TNF-α Production Research Articles

Design, Synthesis, Anti-inflammatory Evaluation and In silico Molecular Docking of Novel Furan-based Derivatives as Potential TNF-α Production Inhibitors

Introduction: Inflammation is the first response and an alarming signal for the onset of chronic disease. Most of the anti-inflammatory drugs available in the market are reported to have undesirable gastrointestinal toxicities. Therefore, it is of urgent significance to develop anti-inflammatory drugs with low toxicity and good efficacy. Methods: We created a targeted scaffold based on a literature review by combining the different structural characteristics of furan and benzyl amides into a single pharmacophore. A series of eighteen furanbased derivatives (1-18) were designed, synthesized for in vitro and in vivo anti-inflammatory activity. The characterization of synthesized compounds was elucidated by techniques like 1H-NMR, 13C-NMR, FT-IR and MS. Results: The synthetic compounds were examined through molecular docking studies on TNF-α for probable binding mode and interactions with hydrophilic and hydrophobic pocket of TNF-α in comparison to standard drug (Indomethacin). Conclusion: When compared to the standard treatment, compounds 18, 15 and 9 displayed a remarkable inhibitory effect on the production of TNF-α and in vivo inflammatory activity with no damage to stomach and reduction of LPO. The compounds 18, 15 and 9 might be a good consideration for potential antiinflammatory agents.

Selenopolysaccharide Isolated from Lentinula edodes Mycelium Affects Human T-Cell Function

Lentinula edodes polysaccharides are natural immunomodulators. SeLe30, analyzed in this study, is a new mixture of selenium-enriched linear 1,4-α-glucans and 1,3-β- and 1,6-β-glucans isolated from L. edodes mycelium. In the present study, we evaluated its immunomodulatory properties in human T cells. Peripheral blood mononuclear cells (PBMCs) and T cells were isolated from healthy donors’ buffy coats. The effects of SeLe30 on CD25, CD366, and CD279 expression, the subsets of CD8+ T cells, and IFN-γ, IL-6, and TNF-α production were analyzed. SeLe30 downregulated CD25, CD279, and CD366 expression on T cells stimulated by the anti-CD3 antibody (Ab) and upregulated in unstimulated and anti-CD3/CD28-Abs-stimulated T cells. It increased the percentage of central memory CD8+ T cells in unstimulated PBMCs and naïve and central memory T cells in anti-CD3-Ab-stimulated PBMCs. SeLe30 decreased the number of central memory and naïve CD8+ T cells in anti-CD3/CD28-stimulated T cells, whereas, in PBMCs, it reduced the percentage of effector memory CD8+ T cells. Moreover, SeLe30 upregulated cytokine production. SeLe30 exhibits context-dependent effects on T cells. It acts on unstimulated T cells, affecting their activation while increasing the expression of immune checkpoints, which sensitizes them to inhibitory signals that can silence this activation. In the case of a lack of costimulation, SeLe30 exhibits an inhibitory effect, reducing T-cell activation. In cells stimulated by dual signals, its effect is further enhanced, again increasing the “safety brake” of CD366 and CD279. However, the final SeLe30 effect is mediated by its indirect impacts by altering interactions with other immune cells.

Interleukin-6 levels in atrium and onset of spontaneous atrial fibrillation: is there a causal relationship

Abstract Background Cumulative evidence suggests that atrial interleukin-6 (IL-6) may play a significant role in the development of spontaneous postoperative atrial fibrillation (sPOAF) in coronary artery bypass grafting (CABG) patients. Our previous studies found that the IL-6 produced by the atrium is positively related to the development of sPOAF. However, the causal relationship between IL-6 and sPOAF is not established. Purpose This study aims to explore the potential causality between atrial IL-6 and spontaneous AF(sAF), alongside assessing the impact of IL-6 on the electrophysiological properties of atrial myocytes (AM). Methods To determine the causal link between IL-6 and sAF, mice were randomly divided into eight groups: seven receiving injections of varying doses of recombinant IL-6 protein groups (15ng/kg to180ng/kg) into the left atrial wall, and one control group (vehicle). Mice were monitored for sAF for 7 days using implanted telemetry device. At 48 hrs post-injection, the left atrial appendages of another set of mice were collected for analyzing levels of IL-6, Myeloperoxidase (MPO), Transforming Growth Factor-β1 (TGF-β1), and Tumor Necrosis Factor-α (TNF-α). To assess the effect of IL-6 on the electrophysiological characteristics of AM, AMs were isolated from the hearts of 12-week-old mice and divided into experimental and control groups for electrophysiological testing. The experimental group was perfused with extracellular solutions containing 100 ng/mL of IL-6 while the control group was perfused with standard extracellular solutions. The measurements focused on action potential (AP), transient outward potassium current (Ito), and inward rectifier potassium current (Ik1) after a 30-minute perfusion. Results As depicted in Fig. 1A, a dose-response relationship between IL-6 levels and sAF occurrence was observed, with incidence increasing from 0% at 15 ng/kg to 75% at 180 ng/kg (χ²= 16.691, P = 0.005). The injection of IL-6 also induced the production of MPO, TGF-β1, and TNF-α with a dose-dependent effects (Fig. 2). In comparison to control, the AP duration at 90% (APD90) and APD50 of repolarization of atrial myocytes were significantly shortened in IL-6 group (Fig. 1B). For voltages from -10mV to +70mV, atrial myocytes in the IL-6 group showed significantly lower Ito current density than those in the control (Fig. 1C). No significant alterations were observed in the impact of IL-6 on Ik1 current density in atrial myocytes (Fig.1D). Conclusion To the best of our knowledge, this is the first study that experimentally tested the causal relation between IL-6 and sAF. The injection of IL-6 could cause the onset of sAF and produce MPO, TGF-β1, and TNF-α with a significant dose-dependent effect. The effect of IL-6 on inducing sAF might be through its effect on reducing APD and Ito current density in mouse atrial myocytes. This suggests that inhibition of atrial IL-6 might be a potential novel sPOAF prevention strategy.

Differential Adjuvant Activity by Flagellins from Escherichia coli, Salmonella enterica Serotype Typhimurium, and Pseudomonas aeruginosa

(1) Background: The adjuvant properties of flagellin from various bacterial species have been extensively studied; however, a systematic comparison of the immunoadjuvant effects of flagellins from different bacterial species is lacking. This study aims to analyze the amino acid sequences and structural features of flagellins from Escherichia coli (FliCE.C), Salmonella enterica serotype Typhimurium (FliCS.T), and Pseudomonas aeruginosa (FliCP.A), and to evaluate their adjuvant activities in terms of Toll-like receptor 5 (TLR5) activation, antibody production, and cytokine responses in a murine model. (2) Methods: Bioinformatics analysis was conducted to compare the amino acid sequences and structural domains (D0, D1, D2, and D3) of flagellins from the three bacterial species. PyMol atomic models were used to confirm structural differences. Toll-like receptor 5 (TLR5) activation assays were performed to measure IL-8 and TNF-α production in vitro. The IgG antibody titers against the model antigen FaeG and cytokine responses, including IL-4 and TNF-α secretion were evaluated in a murine model. (3) Results: Bioinformatics analysis revealed that the D0 and D1 domains are highly conserved, whereas the D2 and D3 domains exhibit significant variability across the three species. Structural analysis via PyMol confirmed these differences, particularly in the D2 and D3 domains. TLR5 activation assays showed that FliCS.T and FliCP.A induced higher levels of IL-8 and TNF-α production compared to FliCE.C, indicating species-specific variations in TLR5 activation. In the murine model, FliCS.T as an adjuvant produced higher antibody titers against FaeG and increased IL-4 secretion in splenocytes compared to FliCE.C and FliCP.A. FliCP.A induced higher TNF-α expression than FliCS.T and FliCE.C, suggesting FliCS.T and FliCP.A are more effective at inducing T-cell responses. (4) Conclusions: This study highlights the potential of FliCS.T and FliCP.A as potent vaccine adjuvants. The results provide insights into the structure–function relationships of these flagellins and support their application in enhancing immune responses against diverse pathogens.

Brazilian red propolis synergistically with imipenem modulates immunological parameters and the bactericidal activity of human monocytes against methicillin-resistant Staphylococcus aureus (MRSA).

Propolis is a bee product found all over the globe and has a well-known antibacterial activity. Previous findings of our group revealed that the combination of Brazilian red propolis (BRP) with a lower concentration of imipenem (IPM) exerted a bactericidal action against methicillin-resistant Staphylococcus aureus (MRSA) in vitro. Here, we aimed at investigating the effects of BRP in combination or not with IPM on human monocytes to assess a possible immunomodulatory action. Monocyte metabolic activity was analysed by MTT assay, cytokine production (TNF-α, IL-1β, IL-6, IL-8, and IL-10) by ELISA, and the expression of cell markers (TLR-2, TLR-4, HLA-DR, and CD80) by flow cytometry. The bactericidal activity of monocytes over MRSA was determined by colony-forming units' count. BRP alone or in combination with IPM exerted no cytotoxic effects on monocytes. BRP downregulated TLR-2 expression and inhibited TNF-α, IL-1β, and IL-6 production, while BRP + IPM stimulated these parameters. BPR alone or in combination increased the bactericidal activity similarly to LPS-activated monocytes. Data indicated the potential of BRP as an anti-inflammatory agent increasing the bactericidal activity of monocytes against MRSA. The combination of BRP + IPM exhibited a stimulatory profile that may be potentially useful in treating patients with MRSA infection.

Hyperemesis gravidarum is a cytokine storm?

Aim. To study the systemic response of proinflammatory cytokines in women suffering from VP. The main group consisted of 47 women with VP, which was divided into 3 subgroups according to severity: the 1st – included 16 patients with mild VP, the 2nd – 15 with moderate severity, and the 3rd – 16 women with severe VP. The control group consisted of 10 pregnant women with a physiological course without signs of toxicosis. Interleukins (IL-1β, IL-6, TNFα) were determined in the blood serum of pregnant women by solid-phase ELISA using Cytokine test kits (St. Petersburg, Russia). With increasing severity of toxicosis, there is a significant increase in IL-1β in severe VP – 1.9 times higher than the same value of the norm (p 0.05), 2 times higher than in mild vomiting (p 0.05), and in 1.7 times – with moderate VP (p 0.05). The study of the level of IL-6 revealed a directly proportional increase in the indicator with increasing severity of toxicosis in relation to the control indicator: with mild vomiting – 1.9 times (p 0.05), with moderate vomiting – 2.6 times (p 0.001), and in severe VP – 2.9 times (p 0.0001). A significant decrease in the level of TNFα was observed with increasing severity of toxicosis in relation to the control indicator: with mild vomiting – by 1.5 times (p 0.05), with moderate vomiting – by 1.9 times (p 0.05), and with severe VP – 2.1 times (p 0.05). Excessive secretion of proinflammatory cytokines IL-1β and IL-6 (2 and 3 times, respectively), exceeding the norm, with a sharp suppression of TNFα production (2 times) can be considered as predictors of a “cytokine storm”, clinically occurring with multiple organ failure up to death if the pregnancy is not terminated in a timely manner.

Gestational Diabetes Mellitus-Induced Inflammation in the Placenta via IL-1β and Toll-like Receptor Pathways

Gestational diabetes mellitus is characterised by an insufficient insulin response to hyperglycaemia and the development of insulin resistance. This state has adverse effects on the health outcomes of the mother and child. Existing hyperglycaemia triggers a state of inflammation that involves several tissues, including the placenta. In this study, we analysed the putative pathomechanism of GDM, with special emphasis on the role of chronic, sterile, pro-inflammatory pathways. The expression and regulation of the elements of IL-1β and Toll-like receptor (TLR) pathways in GDM maternal blood plasma, healthy placental explants and a choriocarcinoma cell line (BeWo cell line) stimulated with pro-inflammatory factors was evaluated. Our results indicate elevated expression of the IL-1β and TLR pathways in GDM patients. After stimulation with IL-1β or LPS, the placental explants and BeWo cell line showed increased production of pro-inflammatory IL-6, TNFa and IL-1β together with increased expression of the elements of the signalling pathways. The application of selected inhibitors of NF-ĸB, MAPK and recombinant interleukin 1 receptor antagonist (IL1RA) proved the key involvement of the IL-1β pathway and TLRs in the pathogenesis of GDM. Our results show the possible existence of loops of autocrine stimulation and a possible inflammatory pathomechanism in placentas affected by GDM.

Impact of Senescent Cell-Derived Extracellular Vesicles on Innate Immune Cell Function.

Extracellular vesicles (EVs) are components of the senescence-associated secretory phenotype (SASP) that influence cellular functions via their cargo. Here, the interaction between EVs derived from senescent (SEVs) and non-senescent (N-SEVs) fibroblasts and the immune system is investigated. Via endocytosis, SEVs are phagocytosed by monocytes, neutrophils, and B cells. Studies with the monocytic THP-1 cell line find that pretreatment with SEVs results in a 32% (p < 0.0001) and 66% (p < 0.0001) increase in lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-α) production when compared to vehicle control or N-SEVs respectively. Interestingly, relative to vehicle control, THP-1 cells exposed to N-SEVs exhibit a 20% decrease in TNF-α secretion (p < 0.05). RNA sequencing reveals significant differences in gene expression in THP-1 cells treated with SEVs or N-SEVs, with vesicle-mediated transport and cell cycle regulation pathways featuring predominantly with N-SEV treatment, while pathways relating to SLITS/ROBO signaling, cell metabolism, and cell cycle regulation are enriched in THP-1 cells treated with SEVs. Proteomic analysis also reveals significant differences between SEV and N-SEV cargo. These results demonstrate that phagocytes and B cells uptake SEVs and drive monocytes toward a more proinflammatory phenotype upon LPS stimulation. SEVs may therefore contribute to the more proinflammatory immune response seen with aging.

Long-term enzyme replacement therapy in Fabry patients protects against oxidative and inflammatory process.

Fabry disease (FD) is an X-linked recessive lysosomal storage disorder, characterized by a deficiency of α-galactosidase, which causes the progressive accumulation of glycosphingolipids, especially globotriaosylsphingosine (Gb3), in lysosomes across multiple organs. Substrate deposition, associated with tissue damage in FD, also contributes to the emergence of a pro-inflammatory state presented by some patients. We investigated pro- and anti-inflammatory cytokines, and the expression of inflammation-associated genes in treated FD patients, as well as oxidative parameters. We found a decrease in the production of cytokines IL-1β, IL-6, IL-10, and TNF-α in male FD patients and a normalization of redox status in male and female FD patients, once the levels of protein, lipid oxidation, and nitrite and nitrate content were like healthy individuals. Our results suggest that long-term ERT in men with FD contributes to the reduction of a pro-inflammatory scenario and a decrease of oxidative damage in patients, reflecting greater control throughout the disease and in the multisystemic changes characteristic of this disorder. These findings lead us to believe that long-term ERT can improve the redox status and protect these individuals against oxidative and nitrative stress, as well as the inflammatory process.

Role of Vitamin C on methotrexate-induced nephrotoxicity in psoriasis context: A preclinical assessment

Methotrexate (MTX) is the most prescribed drug for systemic treatment of psoriasis. However, its clinical use is limited by its nephrotoxicity, which antioxidants can attenuate. This study evaluates the impact of vitamin C (vitC), a well-known antioxidant, on nephrotoxicity induced by high MTX doses in the context of psoriasis. To achieve this purpose, the kidney injury triggered by acute MTX exposure was established in an imiquimod-induced psoriasis-like mouse model. Mice were randomly divided into six groups: group 1 (control); group 2 (Imiquimod, IMQ), group 3 (IMQ+vitC 175 mg/kg/day); group 4 (MTX 20 mg/kg i.p); group 5 (IMQ+MTX 20 mg/kg) and group 6 (IMQ+MTX 20 mg/kg + vitC 175 mg/kg/day). The effects of these treatments were determined by considering the evolution of IMQ-induced skin lesions and serum creatinine levels. Moreover, histopathological analysis, lipid peroxidation, oxidative stress, and TNF-α production were determined in kidney tissue. Results showed that vitC attenuates renal damage in the context of IMQ-induced psoriasis. However, the opposite occurs when administered with IMQ+MTX, worsening skin psoriasis lesions and exacerbating acute renal tubular necrosis and oxidative DNA damage. These results establish new clues about the MTX-induced nephrotoxicity in the psoriasis context and the putative protective effects of vitC. It suggests that vitC supplementation could help attenuate the renal damage promoted by the psoriatic pathological environment. However, it should be avoided in psoriasis patients with renal dysfunction treated with MTX.

Regulatory T cells modulate monocyte functions in immunocompetent antiretroviral therapy naive HIV-1 infected people

We previously demonstrated that the overall number of regulatory T (Treg) cells decrease proportionately with helper CD4+ T cells and their frequencies increase in antiretroviral therapy (ART)-naive human immunodeficiency virus type-1 (HIV-1) infected individuals. The question now is whether the discrepancies in Treg cell numbers and frequencies are synonymous to an impairment of their functions. To address this, we purified Treg cells and assessed their ability to modulate autologous monocytes functions. We observed that Treg cells were able to down modulate autologous monocytes activation as well as interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) production during stimulation with polyinosinic-polycytidylic acid stabilized with poly-L-lysine and carboxymethylcellulose (poly-ICLC). This activity of Treg cells has been shown to be influenced by immunocompetence including but not limited to helper CD4+ T cell counts, in individuals with HIV-1 infection. Compared to immunosuppressed participants (CD4 < 500 cells/µL), immunocompetent participants (CD4 ≥ 500 cells/µL) showed significantly higher levels of transforming growth factor beta (TGF-β) and IL-10 (p < 0.001 and p < 0.05, respectively), key cytokines used by Treg cells to exert their immunosuppressive functions. Our findings suggest the contribution of both TGF-β and IL-10 in the suppressive activity of Treg cells.

Efficacy of Clindamycin in Preventing Abortion and Vertical Transmission of Toxoplasma gondii (PRU Strain) Infection in Pregnant BALB/c Mice

Background: Toxoplasma gondii transmission can occur during pregnancy if the mother contracts the infection for the first time. Treatment strategies include the use of antimicrobial medications and providing supportive care. Spiramycin is commonly used to treat toxoplasmosis in pregnant women and to hinder the disease's transmission. However, its ability to treat the fetus is questionable due to its limited capacity to cross the placental barrier. Additionally, economic constraints and sanctions may impede access to this medication. Objectives: Consequently, in search of an effective treatment, for the first time in Iran, the effectiveness of clindamycin in preventing abortion and vertical transmission of the PRU strain of T. gondii infection in pregnant mice was evaluated. Methods: On the twelfth day of gestation, pregnant mice were exposed to T. gondii and subsequently received treatment with either clindamycin or spiramycin. This resulted in the establishment of four distinct groups: A normal control group, an infected group without treatment, an infected group treated with clindamycin, and another infected group treated with spiramycin. Following these interventions, a series of parasitological evaluations (including microscopic examination and real-time PCR), histopathological evaluations, and immunological assessments were conducted. Results: The findings showed a significant reduction in the number of cysts in the eye and brain (ranging from 77.32% to 90.72%) among the groups treated with clindamycin and spiramycin compared to the control group. Furthermore, treatment with clindamycin, like treatment with spiramycin, was able to suppress inflammatory changes, prevent cell death, and reduce vascular cuffs in the brain, as well as decrease bleeding, placental thrombosis, and the accumulation of inflammatory cells in the placenta. Clindamycin was also effective in diminishing retinal folds, tiny retinal bleeds, and cell vacuolation in eye tissues. Immunologically, treatment in both the spiramycin and clindamycin groups resulted in a decrease in the level of the cytokine TNF-α, indicating an increase in the cellular immune response. In addition, increased levels of IL-10 in the treated infected groups could contribute to the reduction of TNF-α production. Conclusions: Typically, spiramycin is the first choice for treating congenital toxoplasmosis, but clindamycin can be a useful substitute or additional treatment when resistance to primary medications occurs, when there is intolerance, or when access to the main drugs is restricted.

Selenomethionine Mitigates Effects of Nocardia cyriacigeorgica-Induced Inflammation, Oxidative Stress, and Apoptosis in Bovine Mammary Epithelial Cells

Nocardia cyriacigeorgica causes bovine mastitis, reduces milk quantity and quality, and is often resistant to antimicrobials. Selenomethionine (SeMet) is a form of selenium, which reduces reactive oxygen species (ROS)-mediated apoptosis and intramammary infections. However, the protective effects of SeMet on N. cyriacigeorgica-infected bovine mammary epithelial cells (bMECs) are unclear. The objective of this study was to evaluate whether SeMet mitigated N. cyriacigeorgica-induced inflammatory injury, oxidative damage and apoptosis in bMECs. Cells were cultured with or without being pretreated with 40 µM of SeMet for 12 h, then challenged with N. cyriacigeorgica (multiplicity of infection = 5:1) for 6 h. Although N. cyriacigeorgica was resistant to lincomycin, erythromycin, enrofloxacin, penicillin, amoxicillin, cephalonium, cephalexin, and ceftriaxone, 40 μM SeMet increased cell viability and inhibited lactate dehydrogenase release in infected bMECs. Furthermore, N. cyriacigeorgica significantly induced mRNA production and protein expression of TNF-α, IL-1β, IL-6, and IL-8 at 6 h. Cell membrane rupture, cristae degeneration and mitochondria swelling were evident with transmission electron microscopy. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) activities were down-regulated after 3, 6, or 12 h, whereas malondialdehyde (MDA) and ROS contents were significantly upregulated, with cell damage and apoptosis rapidly evident (the latter increased significantly in a time-dependent manner). In contrast, bMECs pretreated with 40 μM SeMet before infection, SOD, and GSH-px activities were upregulated (p &lt; 0.05); MDA and ROS concentrations were downregulated (p &lt; 0.05), and apoptosis was reduced (p &lt; 0.05). In conclusion, 40 μM SeMet alleviated inflammation, oxidative stress and apoptosis induced by N. cyriacigeorgica in bMECs cultured in vitro.

Evaluation of the Anti-Inflammatory/Immunomodulatory Effect of Teucrium montanum L. Extract in Collagen-Induced Arthritis in Rats.

The anti-inflammatory/immunomodulatory effects of Teucrium montanum L. (TM), a plant distributed in the Mediterranean region, have been insufficiently examined. The effects of the TM ethanol extract were tested in a rat collagen-induced arthritis (CIA) model of rheumatoid arthritis. LC-MS was used for the phytochemical analysis of the TM extract. Dark Agouti rats were immunized with bovine type II collagen (CII) in incomplete Freund's adjuvant for CIA, and treated with 100 or 200 mg/kg of TM extract daily via oral administration. Clinical and histopathological evaluations and a flow cytometric analysis of the phenotypic and functional characteristics of splenocytes and draining lymph node cells were performed. The cytokines in the paw tissue culture supernatants and anti-CII antibodies in serum were determined by ELISA. The TM extract, with the dominant components verbascoside and luteolin 7-O-rutinoside, reduced the arthritic score and ankle joint inflammation in CIA rats, promoted the antioxidant profile in serum, and lowered pro-inflammatory TNF-α, IL-6 and IL-1β production. It suppressed the activation status of CD11b+ cells by lowering CD86, MHCII and TLR-4 expression, and promoted the Th17/T regulatory cell (Tregs) balance towards Tregs. A lower frequency of B cells was accompanied by a lower level of anti-CII antibodies in treated rats. These findings imply the favorable effect of TM extract on the clinical presentation of CIA, suggesting its anti-inflammatory/immunomodulatory action and potential therapeutic effect.

CD4+ T cell help during early acute hepacivirus infection is critical for viral clearance and the generation of a liver-homing CD103+CD49a+ effector CD8+ T cell subset.

In hepatitis C virus (HCV) infection, CD4+ and CD8+ T cells are crucial for viral control. However, a detailed understanding of the kinetic of CD4+ T cell help and its role in the generation of different CD8+ T cell subsets during acute infection is lacking. The absence of a small HCV animal model has impeded mechanistic studies of hepatic antiviral T cell immunity and HCV vaccine development. In this study, we used a recently developed HCV-related rodent hepacivirus infection mouse model to investigate the impact of CD4+ T cell help on the hepatic CD8+ T cell response and viral clearance during hepacivirus infection in vivo. Our results revealed a specific kinetic of CD4+ T cell dependency during acute infection. Early CD4+ T cell help was essential for CD8+ T cell priming and viral clearance, while CD4+ T cells became dispensable during later stages of acute infection. Effector CD8+ T cells directly mediated timely hepacivirus clearance. An analysis of hepatic CD8+ T cells specific for two different viral epitopes revealed the induction of subsets of liver-homing CD103+CD49a+ and CD103-CD49a+ effector CD8+ T cells with elevated IFN-γ and TNF-α production. CD103+CD49a+ T cells further persisted as tissue-resident memory subsets. A lack of CD4+ T cell help and CD40L-CD40 interactions resulted in reduced effector functions and phenotypical changes in effector CD8+ T cells and a specific loss of the CD103+CD49a+ subset. In summary, our study shows that early CD4+ T cell help through CD40L signaling is essential for priming functional effector CD8+ T cell subsets, including unique liver-homing subsets, and hepacivirus clearance.

Myeloid-Specific JAK2 Contributes to Inflammation and Salt Sensitivity of Blood Pressure.

Salt sensitivity of blood pressure (SSBP), characterized by acute changes in blood pressure with changes in dietary sodium intake, is an independent risk factor for cardiovascular disease and mortality in people with and without hypertension. We previously found that elevated sodium concentration activates antigen-presenting cells (APCs), resulting in high blood pressure, but the mechanisms are unknown. Here, we hypothesized that APC-specific JAK2 (Janus kinase 2) through STAT3 (signal transducer and activator of transcription 3) and SMAD3 (small mothers against decapentaplegic homolog 3) contributes to SSBP. We performed bulk or single-cell transcriptomic analyses following in vitro monocytes exposed to high salt and in vivo high sodium treatment in humans using a rigorous salt-loading/depletion protocol to phenotype SSBP. We also used a myeloid cell-specific CD11c+ JAK2 knockout mouse model and measured blood pressure with radiotelemetry after N-omega-nitro-L-arginine-methyl ester and a high salt diet treatment. We used flow cytometry for immunophenotyping and measuring cytokine levels. Fluorescence in situ hybridization and immunohistochemistry were performed to spatially visualize the kidney's immune cells and cytokine levels. Echocardiography was performed to assess cardiac function. We found that high salt treatment upregulates gene expression of the JAK/STAT/SMAD pathway while downregulating inhibitors of this pathway, such as suppression of cytokine signaling and cytokine-inducible SH2, in human monocytes. Expression of the JAK2 pathway genes mirrored changes in blood pressure after salt loading and depletion in salt-sensitive but not salt-resistant humans. Ablation of JAK2, specifically in CD11c+ APCs, attenuated salt-induced hypertension in mice with SSBP. Mechanistically, we found that SMAD3 acted downstream of JAK2 and STAT3, leading to increased production of highly reactive isolevuglandins and proinflammatory cytokine IL (interleukin)-6 in renal APCs, which activate T cells and increase production of IL-17A, IL-6, and TNF-α (tumor necrosis factor-alpha). Our findings reveal the APC JAK2 signaling pathway as a potential target for the diagnosis and treatment of SSBP in humans.

Tropisetron attenuates high glucose-induced oxidative stress and inflammation in ARPE-19 cells in vitro via regulating SIRT1/ROCK1 signaling.

Diabetic retinopathy (DR) is the leading cause of acquired blindness in diabetic patients. Tropisetron (TRO) exerts potent therapeutic effects against diabetic tissues. The present study aimed to investigate the effects of TRO on retinal injury under diabetic condition. Human retinal pigment epithelial cell line ARPE-19 was treated with high glucose (HG) for 48 h to mimic hyperglycemia-induced retinal damage and subsequently treated with multiple concentrations of TRO for therapeutic intervention. Cell viability and lactate dehydrogenase (LDH) release were detected to assess cell damage. The production of inflammatory cytokines and oxidative stress-related factors was evaluated by corresponding commercial kits. Cell apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The expression of inflammation-, apoptosis-, and SIRT1/ROCK1-related proteins was examined using western blot analysis. Additionally, ARPE-19 cells were transfected with over-express ROCK1 (Ov-ROCK1) or pretreatment with SIRT1 inhibitor EX527 to perform the rescue experiments. TRO alleviated cell damage in HG-induced ARPE-19 cells through elevating cell viability and reducing LDH release. HG-caused excessive production of TNF-α, IL-1β and IL-6, ROS, malondialdehyde and decreased superoxide dismutase activity were partly inhibited by TRO treatment. HG-induced cell apoptosis, accompanied with the upregulation of proapoptotic proteins and the downregulation of antiapoptotic proteins, was hindered by TRO treatment. HG led to the loss of SIRT1 and an elevation of ROCK1 in ARPE-19 cells, which was reversed following TRO treatment. Furthermore, pretreatment with EX527 or transfected with Ov-ROCK1 partially abolished the protective role of TRO against inflammation, oxidative stress and cell apoptosis in HG-challenged ARPE-19 cells. TRO exerted a protective role against HG-caused ARPE-19 cells inflammation, oxidative stress and cell apoptosis by regulating SIRT1/ROCK1 axis, suggesting that TRO might be therapeutic agent for alleviating retinal pigment epithelial cell damage in DR.

Novel Peptides LFLLP and DFFL from Jack Bean Protein Hydrolysates Suppress the Inflammatory Response in Lipopolysaccharide-Stimulated RAW 264.7 Cells.

The production of inflammatory cytokines such as tumor necrosis factor (TNF)-α by activated macrophage cells plays an important role in the development of intestinal inflammation. The present study investigated the anti-inflammatory effect of the protein hydrolysates prepared from the jack bean (JBPHs), Canavalia ensiformis (L.) DC, using the enzyme Alcalase, in a murine macrophage model, RAW 264.7 cells, which were stimulated by lipopolysaccharides. JBPHs reduced the TNF-α expression at the protein and mRNA levels through the downregulation of cellular signaling pathways involved in nuclear factor kappa B (NF-κB), extracellular signal-regulated kinase (ERK), and p38. A combination of mass spectrometry and in silico approaches identified 10 potential anti-inflammatory peptides in the JBPHs, including LFLLP and DFFL. Interestingly, while LFLLP targeted the NF-κB pathway, DFFL targeted p38 and ERK to suppress the TNF-α production in the RAW 264.7 cells. In addition, LFLLP and DFFL were localized in the cytosol of the cells. These results demonstrated that LFLLP and DFFL were incorporated by RAW 264.7 cells and, at least in part, contributed to the reduction in TNF-α by JBPHs. These peptides isolated from JBPHs may well be utilized as new alternatives to alleviate intestinal inflammation.

Exploring the complex immunomodulatory effects and gut defense via oral administration of Astragali radix water extract to normal mice

BackgroundAstragali radix (AR) is one of the most widely used traditional Chinese herbal medicines. It exhibits diverse biological activities, including immunomodulatory and anti-inflammatory properties; however, some of its activities have only been demonstrated in vitro.ObjectiveTo examine the effects of orally administered AR extract on immune cells and the intestine under physiological conditions, which bridges the gap between previously observed in vitro outcomes and in vivo results.MethodsAR extract was prepared by hot water extraction. Three separate animal experiments were conducted to isolate macrophages, splenocytes, and the small intestine epithelium. For the macrophage preparation experiment, an intraperitoneal injection of sterile thioglycolate was administered. The mice received oral AR extract at doses of 0.1, 0.5, or 2.5 g/kg for ten days. At the end of each experiment, cells or tissues were isolated. A portion of macrophages and splenocytes were analyzed for the phenotypic changes. The remaining cells were cultured and stimulated with lipopolysaccharide (LPS) or mitogen ex vivo to assess activation status, proliferation, and cytokine production. Samples of the intestine were subjected to real-time RT-PCR.ResultsPeritoneal macrophages from AR-treated mice exhibited increased expression of scavenger receptors, including SRA and CD36. Stimulation of these macrophages ex vivo with LPS selectively modulated the inflammatory response, including reduced expression of the costimulatory molecules CD40 and CD86, which are important for T cell responses, without affecting TNF-α and IL-6 production. Splenocytes from AR-treated mice exhibited a dose-dependent increase in CD4 and CD8 T cells; however, stimulation with mitogen decreased T cell proliferation and reduced IFN-γ production, which is essential for macrophage activation. An analysis of the small intestinal epithelium revealed an attenuated antimicrobial response, including reduced IgA content in the lumen and decreased expression of mucin-2 and polymeric Ig receptor genes.ConclusionThe response of immune cells following oral treatment with AR extract did not replicate the previously documented in vitro findings. Immune cells and intestinal epithelium from mice administered oral AR extract exhibited a selective anti-inflammatory phenotype. The overall findings indicate that the systemic effects after oral administration of AR extract include reduced sensitivity to inflammatory insults.

Phthalocyanine derivative attenuates TNF-α production in macrophage culture and prevents alveolar bone loss in experimental periodontitis.

This study investigated the activity and mechanism of action of the iron tetracarboxyphthalocyanine (FeTcPc) on tumor necrosis factor alpha (TNF-α) production and its impact on experimental periodontitis. RAW 264.7 macrophages were treated with FeTcPc, activated with lipopolysaccharide (LPS) at 10 ng/mL, and the TNF-α levels were measured, as well as the nuclear factor kappa B (NF-κB) activation. Subsequently, a mouth gel containing 1% FeTcPc was topically administered to the gingival tissue of mice with periodontitis-induced ligatures. Bone loss and the gene expression of Tnfα, p65 (NF-κB), and receptor-activating nuclear factor kappa B ligand (Rankl) were quantified in gingival tissue. Finally, the systemic toxicity of FeTcPc was estimated in Galleria mellonella larvae. In an activated RAW 264.7 macrophage culture, 100 μM FeTcPc reduced TNF-α release and NF-κB activation. Regarding experimental periodontitis, topical application of mouth gel containing 1% FeTcPc blocked alveolar bone loss. Additionally, 1% FeTcPc reduced the expression of Tnfα, p65 (NF-κB), and Rankl in gingival tissue. Finally, administration FeTcPc at doses ranging from 1 to 1000 mg/kg did not cause acute systemic toxicity in G. mellonella. Overall, we demonstrated the potential of mouth gel containing FeTcPc as a therapeutic strategy for managing osteolytic inflammatory disorders, such as periodontitis.