilitates cellular and paracrine interactions between a vascular component, which contains endothelial cells and monocytes, and a tissue hydrogel component that houses tendon cells and macrophages. It is found that the hToC replicates some aspects of in vivo inflammatory and fibrotic phenotypes in preclinical and clinical samples, including activated mTOR signaling, vascular inflammation, tissue contraction induced by myofibroblast activation, inflammatory cytokines secretion, and transendothelial migration of monocytes to the tissue hydrogel. Transcriptional analysis demonstrates significant overlap in enriched pathways in the hToC with human tenolysis samples, including the activation of mTOR signaling. Rapamycin suppresses the vascular inflammation and fibrotic phenotype in the hToC, which provides proof-of-concept of its utility as an in vitro tool for investigating multicellular crosstalk in fibrosis and testing therapeutics to mitigate it.

Abstract 4132968: CCR5 modulation enhances M-MDSCs functionality in early atherosclerosis

Background: The role of immune suppressive microenvironment, promulgated by the presence of Myeloid Derived Suppressor Cells (MDSC) have been the major focus of research delineating the underlying mechanisms of cardiovascular diseases (CVD). CCR5, a chemokine receptor, has been associated with both immunosuppressive and inflammatory phenotypes, however, the possible role of CCR5 pertaining to MDSCs in the development of atherosclerosis has not been elucidated yet. Therefore, we investigated the therapeutic potential of CCR5 modulation in early atherosclerosis progression. Methods: CCR5 receptor-ligand expression was studied with flow cytometry, real time PCR and immunocytochemistry in 65 individuals (25 Healthy controls, 20 young hypercholesterolemic individuals, and 20 stable [Coronary Artery Disease] CAD patients). To induce atherosclerosis, mice were fed high fat diet (HFD) for 6 weeks following partial ligation of left carotid artery in C57BL6 mice. MDSCs phenotype and functionality were assessed using CCR5 inhibitor in vitro (10 -8 M) and in vivo (i.p 3ng/day for 15 days) . M-MDSCs phenotype switch, atherosclerotic lesion development and plaque phenotype was studied in vivo . Results: We observed CCR5 elevation on Monocytic-MDSCs in the early phase of atherosclerosis. M-MDSCs displayed an intermediate phenotype with expression of both pro and anti-inflammatory mediators in early atherosclerosis, a similar trend was observed in inflammatory cytokines stimulated M-MDSCs ( in vitro ). Notably, DAPTA treatment shifted phenotype of M-MDSCs to anti-inflammatory profile with elevated expression of IL-10 and reduced expression of IFNγ, IL4, and IL22. Further, CCR5 modulation expanded IL10+M-MDSC in vivo . Additionally, DAPTA reduced the migratory potential, reduced cholesterol uptake and improved the functionality (suppression of T cell) of M-MDSCs. Blocking CCR5 with DAPTA led to the formation of stable plaque in HFD-fed mice. Conclusion: Increased expression of CCR5 coupled with inflammatory cytokine secretion by regulatory cells lead to dysfunctional M-MDSCs in CVD risk factor population. Possibly this dysfunctionality contributes to the development and progression of CVD including atherosclerosis. Our findings show targeting of CCR5 with DAPTA reduces atherogenesis, increases plaque stabilization and corrects phenotypic alterations in immune suppressive M-MDSCs. The functionality and phenotype of M-MDSCs in atherosclerosis, may be ameliorated by targeting CCR5.

Construction of a Myeloid Gene Signature Revealing Novel Therapeutic Targets for Myeloid Malignancies

FH, SL and BG equal contributors Myeloproliferative neoplasms (MPN) and acute myeloid leukemia (AML) exhibit shared hallmarks of hyperinflammation and aberrant clonal expansion. Chronic MPNs exhibit a propensity for transformation to secondary AML (sAML), which imparts a dismal prognosis with limited treatment options. However, the pathogenesis of leukemia transition for MPN and the bidirectional relationship between MPN and AML remain incompletely understood. In this study, we investigated commonly-featured genes and pathways of these two diseases to explore novel therapeutic strategies. To dissect the causal association between MPN and AML, we first performed two-sample bidirectional mendelian randomization (MR) analysis which revealed that MPN predicts the risk of AML across multiple statistical methods with consistent directionality and significance. Heterogeneity, pleiotropy, or outlier effects were ruled out by sensitivity analyses with no reverse causality from AML to MPN. To identify a potential myeloid signature, we estimated causal effects of 2,940 plasma proteins on MPN via a two-sample MR framework and identified 55 candidates significantly associated with increased risk of MPN (P &amp;lt; 0.05, OR &amp;gt; 1). Utilizing data from the BEAT-AML cohort, we further screened the prognostic value of these candidates using Cox and log-rank test survival analyses and identified a 4-gene myeloid signature comprising MPO, CDCP1, CRISP3 and DXCR, with each conferring prognostic significance individually. Our scRNA-seq results confirmed enrichment of myeloid signature genes in myeloid progenitor cells, with further elevation in MPN. Of note, expression of myeloid signature genes was induced by Jak2V617F and MPLW515L in Ba/F3 cells. We then investigated the combined prognostic significance of these four genes by constructing a risk score via LASSO regression modeling and divided patients into high- and low-risk groups accordingly. Cox analysis validated the risk score as an independent prognostic factor (HR: 3.01 (1.2 - 7.6), P = 0.019) and Kaplan-Meier survival analysis demonstrated that low-risk AML patients had a significantly better survival than high-risk AML patients with median survival of 21.9 months vs. 11.9 months (P &amp;lt; 0.0001). The prediction accuracy of the myeloid signature was successfully validated in the TCGA-LAML cohort. The risk score significantly correlated with several key clinical parameters, including age, platelet count, ELN2017 staging, monocyte percentage and bone marrow cellularity. To interrogate the underlying mechanism of the myeloid signature, we performed gene set enrichment analysis (GSEA) and observed enrichment of several inflammatory pathways in high-risk AML patients. A similar enrichment of inflammatory pathways in MPN samples compared to healthy donors observed in our previous scRNA-seq results reinforced hyperinflammation as a shared etiology of myeloid malignancies. To explore potential therapeutic strategies, we performed in silico drug sensitivity screening which predicted specific vulnerability of high-risk AML to compounds targeting PI3K/AKT/mTOR pathway signaling. Activation of PI3K/AKT/mTOR signaling in both MPN and AML samples observed across multiple single cell and bulk RNA-seq datasets further prompted us to evaluate targeted therapies against this pathway in myeloid malignancies. MPN and AML cells exhibited sensitivity to mTOR inhibitors as indicated by suppression of cellular proliferation, metabolism, colony formation, and inflammatory cytokine secretion in conjunction with induction of apoptosis. We further evaluated the therapeutic effects of mTOR inhibition in vivo via administration of omipalisib, a potent dual PI3K/mTOR inhibitor, to JAK2V617F knock-in mice. Omipalisib treatment significantly ameliorated features of myeloid malignancies including splenomegaly and leukocytosis which were both significantly reduced following 4 weeks of treatment, without affecting body weight. In summary, our MR analyses reveal that MPN predicts the risk of AML and enabled the construction of a novel myeloid signature which risk stratified AML patients across two different cohorts. We further demonstrate shared activation of PI3K/AKT/mTOR pathway across myeloid malignancies, with in vitro and in vivo data providing a rationale for therapeutic targeting of PI3K/AKT/mTOR pathway in these diseases.

Mesenchymal stem cell-derived extracellular vesicles reduce inflammatory responses to SARS-CoV-2 and Influenza viral proteins via miR-146a/NF-κB pathway

The risk of severe disease caused by co-infection with SARS-CoV-2 and influenza virus (IAV) raises an annual concern for global public health. Extracellular vesicles (EV) derived from mesenchymal stem cells (MSC) possess anti-inflammatory properties that can attenuate the inflammatory cytokine levels induced by viral infection. However, the effects of MSC-EV treatment on SARS-CoV-2 and IAV co-infection have not been elucidated. In the present study, we co-induced lung epithelial cells (EpiC) with SARS-CoV-2 Spike protein (S) and H1N1 influenza viral HA protein (HA) and found robust upregulation of inflammatory cytokines in comparison to those induced by either S or HA protein. Consequently, treatment of lung endothelial cells (EC) with conditioned medium from EpiC co-induced by both S and HA proteins resulted in increased apoptosis and impaired angiogenic ability, suggesting the effects of co-induction on epithelial-endothelial crosstalk. In addition, lung EpiC co-induced by both S and HA proteins showed paracrine effects on the recruitment of immune cells, including monocytes, macrophages and neutrophils. Of Note, EV derived from Wharton Jelly’s MSC (WJ-EV) transferred miR-146a to recipient lung EpiC, which impaired TRAF6 and IRAK1, resulting in the downregulation of NF-κB pathway and secretion of inflammatory cytokines, rescuing the epithelial-endothelial crosstalk, and reducing the elevation of immune cell recruitment. Moreover, the anti-inflammatory properties of WJ-EV are affected by type 2 Diabetes Mellitus. WJ-EV derived from donors with type 2 Diabetes Mellitus contained less miR-146a and showed impaired ability to downregulate the NF-κB pathway and inflammatory cytokines in recipient cells. Taken together, our findings demonstrate the role of miR-146a in targeting the NF-κB pathway in the anti-inflammatory abilities of WJ-EV, which is a promising strategy to rescue the epithelial-endothelial crosstalk altered by co-infection with SARS-CoV-2 and IAV.

Practical supplements for prevention and management of migraine attacks: a narrative review.

Migraine is one of the most debilitating neurological disorders that causes frequent attacks of headaches and affects approximately 11% of the global population. Deficient or even insufficient levels of vital nutrients would increase the severity and frequency of migraine attacks. Therefore, we aimed to examine the practical supplements for the prevention and management of migraine attacks. This narrative review study was conducted by searching PubMed, ISI web of science, EMBASE, Google Scholar, and Scopus using the keywords of "dietary supplement" and "migraine" plus their MeSH terms. Original articles published in English language from their inception to July 27th, 2024, studies that investigated adult population (aged >18 years), and those assessing the impact of intended nutrient supplementation on clinical symptoms of migraine were included in the study. Oxidative stress and low intake of antioxidants would be risk factors for migraine attacks by inducing inflammation. The secretion of inflammatory cytokines, such as tumor necrosis factor (TNF)-a, would lead to neuroinflammation and migraine episodes by increasing the cellular permeability and interactions. Evidence also indicated a direct association between phases of migraine attacks and calcitonin gene-related peptide (CGRP), mitochondrial disorders, monoaminergic pathway, disruption in brain energy metabolism, and higher serum levels of glutamate and homocysteine. Therefore, supplementation with nutrients involved in mitochondrial function, brain energy metabolism, and even methyl donors would relieve migraine attacks. Evidence indicated that supplementation with riboflavin, omega-3 fatty acids, alpha lipoic acid, magnesium, probiotics, coenzyme Q10, ginger, and caffeine would have favorable effects on migraine patients. However, more prospective studies are required to evaluate the effect of other nutrients on migraine patients.

Soluble immune checkpoints associated with disease activity and treatment response in GD and TED.

Soluble immune checkpoints play an important role in peripheral tolerance that has seldom been investigated in Graves' disease (GD) and thyroid eye disease (TED). The objective of this work is to examine the alteration of soluble immune checkpoints in GD and TED. We performed a quantitative multiplex analysis of 17 immune checkpoint proteins in serum from 50 GD patients without TED, 28 GD patients with TED and 40 healthy controls. The association with demographical, serological, clinical features and 27 cytokines was analyzed. A follow-up was conducted in GD patients without TED. Functional outcomes of sLAG-3 and sGITR were assessed in cell cultures using rh-LAG3, rh-GITR, an antagonistic LAG-3 antibody and an antagonistic GITR antibody. GD patients with TED had distinct sICP and cytokine profiles compared with GD patients without TED. Active TED patients exhibited elevation in the levels of sBTLA, sLAG-3, sGITR, sCD80, sCD86 and sPD-L1. Further, GD patients without TED with high sBTLA, sCD27 and sCD40 levels at baseline showed a better improvement in thyrotropin receptor antibody (TRAb) titers after antithyroid drug (ATD) treatment. Adding recombinant human GITR and LAG-3 to PBMC cultures resulted in increased inflammatory cytokine secretion and decreased anti-inflammatory cytokine secretion. The present study uncovers disturbed soluble immune checkpoints and cytokines in GD patients with and without TED, and may pave the way for novel immunological screening, allowing for identification of TED patients at higher risk of developing active disease and GD patients with better treatment response after ATD treatment.

TRIM7 knockdown protects against LPS-induced autophagy, ferroptosis, and inflammatory responses in human bronchial epithelial cells.

Asthma is one of the most common respiratory diseases in pediatric department. Several asthma-associated events including inflammatory responses, autophagy, and ferroptosis have been identified as typical pathological processes. TRIM7 is a member of TRIM proteins family associated with several types of diseases. Nevertheless, its role in asthma is still elusive. The current research showed that TRIM7 was involved in the pathogenesis of asthma mainly by regulating the Akt signaling pathway. In detail, we found that TRIM7 was highly expressed in patients with asthma and in an in vitro model of asthma. The following analysis indicated that TRIM7 knockdown attenuated the expression and secretion of inflammatory cytokines including TNF-α, IL-1β and IL-6 in lipopolysaccharide (LPS)-exposed human bronchial epithelial cells (HBECs). Meanwhile, knockdown of TRIM7 exerted inhibitory effects on LPS-induced autophagy and ferroptosis. Further mechanistic studies showed that TRIM7 knockdown inhibited LPS-induced activation of Akt pathway, while overexpression of Akt attenuated the inhibitory effects of TRIM7 knockdown on LPS-exposed HBECs. Collectively, we reported here that TRIM7 knockdown inhibited LPS-induced autophagy, ferroptosis, and inflammatory cytokine secretion in HBECs via regulating the Akt pathway, providing a new insight into the strategies for improving asthma treatments.

Combining ERAP1 silencing and entinostat therapy to overcome resistance to cancer immunotherapy in neuroblastoma

BackgroundCheckpoint immunotherapy unleashes tumor control by T cells, but it is undermined in non-immunogenic tumors, e.g. with low MHC class I expression and low neoantigen burden, such as neuroblastoma (NB). Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an enzyme that trims peptides before loading on MHC class I molecules. Inhibition of ERAP1 results in the generation of new antigens able of inducing potent anti-tumor immune responses. Here, we identify a novel non-toxic combinatorial strategy based on genetic inhibition of ERAP1 and administration of the HDAC inhibitor (HDACi) entinostat that increase the immunogenicity of NB, making it responsive to PD-1 therapy.MethodsCRISPR/Cas9-mediated gene editing was used to knockout (KO) the ERAP1 gene in 9464D NB cells derived from spontaneous tumors of TH-MYCN transgenic mice. The expression of MHC class I and PD-L1 was evaluated by flow cytometry (FC). The immunopeptidome of these cells was studied by mass spectrometry. Cocultures of splenocytes derived from 9464D bearing mice and tumor cells allowed the assessment of the effect of ERAP1 inhibition on the secretion of inflammatory cytokines and activation and migration of immune cells towards ERAP1 KO cells by FC. Tumor cell killing was evaluated by Caspase 3/7 assay and flow cytometry analysis. The effect of ERAP1 inhibition on the immune content of tumors was analyzed by FC, immunohistochemistry and multiple immunofluorescence.ResultsWe found that inhibition of ERAP1 makes 9464D cells more susceptible to immune cell-mediated killing by increasing both the recall and activation of CD4+ and CD8+ T cells and NK cells. Treatment with entinostat induces the expression of MHC class I and PD-L1 molecules in 9464D both in vitro and in vivo. This results in pronounced changes in the immunopeptidome induced by ERAP1 inhibition, but also restrains the growth of ERAP1 KO tumors in vivo by remodelling the tumor-infiltrating T-cell compartment. Interestingly, the absence of ERAP1 in combination with entinostat and PD-1 blockade overcomes resistance to PD-1 immunotherapy and increases host survival.ConclusionsThese findings demonstrate that ERAP1 inhibition combined with HDACi entinostat treatment and PD-1 blockade remodels the immune landscape of a non-immunogenic tumor such as NB, making it responsive to checkpoint immunotherapy.

Deleting fibroblast growth factor 2 in macrophages aggravates septic acute lung injury by increasing M1 polarization and inflammatory cytokine secretion.

Septic lung injury is strongly associated with polarization of M1 macrophages and excessive cytokine release. Fibroblast growth factor (FGF) signaling plays a role in both processes. However, the impact of FGF2 deficiency on macrophage polarization and septic acute lung injury remains unclear. To investigate this, we obtained macrophages from FGF2 knockout mice and examined their polarization and inflammatory cytokine expression. We also eliminated endogenous macrophages using clodronate liposomes and administered FGF2 knockout or WT macrophages intravenously in conjunction with cecal ligation and puncture (CLP) surgery to induce sepsis. In vitro analysis by flow cytometry and real-time PCR analysis demonstrated that FGF2 deficiency resulted in increased expression of M1 markers (iNOS and CD86) and inflammatory cytokines (CXCL1, IL1β, and IL6), especially after LPS stimulation. Additionally, immunofluorescence demonstrated increased nuclear translocation of p65 NF-κB in FGF2 knockout macrophages and RNA-seq analysis showed enrichment of differentially expressed genes in the IL17 and TNFα inflammatory signaling pathways. Furthermore, in vivo experiments revealed that depletion of FGF2 in macrophages worsened sepsis-induced lung inflammation, lung vascular leak, and lung histological injury, accompanied by an increase in CD86-positive cells and apoptosis. Our study suggests that FGF2 deficiency in macrophages plays a critical role in the pathogenesis of septic ALI, possibly because of the enhanced M1 macrophage polarization and production of proinflammatory cytokines. These findings provide empirical evidence for potential therapeutic interventions targeting FGF2 signaling to modulate the polarization of M1 and M2 macrophages in the management of sepsis-induced acute lung injury.

Quercetin suppresses pyroptosis in mouse fibroblasts by inhibiting the NLRP3/caspase-1/GSDMD pathway

To investigate whether quercetin inhibits pyroptosis of mouse fibroblast NIH-3T3 cells by regulating the NLRP3/caspase-1/GSDMD signaling pathway. NIH-3T3 cells were treated with quercetin or MCC950 (a specific inhibitor of NLRP3) before stimulation with lipopolysaccharide (LPS) and ATP to induce cell pyroptosis. The optimal quercetin concentration and duration were screened using the CCK-8assay after testing various concentrations and times. Morphological changes of the treated cells was observed, and the levels of IL-18 and IL-1β in the cell culture supernatant were detected with ELISA; the protein expressions of NLRP3, cleaved caspase-1, and GSDMD-N and the mRNA levels of NLRP3, caspase-1 and GSDMD were detected using Western blotting and qRT-PCR. The changes in cell pyroptosis were examined with TUNEL staining and LDH release assay. The CCK-8 assay indicated that 24-hour treatment with 20 μmol/L quercetin yielded the most favorable results. LPS and ATP stimulation of NIH-3T3 cells induced obvious swelling, cell membrane rupture and leakage of cell contents, significantly increased IL-18 and IL-1β levels, and enhanced protein expressions of NLRP3, cleaved caspase-1 and GSDMD-N and mRNA levels of NLRP3, caspase-1 and GSDMD. LPS and ATP stimulation also caused a significant increment of TUNEL-positive cell counts and LDH release in NIH-3T3 cells. Treatment with quercetin or MCC950 significantly reduced cell pyroptosis induced by LPS and ATP, lowered the concentrations of IL-18 and IL-1β, decreased the expression levels of NLRP3, caspase-1/cleaved caspase-1, GSDMD/GSDMD-N, and reduced the number of TUNEL-positive cells and LDH release. Quercetin suppresses pyroptosis of mouse fibroblasts stimulated with LPS and ATP and reduces secretion of inflammatory cytokines by inhibiting the NLRP3/caspase-1/GSDMD pathway.

Neutrophils with low production of reactive oxygen species are activated during immune priming and promote development of arthritis

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease mediated by immune cell dysfunction for which there is no universally effective prevention and treatment strategy. As primary effector cells, neutrophils are important in the inflammatory joint attack during the development of RA. Here, we used single-cell sequencing technology to thoroughly analyze the phenotypic characteristics of bone marrow-derived neutrophils in type II collagen (COL2)-induced arthritis (CIA) models, including mice primed and boosted with COL2. We identified a subpopulation of neutrophils with high expression of neutrophil cytoplasmic factor 1 (NCF1) in primed mice, accompanied by a characteristic reactive oxygen species (ROS) response, and a decrease in Ncf1 expression in boosted mice with the onset of arthritis. Furthermore, we found that after ROS reduction, arthritis occurred in primed mice but was attenuated in boosted mice. This bidirectional effect of ROS suggested a protective role of ROS during immune priming. Mechanistically, we combined functional assays and metabolomics identifying Ncf1-deficient neutrophils with enhanced migration, chemotactic receptor CXCR2 expression, inflammatory cytokine secretion, and Th1/Th17 differentiation. This alteration was mainly due to the metabolic reprogramming of Ncf1-deficient neutrophils from an energy supply pathway dominated by gluconeogenesis to an inflammatory immune pathway associated with the metabolism of histidine, glycine, serine, and threonine signaling, which in turn induced arthritis. In conclusion, we have systematically identified the functional and inflammatory phenotypic characteristics of neutrophils under ROS regulation, which provides a theoretical basis for understanding the pathogenesis of RA, to further improve prevention strategies and identify novel therapeutic targets.