Sinomenine treats rheumatoid arthritis by inhibiting MMP9 and inflammatory cytokines expression: bioinformatics analysis and experimental validation.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease marked by inflammatory cell infiltration and joint damage. The Chinese government has approved the prescription medication sinomenine (SIN), an effective anti-inflammation drug, for treating RA. This study evaluated the possible anti-inflammatory actions of SIN in RA based on bioinformatics analysis and experiments. Six microarray datasets were acquired from the gene expression omnibus (GEO) database. We used R software to identify differentially expressed genes (DEGs) and perform function evaluations. The CIBERSORT was used to calculate the abundance of 22 infiltrating immune cells. The weighted gene co-expression network analysis (WGCNA) was used to discover genes associated with M1 macrophages. Four public datasets were used to predict the genes of SIN. Following that, function enrichment analysis for hub genes was performed. The cytoHubba and least absolute shrinkage and selection operator (LASSO) were employed to select hub genes, and their diagnostic effectiveness was predicted using the receiver operator characteristic (ROC) curve. Molecular docking was undertaken to confirm the affinity between the SIN and hub gene. Furthermore, the therapeutic efficacy of SIN was validated in LPS-induced RAW264.7 cells line using Western blot and Enzyme-linked immunosorbent assay (ELISA). The matrix metalloproteinase 9 (MMP9) was identified as the hub M1 macrophages-related biomarker in RA using bioinformatic analysis and molecular docking. Our study indicated that MMP9 took part in IL-17 and TNF signaling pathways. Furthermore, we found that SIN suppresses the MMP9 protein overexpression and pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the LPS-induced RAW264.7 cell line. In conclusion, our work sheds new light on the pathophysiology of RA and identifies MMP9 as a possible RA key gene. In conclusion, the above findings demonstrate that SIN, from an emerging research perspective, might be a potential cost-effective anti-inflammatory medication for treating RA.

The beneficial effects of ethanolic extract of Sargassum serratifolium in DNCB-induced mouse model of atopic dermatitis.

Atopic dermatitis is a chronic complex inflammatory skin disorder that requires sustainable treatment methods due to the limited efficacy of conventional therapies. Sargassum serratifolium, an algal species with diverse bioactive substances, is investigated in this study for its potential benefits as a therapeutic agent for atopic dermatitis. RNA sequencing of LPS-stimulated macrophages treated with ethanolic extract of Sargassum serratifolium (ESS) revealed its ability to inhibit a broad range of inflammation-related signaling, which was proven in RAW 264.7 and HaCaT cells. In DNCB-induced BALB/c or HR-1 mice, ESS treatment improved symptoms of atopic dermatitis within the skin, along with histological improvements such as reduced epidermal thickness and infiltration of mast cells. ESS showed a tendency to improve serum IgE levels and inflammation-related cytokine changes, while also improving the mRNA expression levels of Chi3l3, Ccr1, and Fcεr1a genes in the skin. Additionally, ESS compounds (sargachromanol (SCM), sargaquinoic acid (SQA), and sargahydroquinoic acid (SHQA)) mitigated inflammatory responses in LPS-treated RAW264.7 macrophages. In summary, ESS has an anti-inflammatory effect and improves atopic dermatitis, ESS may be applied as a therapeutics for atopic dermatitis.

Parallel detection of multiple biomarkers in a point-of-care-competent device for the prediction of exacerbations in chronic inflammatory lung disease.

Sudden aggravations of chronic inflammatory airway diseases are difficult-to-foresee life-threatening episodes for which advanced prognosis-systems are highly desirable. Here we present an experimental chip-based fluidic system designed for the rapid and sensitive measurement of biomarkers prognostic for potentially imminent asthma or COPD exacerbations. As model biomarkers we chose three cytokines (interleukin-6, interleukin-8, tumor necrosis factor alpha), the bacterial infection marker C-reactive protein and the bacterial pathogen Streptococcus pneumoniae-all relevant factors in exacerbation episodes. Assay protocols established in laboratory environments were adapted to 3D-printed fluidic devices with emphasis on short processing times, low reagent consumption and a low limit of detection in order to enable the fluidic system to be used in point-of-care settings. The final device demonstrator was validated with patient sample material for its capability to detect endogenous as well as exogenous biomarkers in parallel.

Effects of intravenous inflammasome inhibitor (NuSepin) on suppression of proinflammatory cytokines release induced by cardiopulmonary bypass in swine model: a pilot study.

The systemic inflammatory response syndrome can occur due to an inflammatory reaction to the release of cytokines, and it has been linked to the circulation of pro- and anti-inflammatory cytokines. The cardiopulmonary bypass (CPB) system is known to activate numerous inflammatory pathways. Applying CPB in large animals for an extended period may be useful as a controlled experimental model for systemic inflammatory responses. The authors hypothesized that 0.2 mg/kg NuSepin® would inhibit CBP-induced proinflammatory cytokine release, and attenuate CPB-induced vasoplegia. CPB was maintained for 2 h in 8 male Yorkshire pigs. Ten ml of saline was administered intravenously to the control group, while the study group received 10 ml of NuSepin® (0.2 mg/kg), before start of CPB. Blood samples were collected at four different time points to evaluating the level of cytokine (TNF-α, IL-1ß, IL-6, IL-8) release during and after CBP. All vital signals were recorded as continuous waveforms using the vital recorder®. Our study demonstrated that IL-6 increased in both groups during CPB remained unchanged. However, in the Nusepin group, IL-6 levels rapidly decreased when CPB was stopped and the proinflammatory reaction subsided. Furthermore, the dose of norepinephrine required to maintain a mean pressure of 60 mmHg was also lower in the Nusepin group.

Neutrophils cultured ex vivo from CD34+ stem cells are immature and genetically tractable.

BACKGROUND: Neutrophils are granulocytes with essential antimicrobial effector functions and short lifespans. During infection or sterile inflammation, emergency granulopoiesis leads to release of immature neutrophils from the bone marrow, serving to boost circulating neutrophil counts. Steady state and emergency granulopoiesis are incompletely understood, partly due to a lack of genetically amenable models of neutrophil development. METHODS: We optimised a method for ex vivo production of human neutrophils from CD34+ haematopoietic progenitors. Using flow cytometry, we phenotypically compared cultured neutrophils with native neutrophils from donors experiencing emergency granulopoiesis, and steady state neutrophils from non-challenged donors. We carry out functional and proteomic characterisation of cultured neutrophils and establish genome editing of progenitors. RESULTS: We obtain high yields of ex vivo cultured neutrophils, which phenotypically resemble immature neutrophils released into the circulation during emergency granulopoiesis. Cultured neutrophils have similar rates of ROS production and bacterial killing but altered degranulation, cytokine release and antifungal activity compared to mature neutrophils isolated from peripheral blood. These differences are likely due to incomplete synthesis of granule proteins, as demonstrated by proteomic analysis. CONCLUSION: Ex vivo cultured neutrophils are genetically tractable via genome editing of precursors and provide a powerful model system for investigating the properties and behaviour of immature neutrophils.

Anti-inflammatory effects of steroidal alkaloids of Solanum lyratum Thunb.

Solanum lyratum Thunb., a traditional Chinese herbal medicine, has a promising background. However, the anti-inflammatory effects of its component steroid alkaloid have not been explored. In this study, animal and cell experiments were performed to investigate the anti-inflammatory effects and mechanism of action of Solanum lyratum Thunb steroid alkaloid (SLTSA), in order to provide evidence for its potential utilization. SLTSA effectively inhibited ear swelling and acute abdominal inflammation of mice. We observed concentration-dependent inhibition of pro-inflammatory cytokines by SLTSA, as confirmed by the ELISA and RT-qPCR results. Flow cytometry, immunofluorescence and RT-qPCR analyses revealed that SLTSA suppressed TLR4 expression. Western blot results indicated that SLTSA inhibited the activation of the TLR4/MyD88/NF-κB signaling pathway. Our study demonstrated that SLTSA possesses anti-inflammatory properties.

S1PR3 inhibition protects against LPS-induced ARDS by inhibiting NF-κB and improving mitochondrial oxidative phosphorylation.

BACKGROUND: Inflammation and endothelial barrier dysfunction are the major pathophysiological changes in acute respiratory distress syndrome (ARDS). Sphingosine-1-phosphate receptor 3 (S1PR3), a G protein-coupled receptor, has been found to mediate inflammation and endothelial cell (EC) integrity. However, the function of S1PR3 in ARDS has not been fully elucidated. METHODS: We used a murine lipopolysaccharide (LPS)-induced ARDS model and an LPS- stimulated ECs model to investigate the role of S1PR3 in anti-inflammatory effects and endothelial barrier protection during ARDS. RESULTS: We found that S1PR3 expression was increased in the lung tissues of mice with LPS-induced ARDS. TY-52156, a selective S1PR3 inhibitor, effectively attenuated LPS-induced inflammation by suppressing the expression of proinflammatory cytokines and restored the endothelial barrier by repairing adherens junctions and reducing vascular leakage. S1PR3 inhibition was achieved by an adeno-associated virus in vivo and a small interfering RNA in vitro. Both the in vivo and in vitro studies demonstrated that pharmacological or genetic inhibition of S1PR3 protected against ARDS by inhibiting the NF-κB pathway and improving mitochondrial oxidative phosphorylation. CONCLUSIONS: S1PR3 inhibition protects against LPS-induced ARDS via suppression of pulmonary inflammation and promotion of the endothelial barrier by inhibiting NF-κB and improving mitochondrial oxidative phosphorylation, indicating that S1PR3 is a potential therapeutic target for ARDS.

Celastrol reduces lung inflammation induced by multiwalled carbon nanotubes in mice via NF-κb-signaling pathway.

Multiwalled carbon nanotubes (MWCNTs) have numerous applications in the field of carbon nanomaterials. However, the associated toxicity concerns have increased significantly because of their widespread use. The inhalation of MWCNTs can lead to nanoparticle deposition in the lung tissue, causing inflammation and health risks. In this study, celastrol, a natural plant medicine with potent anti-inflammatory properties, effectively reduced the number of inflammatory cells, including white blood cells, neutrophils, and lymphocytes, and levels of inflammatory cytokines, such as IL-1ß, IL-6, and TNF-α, in mice lungs exposed to MWCNTs. Moreover, celastrol inhibited the activation of the NF-κB-signaling pathway. This study confirmed these findings by demonstrating comparable reductions in inflammation upon exposure to MWCNTs in mice with the deletion of NF-κB (P50-/-). These results indicate the utility of celastrol as a promising pharmacological agent for preventing MWCNT-induced lung tissue inflammation.

Physical training minimizes immunological dysfunction, oxidative stress and tissue destruction on experimental periodontitis in rats.

The objective of this study is to investigate the effects of a moderate intensity physical training protocol, on alveolar bone morphology of rats submitted to ligature-induced periodontitis. Twenty-eight male Wistar rats were divided into four groups, considering the presence/absence of periodontitis and presence/absence of training. The training protocol was performed on a treadmill, 30 min/day, 5 days a week, for 4 weeks. In the experimental periodontal breakdown, with/without training, ligatures were placed on the lower first molars on the 14th day of the experiment, and were followed until the end of the protocol. At the end of the experiment, animals were euthanized and samples of plasma and mandibles were collected for immunoenzymatic evaluation of interleukins (IL)-1ß, IL-6, TNF-α and IL-10, evaluation of serum concentrations of C-reactive protein, analysis of lipid peroxidation (LPO) and reduced glutathione, histological and microtomographic analyses were performed. Physical training resulted in a reduced levels of IL-1ß, IL-6, TNF-α C-reactive protein and LPO and an increase in the levels of IL-10 in rats with periodontitis (p<0.05); a reduction in the inflammatory infiltrate and decreased fiber degradation was identified in histological analysis. Additionally, it was shown a decrease in vertical bone loss and an increase in the bone volume/trabecular volume ratio was identified in periodontitis+physical training group (p<0.05). Based on the results, the practice of frequent physical exercise, at moderate intensity, can contribute to the reduction of damage related to the disproportionate inflammatory response in periodontitis.

Effects of Antrodia cinnamomea solid culture mycelium by-products on growth performance and immune response in weaning black piglets.

This study evaluated the effects of supplementation with Antrodia cinnamomea mycelium by-product (ACBP) on growth performance and immune response in weaning piglets. Total available content and antioxidant capacity of ACBP were determined. Ninety-six black pigs were randomly distributed to 24 pens. Study compared four groups which were supplemented with ACBP at 0%, 2.5%, 5%, or 10% for 6 weeks after weaning at 4 weeks. Results showed that ACBP on total phenolic, total flavonoid, and total triterpenoids contents were 13.68 mg GAE/g DW, 1.67 µg QE/g DW, and 15.6 mg/g, respectively. Weaning piglets fed 2.5% ACBP showed a significant decreased body weight gain compared with those supplemented with 5% ACBP, 10% ACBP, and control groups. Results showed that all ACBP groups increased the villi height of jejunum significantly. Incidence of diarrhea in 11 weeks with supplementation with 5% and 10% ACBP diets were lower than in control group. The 10% ACBP group showed significantly lower expression of immune response genes (IL-1ß, IL-6, IL-8, TNF-α, and IFN-γ) than the 2.5% and 5% ACBP groups. Based on results, dietary supplementation with 10% ACBP did not significantly affect body weight but could decrease piglet diarrhea condition and expression of IL-1ß and IL-6 genes.

Mitigating polyethylene-mediated periprosthetic tissue inflammation through MEDSAH-grafting.

Periprosthetic tissue inflammation is a challenging complication arising in joint replacement surgeries, which is often caused by wear debris from polyethylene (PE) components. In this study, we examined the potential biological effects of grafting a [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (MEDSAH) polymer onto the surface of PE through a solvent-evaporation technique. J774A.1 macrophage-like cells and primary cultured mouse osteoblasts were treated with PE powder with or without the MEDSAH coating. MEDSAH grafting on PE substantially reduced the expression of pro-inflammatory cytokines and other mediators in primary cultured mouse osteoblasts, but did not significantly impact macrophage-mediated inflammation. Our findings suggest that a MEDSAH coating on PE-based materials has potential utility in mitigating periprosthetic tissue inflammation and osteolysis and preventing aseptic loosening in total joint replacements. Further research, including large-scale clinical trials and biomechanical analyses, is needed to assess the long-term performance and clinical implications of MEDSAH-coated PE-based materials in total joint arthroplasty.

Association of Cytokines with Clinical Indicators in Patients with Drug-Induced Liver Injury.

Objective: To explore characteristics of clinical parameters and cytokines in patients with drug-induced liver injury (DILI) caused by different drugs and their correlation with clinical indicators. Method: The study was conducted on patients who were up to Review of Uncertainties in Confidence Assessment for Medical Tests (RUCAM) scoring criteria and clinically diagnosed with DILI. Based on Chinese herbal medicine, cardiovascular drugs, non-steroidal anti-inflammatory drugs (NSAIDs), anti-infective drugs, and other drugs, patients were divided into five groups. Cytokines were measured by Luminex technology. Baseline characteristics of clinical biochemical indicators and cytokines in DILI patients and their correlation were analyzed. Results: 73 patients were enrolled. Age among five groups was statistically different ( P = 0.032). Alanine aminotransferase (ALT) ( P = 0.033) and aspartate aminotransferase (AST) ( P = 0.007) in NSAIDs group were higher than those in chinese herbal medicine group. Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in patients with Chinese herbal medicine (IL-6: P < 0.001; TNF-α: P < 0.001) and cardiovascular medicine (IL-6: P = 0.020; TNF-α: P = 0.001) were lower than those in NSAIDs group. There was a positive correlation between ALT ( r = 0.697, P = 0.025), AST ( r = 0.721, P = 0.019), and IL-6 in NSAIDs group. Conclusion: Older age may be more prone to DILI. Patients with NSAIDs have more severe liver damage in early stages of DILI, TNF-α and IL-6 may partake the inflammatory process of DILI.

Changes of aqueous humor cytokine profiles of patients with high intraocular pressure after PPV for retinal detachment.

High intraocular pressure (IOP) is one of the early complications after pars plana vitrectomy (PPV), which may cause glaucoma and poor visual prognosis secondary to surgery. Proliferative vitreoretinopathy (PVR) is one of the complications of retinal detachment (RD) and is the main reason for the poor prognosis, which is related to different kinds of cytokines. It's essential for the basic mechanism to analyze the relative aqueous humor cytokine profiles with IOP after PPV for RD. In this study, we have collected the aqueous humor of 16 patients and qualified 27 cytokines using Luminex and compared biomarkers with the high IOP group and the normal group. As a result, the concentrations of VEGF, IL-6, FGF2, and G-CSF upregulated significantly (P < 0.05), while VEGFR2 downregulated significantly (P < 0.05) in the high IOP group. IL-6 was positively correlated with high IOP (r = 0.561, P = 0.041). Meanwhile, the concentrations of IL-6 (r = 0.543, P = 0.03), IL-5 (r = 0.576, P = 0.019), IL-15 (r = 0.614, P = 0.011), IL-4 (r = 0.517, P = 0.04), ICAM-1 (r = 0.611, P = 0.012), and G-CSF (r = 0.636, P = 0.008) were significantly associated with preoperative PVR classification, and the aqueous humor levels of IL-4 (r = 0.567, P = 0.022), HGF (r = 0.701, P = 0.005), and MCP-1 (r = 0.565, P = 0.035) are significant relative to laser points. Hence, cytokines might potentially be the therapeutic target of high IOP after PPV.

Factor of time in dendritic cell (DC) maturation: short-term activation of DCs significantly improves type 1 cytokine production and T cell responses.

Dendritic cells (DCs) have been intensively studied in correlation to tumor immunology and for the development DC-based cancer vaccines. Here, we present the significance of the temporal aspect of DC maturation for the most essential subsequent timepoint, namely at interaction with responding T cells or after CD40-Ligand restimulation. Mostly, DC maturation is still being achieved by activation processes which lasts 24 h to 48 h. We hypothesized this amount of time is excessive from a biological standpoint and could be the underlying cause for functional exhaustion. Indeed, shorter maturation periods resulted in extensive capacity of monocyte-derived DCs to produce inflammatory cytokines after re-stimulation with CD40-Ligand. This effect was most evident for the primary type 1 polarizing cytokine, IL-12p70. This capacity reached peak at 6 h and dropped sharply with longer exposure to initial maturation stimuli (up to 48 h). The 6 h maturation protocol reflected superiority in subsequent functionality tests. Namely, DCs displayed twice the allostimulatory capacity of 24 h- and 48 h-matured DCs. Similarly, type 1 T cell response measured by IFN-γ production was 3-fold higher when CD4+ T cells had been stimulated with shortly matured DC and over 8-fold greater in case of CD8+ T cells, compared to longer matured DCs. The extent of melanoma-specific CD8+ cytotoxic T cell induction was also greater in case of 6 h DC maturation. The major limitation of the study is that it lacks in vivo evidence, which we aim to examine in the future. Our findings show an unexpectedly significant impact of temporal exposure to activation signals for subsequent DC functionality, which we believe can be readily integrated into existing knowledge on in vitro/ex vivo DC manipulation for various uses. We also believe this has important implications for DC vaccine design for future clinical trials.

Organ-specific Toxocara canis larvae migration and host immune response in experimentally infected mice.

We investigated organ specific Toxocara canis larval migration in mice infected with T. canis larvae. We observed the worm burden and systemic immune responses. Three groups of BALB/c mice (n=5 each) were orally administered 1,000 T. canis 2nd stage larvae to induce larva migrans. Mice were sacrificed at 1, 3, and 5 weeks post-infection. Liver, lung, brain, and eye tissues were collected. Tissue from 2 mice per group was digested for larval count, while the remaining 3 mice underwent histological analysis. Blood hematology and serology were evaluated and compared to that in a control uninfected group (n=5) to assess the immune response. Cytokine levels in bronchoalveolar lavage (BAL) fluid were also analyzed. We found that, 1 week post-infection, the mean parasite load in the liver (72±7.1), brain (31±4.2), lungs (20±5.7), and eyes (2±0) peaked and stayed constant until the 3 weeks. By 5-week post-infection, the worm burden in the liver and lungs significantly decreased to 10±4.2 and 9±5.7, respectively, while they remained relatively stable in the brain and eyes (18±4.2 and 1±0, respectively). Interestingly, ocular larvae resided in all retinal layers, without notable inflammation in outer retina. Mice infected with T. canis exhibited elevated levels of neutrophils, monocytes, eosinophils, and immunoglobulin E. At 5 weeks post-infection, interleukin (IL)-5 and IL-13 levels were elevated in BAL fluid. Whereas IL-4, IL-10, IL-17, and interferon-γ levels in BAL fluid were similar to that in controls. Our findings demonstrate that a small portion of T. canis larvae migrate to the eyes and brain within the first week of infection. Minimal tissue inflammation was observed, probably due to increase of anti-inflammatory cytokines. This study contributes to our understanding of the histological and immunological responses to T. canis infection in mice, which may have implications to further understand human toxocariasis.

Canine peripheral non-conventional TCRαß+ CD4-CD8α- double-negative T cells show T helper 2-like and regulatory properties.

The dog is an important companion animal and also serves as model species for human diseases. Given the central role of T cells in immune responses, a basic understanding of canine conventional T cell receptor (TCR)αß+ T cells, comprising CD4+ single-positive (sp) T helper (Th) and CD8α+ sp cytotoxic T cell subsets, is available. However, characterization of canine non-conventional TCRαß+ CD4+CD8α+ double-positive (dp) and TCRαß+ CD4-CD8α- double-negative (dn) T cells is limited. In this study, we performed a comprehensive analysis of canine dp and dn T cells in comparison with their conventional counterparts. TCRαß+ T cells from peripheral blood of healthy dogs were sorted according to their CD4/CD8α phenotype into four populations (i.e. CD4+ sp, CD8α+ sp, dp, and dn) and selected surface markers, transcription factors and effector molecules were analyzed ex vivo and after in vitro stimulation by RT-qPCR. Novel characteristics of canine dp T cells were identified, expanding the previously characterized Th1-like phenotype to Th17-like and Th2-like properties. Overall, mRNA expression of various Th cell-associated cytokines (i.e. IFNG, IL17A, IL4, IL13) in dp T cells upon stimulation highlights their versatile immunological potential. Furthermore, we demonstrated that the CD4-CD8α- dn phenotype is stable during in vitro stimulation. Strikingly, dn T cells were found to express highest mRNA levels of type 2 effector cytokines (IL4, IL5, and IL13) upon stimulation. Their strong ability to produce IL-4 was confirmed at the protein level. Upon stimulation, the percentage of IL-4-producing cells was even higher in the non-conventional dn than in the conventional CD4+ sp population. Constitutive transcription of IL1RL1 (encoding IL-33Rα) further supports Th2-like properties within the dn T cell population. These data point to a role of dn T cells in type 2 immunity. In addition, the high potential of dn T cells to transcribe the gene encoding the co-inhibitory receptor CTLA-4 and to produce the inhibitory cytokine IL-10 indicates putative immunosuppressive capacity of this population. In summary, this study reveals important novel aspects of canine non-conventional T cells providing the basis for further studies on their effector and/or regulatory functions to elucidate their role in health and disease.

COVID-19 PBMCs are doubly harmful, through LDN-mediated lung epithelial damage and monocytic impaired responsiveness to live Pseudomonas aeruginosa exposure.

Introduction: Although many studies have underscored the importance of T cells, phenotypically and functionally, fewer have studied the functions of myeloid cells in COVID disease. In particular, the potential role of myeloid cells such as monocytes and low-density neutrophils (LDNs) in innate responses and particular in the defense against secondary bacterial infections has been much less documented. Methods: Here, we compared, in a longitudinal study, healthy subjects, idiopathic fibrosis patients, COVID patients who were either hospitalized/moderate (M-) or admitted to ICU (COV-ICU) and patients in ICU hospitalized for other reasons (non-COV-ICU). Results: We show that COVID patients have an increased proportion of low-density neutrophils (LDNs), which produce high levels of proteases (particularly, NE, MMP-8 and MMP-9) (unlike non-COV-ICU patients), which are partly responsible for causing type II alveolar cell damage in co-culture experiments. In addition, we showed that M- and ICU-COVID monocytes had reduced responsiveness towards further live Pseudomonas aeruginosa (PAO1 strain) infection, an important pathogen colonizing COVID patients in ICU, as assessed by an impaired secretion of myeloid cytokines (IL-1, TNF, IL-8,…). By contrast, lymphoid cytokines (in particular type 2/type 3) levels remained high, both basally and post PAO1 infection, as reflected by the unimpaired capacity of T cells to proliferate, when stimulated with anti-CD3/CD28 beads. Discussion: Overall, our results demonstrate that COVID circulatory T cells have a biased type 2/3 phenotype, unconducive to proper anti-viral responses and that myeloid cells have a dual deleterious phenotype, through their LDN-mediated damaging effect on alveolar cells and their impaired responsiveness (monocyte-mediated) towards bacterial pathogens such as P. aeruginosa.

Cynaroside ameliorates methotrexate-induced enteritis in rats through inhibiting NLRP3 inflammasome activation.

Introduction: Cynaroside exhibits various biological properties, including anti-inflammatory, antiviral, antitumor, and cardioprotective effects. However, its involvement in methotrexate (MTX)-induced intestinal inflammation remains inadequately understood. Thus, we investigated the impact of cynaroside on MTX-induced intestinal inflammation and its potential mechanisms. Methods: To assess the protective potential of cynaroside against intestinal inflammation, Sprague-Dawley rats were subjected to a regimen of 7 mg/kg MTX for 3 days, followed by treatment with cynaroside at varying doses (10, 20, or 40 mg/kg). Histopathological evaluations were conducted alongside measurements of inflammatory mediators to elucidate the involvement of the NLRP3 inflammasome in alleviating intestinal inflammation. Results: Administration of 7 mg/kg MTX resulted in decreased daily food intake, increased weight loss, and elevated disease activity index in rats. Conversely, treatment with cynaroside at 20 or 40 mg/kg ameliorated the reductions in body weight and daily food intake and suppressed the MTX-induced elevation in the disease activity index. Notably, cynaroside administration at 20 or 40 mg/kg attenuated inflammatory cell infiltration, augmented goblet cell numbers and lowered serum levels of tumor necrosis factor-α, interleukin (IL)-1ß, and IL-18, as well as the CD68-positive cell rate in the intestines of MTX-induced rats. Furthermore, cynaroside downregulated the expression levels of NLRP3, cleaved caspase 1, and cleaved IL-1ß in MTX-induced rats. Discussion: Collectively, our findings indicated that cymaroside alleviates intestinal inflammatory injury by inhibiting the activation of NLRP3 inflammasome in MTX-induced rats.

Bacillus Calmette-Guérin immunotherapy induces an efficient antitumor response to control murine melanoma depending on MyD88 signaling.

Bacillus Calmette-Guérin (BCG) is the first line treatment for bladder cancer and it is also proposed for melanoma immunotherapy. BCG modulates the tumor microenvironment (TME) inducing an antitumor effective response, but the immune mechanisms involved still poorly understood. The immune profile of B16-F10 murine melanoma cells was assessed by infecting these cells with BCG or stimulating them with agonists for different innate immune pathways such as TLRs, inflammasome, cGAS-STING and type I IFN. B16-F10 did not respond to any of those stimuli, except for type I IFN agonists, contrasting with bone marrow-derived macrophages (BMDMs) that showed high production of proinflammatory cytokines. Additionally, we confirmed that BCG is able to infect B16-F10, which in turn can activate macrophages and spleen cells from mice in co-culture experiments. Furthermore, we established a subcutaneous B16-F10 melanoma model for intratumoral BCG treatment and compared wild type mice to TLR2-/-, TLR3-/-, TLR4-/-, TLR7-/-, TLR3/7/9-/-, caspase 1-/-, caspase 11-/-, IL-1R-/-, cGAS-/-, STING-/-, IFNAR-/-, MyD88-/-deficient animals. These results in vivo demonstrate that MyD88 signaling is important for BCG immunotherapy to control melanoma in mice. Also, BCG fails to induce cytokine production in the co-culture experiments using B16-F10 and BMDMs or spleen cells derived from MyD88-/- compared to wild-type (WT) animals. Immunotherapy with BCG was not able to induce the recruitment of inflammatory cells in the TME from MyD88-/- mice, impairing tumor control and IFN-γ production by T cells. In conclusion, MyD88 impacts on both innate and adaptive responses to BCG leading to an efficient antitumor response against melanoma.

Plasticity and lineage commitment of individual TH1 cells are determined by stable T-bet expression quantities.

T helper 1 (TH1) cell identity is defined by the expression of the lineage-specifying transcription factor T-bet. Here, we examine the influence of T-bet expression heterogeneity on subset plasticity by leveraging cell sorting of distinct in vivo-differentiated TH1 cells based on their quantitative expression of T-bet and interferon-γ. Heterogeneous T-bet expression states were regulated by virus-induced type I interferons and were stably maintained even after secondary viral infection. Exposed to alternative differentiation signals, the sorted subpopulations exhibited graded levels of plasticity, particularly toward the TH2 lineage: T-bet quantities were inversely correlated with the ability to express the TH2 lineage-specifying transcription factor GATA-3 and TH2 cytokines. Reprogramed TH1 cells acquired graded mixed TH1 + TH2 phenotypes with a hybrid epigenetic landscape. Continuous presence of T-bet in differentiated TH1 cells was essential to ensure TH1 cell stability. Thus, innate cytokine signals regulate TH1 cell plasticity via an individual cell-intrinsic rheostat to enable T cell subset adaptation to subsequent challenges.