Proinflammatory Cytokines TNF Research Articles

Anti-inflammatory phenolic glycosides from endemic Marrubium rotundifolium Boiss.

Marrubium rotundifolium Boiss. is an endemic plant distributed in Aegean Region of Türkiye. It’s traditionally used in Anatolian medicine for treatment of cold and flu, dyspepsia, costiveness and intestinal spasms. However, phytochemical studies on the plant are limited. In the present study, five phenolic glycosides 4’,5,6,7-tetramethoxy scutellarein (1), apigenin-7-O-(3”-O-E-p-coumaroyl)-β-d-glucopyranoside (2), tiliroside (3), 4-(β-d-glucopyranosyloxy) benzoic acid (4), and astragalin (5) were isolated from the aerial parts of M. rotundifolium and their structures were elucidated on the basis of spectroscopic methods (1D and 2D NMR, and MALDI-TOF/MS). Moreover, anti-­inflammatory activities of the isolated compounds were evaluated by measurements of interleukins (IL-1β, IL-6) and tumour necrosis factor-alpha (TNF-α) levels. Tiliroside exhibited the highest potency in all pathways. At concentrations of 3 and 6 µg/mL, it significantly decreased the levels of pro-inflammatory cytokines IL-1β (192.53 and 175.54 pg/mL), IL-6 (925.52 and 946.81 pg/mL), and TNF- α (6465.86 and 6267.67 pg/mL). This is the first report on phenolic profile of endemic M. rotundifolium. The anti-inflammatory and antidiabetic activity potential of the plant should be investigated in further studies.

Retroductal dexamethasone administration promotes the recovery from obstructive and inflammatory salivary gland dysfunction

IntroductionSalivary gland dysfunction, often resulting from salivary gland obstruction-induced inflammation, is a prevalent condition. Corticosteroid, known for its anti-inflammatory and immunomodulatory properties, is commonly prescribed in clinics. This study investigates the therapeutic implications and potential side effects of dexamethasone on obstructive sialadenitis recovery using duct ligation mice and salivary gland organoid models.MethodsFunctional and pathological changes were assessed after administering dexamethasone to the duct following deligation 2 weeks after maintaining ligation of the mouse submandibular duct. Additionally, lipopolysaccharide- and tumor necrosis factor-induced salivary gland organoid inflammation models were established to investigate the effects and underlying mechanisms of action of dexamethasone.ResultsDexamethasone administration facilitated SG function restoration, by increasing salivary gland weight and saliva volume while reducing saliva lag time. Histological evaluation revealed, reduced acinar cell atrophy and fibrosis with dexamethasone treatment. Additionally, dexamethasone suppressed pro-inflammatory cytokines IL-1β and TNF expression. In a model of inflammation in salivary gland organoids induced by inflammatory substances, dexamethasone restored acinar markers such as AQP5 gene expression levels, while inhibiting pro-inflammatory cytokines TNF and IL6, as well as chemokines CCL2, CXCL5, and CXCL12 induction. Macrophages cultured in inflammatory substance-treated media from salivary gland organoid cultures exhibited pro-inflammatory polarization. However, treatment with dexamethasone shifted them towards an anti-inflammatory phenotype by reducing M1 markers (Tnf, Il6, Il1b, and Cd86) and elevating M2 markers (Ym1, Il10, Cd163, and Klf4). However, high-dose or prolonged dexamethasone treatment induced acino-ductal metaplasia and had side effects in both in vivo and in vitro models.ConclusionsOur findings suggest the effectiveness of corticosteroids in treating obstructive sialadenitis-induced salivary gland dysfunction by regulating pro-inflammatory cytokines.

Retroductal dexamethasone administration promotes the recovery from obstructive and inflammatory salivary gland dysfunction.

Salivary gland dysfunction, often resulting from salivary gland obstruction-induced inflammation, is a prevalent condition. Corticosteroid, known for its anti-inflammatory and immunomodulatory properties, is commonly prescribed in clinics. This study investigates the therapeutic implications and potential side effects of dexamethasone on obstructive sialadenitis recovery using duct ligation mice and salivary gland organoid models. Functional and pathological changes were assessed after administering dexamethasone to the duct following deligation 2 weeks after maintaining ligation of the mouse submandibular duct. Additionally, lipopolysaccharide- and tumor necrosis factor-induced salivary gland organoid inflammation models were established to investigate the effects and underlying mechanisms of action of dexamethasone. Dexamethasone administration facilitated SG function restoration, by increasing salivary gland weight and saliva volume while reducing saliva lag time. Histological evaluation revealed, reduced acinar cell atrophy and fibrosis with dexamethasone treatment. Additionally, dexamethasone suppressed pro-inflammatory cytokines IL-1β and TNF expression. In a model of inflammation in salivary gland organoids induced by inflammatory substances, dexamethasone restored acinar markers such as AQP5 gene expression levels, while inhibiting pro-inflammatory cytokines TNF and IL6, as well as chemokines CCL2, CXCL5, and CXCL12 induction. Macrophages cultured in inflammatory substance-treated media from salivary gland organoid cultures exhibited pro-inflammatory polarization. However, treatment with dexamethasone shifted them towards an anti-inflammatory phenotype by reducing M1 markers (Tnf, Il6, Il1b, and Cd86) and elevating M2 markers (Ym1, Il10, Cd163, and Klf4). However, high-dose or prolonged dexamethasone treatment induced acino-ductal metaplasia and had side effects in both in vivo and in vitro models. Our findings suggest the effectiveness of corticosteroids in treating obstructive sialadenitis-induced salivary gland dysfunction by regulating pro-inflammatory cytokines.

Amiloride lowers plasma TNF and interleukin-6 but not interleukin-17A in patients with hypertension and type 2 diabetes.

Interleukin (IL)-17A contributes to hypertension in preclinical models. T helper 17 and dendritic cells are activated by NaCl, which could involve the epithelial Na+ channel (ENaC). We hypothesized that the ENaC blocker amiloride reduces plasma IL-17A and related cytokines in patients with hypertension. Concentrations of IL-17A, IFN-γ, TNF, IL-6, IL-1β, and IL-10 were determined by immunoassays in plasma from two patient cohorts before and after amiloride treatment: 1) patients with type 2 diabetes mellitus (T2DM) and treatment-resistant hypertension (n = 69, amiloride 5-10 mg/day for 8 wk) and 2) patients with hypertension and type 1 diabetes mellitus (T1DM) (n = 29) on standardized salt intake (amiloride 20-40 mg/day, 2 days). Plasma and tissue from ANG II-hypertensive mice with T1DM treated with amiloride (2 mg/kg/day, 4 days) were analyzed. The effect of amiloride and benzamil on macrophage cytokines was determined in vitro. Plasma cytokines showed higher concentrations (IL-17A ∼40-fold) in patients with T2DM compared with T1DM. In patients with T2DM, amiloride had no effect on IL-17A but lowered TNF and IL-6. In patients with T1DM, amiloride had no effect on IL-17A but increased TNF. In both cohorts, blood pressure decline and plasma K+ increase did not relate to plasma cytokine changes. In mice, amiloride exerted no effect on IL-17A in the plasma, kidney, aorta, or left cardiac ventricle but increased TNF in cardiac and kidney tissues. In lipopolysaccharide-stimulated human THP-1 macrophages, amiloride and benzamil (from 1 nmol/L) decreased TNF, IL-6, IL-10, and IL-1β. In conclusion, inhibition of ENaC by amiloride reduces proinflammatory cytokines TNF and IL-6 but not IL-17A in patients with T2DM, potentially by a direct action on macrophages.NEW & NOTEWORTHY ENaC activity may contribute to macrophage-derived cytokine release, since amiloride exerts anti-inflammatory effects by suppression of TNF and IL-6 cytokines in patients with resistant hypertension and type 2 diabetes and in THP-1-derived macrophages in vitro.

Tert-Butylhydroquinone Mitigates T-2-Toxin-Induced Testicular Dysfunction by Targeting Oxidative Stress, Inflammation, and Apoptosis in Rats.

Tert-butylhydroquinone (tBHQ) has emerged as a promising candidate for mitigating the adverse effects of T-2-induced reproductive toxicity. The protective effects of tBHQ on rat sperm quality, testicular injury, apoptosis, and inflammation induced by T-2 toxin exposure were investigated. Histopathological examination of testicular tissues revealed severe damage in the T-2-treated group, characterized by disorganized germ cell arrangement, thinning of the convoluted seminiferous tubule walls, and significant cellular necrosis. However, tBHQ administration, either as a preventive or therapeutic measure, mitigated this structural damage. Image analysis confirmed an increase in the cross-sectional area and height of the convoluted seminiferous tubules in the tBHQ-treated groups compared to the T-2-treated group (p < 0.05), indicating tBHQ's efficacy in alleviating testicular damage. Additionally, tBHQ treatment significantly inhibited T-2-induced apoptosis of testicular tissue cells, as evidenced by the results showing reduced apoptotic cell counts and downregulation of the BAX/BCL2 ratio and caspase-3 expression (p < 0.05). tBHQ significantly increased the concentrations of the antioxidant factors SOD, CAT, TAC, and GSH-PX. Furthermore, tBHQ attenuated the inflammatory response induced by T-2 exposure, as indicated by the decreased mRNA expression of the proinflammatory cytokines Tnf, Il1, and Il10 in testicular tissue (p < 0.05). Additionally, tBHQ treatment alleviated the decline in serum testosterone induced by the T-2 and promoted testosterone synthesis gene expression, including for the genes 17β-HSD and Cyp11a1, in rat testes (p < 0.05). These findings underscore tBHQ's role as a therapeutic agent combatting T-2-induced reproductive toxicity, highlighting its antioxidative, anti-apoptotic, and anti-inflammatory properties. Further elucidation of tBHQ's mechanisms of action may offer novel strategies for preventing and treating reproductive disorders induced by environmental toxins.

Rv2231c, a unique histidinol phosphate aminotransferase from Mycobacterium tuberculosis, supports virulence by inhibiting host-directed defense

Nitrogen metabolism of M. tuberculosis is critical for its survival in infected host cells. M. tuberculosis has evolved sophisticated strategies to switch between de novo synthesis and uptake of various amino acids from host cells for metabolic demands. Pyridoxal phosphate-dependent histidinol phosphate aminotransferase-HspAT enzyme is critically required for histidine biosynthesis. HspAT is involved in metabolic synthesis of histidine, phenylalanine, tyrosine, tryptophan, and novobiocin. We showed that M. tuberculosis Rv2231c is a conserved enzyme with HspAT activity. Rv2231c is a monomeric globular protein that contains α-helices and β-sheets. It is a secretory and cell wall-localized protein that regulates critical pathogenic attributes. Rv2231c enhances the survival and virulence of recombinant M. smegmatis in infected RAW264.7 macrophage cells. Rv2231c is recognized by the TLR4 innate immune receptor and modulates the host immune response by suppressing the secretion of the antibacterial pro-inflammatory cytokines TNF, IL-12, and IL-6. It also inhibits the expression of co-stimulatory molecules CD80 and CD86 along with antigen presenting molecule MHC-I on macrophage and suppresses reactive nitrogen species formation, thereby promoting M2 macrophage polarization. Recombinant M. smegmatis expressing Rv2231c inhibited apoptosis in macrophages, promoting efficient bacterial survival and proliferation, thereby increasing virulence. Our results indicate that Rv2231c is a moonlighting protein that regulates multiple functions of M. tuberculosis pathophysiology to increase its virulence. These mechanistic insights can be used to better understand the pathogenesis of M. tuberculosis and to design strategies for tuberculosis mitigation.

Recombinant probiotic Lactococcus lactis delivering P62 mitigates moderate colitis in mice.

p62 is a human multifunctional adaptor protein involved in key cellular processes such as tissue homeostasis, inflammation, and cancer. It acts as a negative regulator of inflammasome complexes. It may thus be considered a good candidate for therapeutic use in inflammatory bowel diseases (IBD), such as colitis. Probiotics, including recombinant probiotic strains producing or delivering therapeutic biomolecules to the host mucosal surfaces, could help prevent and mitigate chronic intestinal inflammation. The objective of the present study was to combine the intrinsic immunomodulatory properties of the probiotic Lactococcus lactis NCDO2118 with its ability to deliver health-promoting molecules to enhance its protective and preventive effects in the context of ulcerative colitis (UC). This study was realized in vivo in which mice were supplemented with the recombinant strain. The intestinal barrier function was analyzed by monitoring permeability, secretory IgA total levels, mucin expression, and tight junction genes. Its integrity was evaluated by histological analyses. Regarding inflammation, colonic cytokine levels, myeloperoxidase (MPO), and expression of key genes were monitored. The intestinal microbiota composition was investigated using 16S rRNA Gene Sequencing. No protective effect of L. lactis NCDO2118 pExu:p62 was observed regarding mice clinical parameters compared to the L. lactis NCDO2118 pExu: empty. However, the recombinant strain, expressing p62, increased the goblet cell counts, upregulated Muc2 gene expression in the colon, and downregulated pro-inflammatory cytokines Tnf and Ifng when compared to L. lactis NCDO2118 pExu: empty and inflamed groups. This recombinant strain also decreased colonic MPO activity. No difference in the intestinal microbiota was observed between all treatments. Altogether, our results show that recombinant L. lactis NCDO2118 delivering p62 protein protected the intestinal mucosa and mitigated inflammatory damages caused by dextran sodium sulfate (DSS). We thus suggest that p62 may constitute part of a therapeutic approach targeting inflammation.

Electrical stimulation of the dorsal motor nucleus of the vagus in male mice can regulate inflammation without affecting the heart rate

BackgroundThe vagus nerve plays an important role in neuroimmune interactions and in the regulation of inflammation. A major source of efferent vagus nerve fibers that contribute to the regulation of inflammation is the brainstem dorsal motor nucleus of the vagus (DMN), as recently shown using optogenetics. In contrast to optogenetics, electrical neuromodulation has broad therapeutic implications. However, the anti-inflammatory effectiveness of electrical stimulation of the DMN (eDMNS) and the possible heart rate (HR) alterations associated with this approach have not been investigated. Here, we examined the effects of eDMNS on HR and cytokine levels in mice administered with lipopolysaccharide (LPS, endotoxin) and in mice subjected to cecal ligation and puncture (CLP) sepsis. MethodsAnesthetized male 8–10-week-old C57BL/6 mice on a stereotaxic frame were subjected to eDMNS using a concentric bipolar electrode inserted into the left or right DMN or sham stimulation. eDMNS (500, 250 or 50 μA at 30 Hz, for 1 min) was performed and HR recorded. In endotoxemia experiments, sham or eDMNS utilizing 250 μA or 50 μA was performed for 5 mins and was followed by LPS (0.5 mg/kg) i.p. administration. eDMNS was also applied in mice with cervical unilateral vagotomy or sham operation. In CLP experiments sham or left eDMNS was performed immediately post CLP. Cytokines and corticosterone were analyzed 90 mins after LPS administration or 24 h after CLP. CLP survival was monitored for 14 days. ResultsEither left or right eDMNS at 500 μA and 250 μA decreased HR, compared with baseline pre-stimulation. This effect was not observed at 50 μA. Left side eDMNS at 50 μA, compared with sham stimulation, significantly decreased serum and splenic levels of the pro-inflammatory cytokine TNF and increased serum levels of the anti-inflammatory cytokine IL-10 during endotoxemia. The anti-inflammatory effect of eDMNS was abrogated in mice with unilateral vagotomy and was not associated with serum corticosterone alterations. Right side eDMNS in endotoxemic mice suppressed serum TNF and increased serum IL-10 levels but had no effects on splenic cytokines. In mice with CLP, left side eDMNS suppressed serum IL-6, as well as splenic IL-6 and increased splenic IL-10 and significantly improved the survival rate of CLP mice. ConclusionsFor the first time we show that a regimen of eDMNS which does not cause bradycardia alleviates LPS-induced inflammation. These eDMNS anti-inflammatory effects require an intact vagus nerve and are not associated with corticosteroid alterations. eDMNS also decreases inflammation and improves survival in a model of polymicrobial sepsis. These findings are of interest for further studies exploring bioelectronic anti-inflammatory approaches targeting the brainstem DMN.

Endoplasmic reticulum stress response in immune cells contributes to experimental autoimmune encephalomyelitis pathogenesis in rats

We examined the role of endoplasmic reticulum (ER) stress and the ensuing unfolded protein response (UPR) in the development of the central nervous system (CNS)-directed immune response in the rat model of experimental autoimmune encephalomyelitis (EAE). The induction of EAE with syngeneic spinal cord homogenate in complete Freund's adjuvant (CFA) caused a time-dependent increase in the expression of ER stress/UPR markers glucose-regulated protein 78 (GRP78), X-box-binding protein 1 (XBP1), C/EBP homologous protein (CHOP), and phosphorylated eukaryotic initiation factor 2α (eIF2α) in the draining lymph nodes of both EAE-susceptible Dark Agouti (DA) and EAE-resistant Albino Oxford (AO) rats. However, the increase in ER stress markers was more pronounced in AO rats. CFA alone also induced ER stress, but the effect was weaker and less sustained compared to full immunization. The ultrastructural analysis of DA lymph node tissue by electron microscopy revealed ER dilatation in lymphocytes, macrophages, and plasma cells, while immunoblot analysis of CD3-sorted lymph node cells demonstrated the increase in ER stress/UPR markers in both CD3+ (T cell) and CD3− (non-T) cell compartments. A positive correlation was observed between the levels of ER stress/UPR markers in the CNS-infiltrated mononuclear cells and the clinical activity of the disease. Finally, the reduction of EAE clinical signs by ER stress inhibitor ursodeoxycholic acid was associated with the decrease in the expression of mRNA encoding pro-inflammatory cytokines TNF and IL-1β, and encephalitogenic T cell cytokines IFN-γ and IL-17. Collectively, our data indicate that ER stress response in immune cells might be an important pathogenetic factor and a valid therapeutic target in the inflammatory damage of the CNS.

Abstract 1410: An effect of Phenylalanine hydroxylase (PAH) deficiency on cancer development in kidney

Abstract Several studies have noted the importance of amino acid metabolism in cancer progression. Preliminary research using public cancer multi-omics data has revealed associations between heterozygous germline mutations in genes associated with amino acid metabolism and cancer development. In particular, the heterozygous status of the PAH gene enhances the risk of developing kidney cancer by more than three times. Phenylalanine hydroxylase (PAH) is an enzyme that converts phenylalanine to tyrosine. It is highly expressed in the liver and kidneys. Analysis of the blood metabolome in prospectively collected PAH heterozygous carriers indicates that heterozygous status causes metabolome fluctuations in the body. By the way, renal cell carcinoma (RCC) is an immunogenic tumor mainly infiltrated by T cells. Accordingly, we suggest that defects in the PAH gene change the metabolites in the body, leading to a reduction in immune function and an increased risk of developing kidney cancer. First, the expression of the PAH protein was confirmed through immunohistochemistry in a tissue microarray from RCC patients, and the results were then compared with clinical information. Next, we simulated the environment of PAH deficiency in vitro by treating the cells with phenylalanine (Phe) and its metabolite, phenylpyruvate (PRA), which accumulates during PAH dysfunction. The cytotoxicity of Phe and PPA was investigated using the WST assay. The Jurkat T cell line was cultured long-term in a medium containing Phe or PPA and then stimulated with PHA/PMA. Cytokine secretion was confirmed in these supernatants. In addition, purified primary human pan T cells were stimulated with Anti-CD3/CD28 in the medium containing either Phe or PPA. Proliferation was evaluated using CFSE staining. The IHC was performed on 467 tissues from RCC patients aged 29 to 101 years. We classified the cases into three scale scores based on the percentage of PAH intensity. Approximately 73% of cases were PAH-negative, and only 27% were positive (score 1+ or 2+). A comparison of IHC results with clinical information showed that lower PAH expression was associated with a lower relapse-free survival rate. Next, Phe and PPA exhibited minimal cytotoxicity. The secretion of representative proinflammatory cytokines IL-2 and TNF- α were dose-dependent and significantly decreased with Phe and PPA treatment. Additionally, cell proliferation, a critical step in acquiring effector functions of T cells, was inhibited by treatment with Phe and PPA, particularly in the CD8+ T cell population compared to CD4+ T cells. These results suggest that metabolite changes within the tissue microenvironment of the kidney, caused by Pah defects, may suppress anti-tumor immunity by impairing the function of immune cells. This novel discovery may contribute to the importance of the PAH gene as a new biomarker for diagnosing kidney cancer and to the development of kidney cancer therapy. Citation Format: Seojeong Kim, Jihyun Park, Jeongmin Park, Chaelin Kang, Kyung Chul Moon, Seung Seok Han, Youngil Koh, Sung-Soo Yoon. An effect of Phenylalanine hydroxylase (PAH) deficiency on cancer development in kidney [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1410.

Tear cytokine levels are reduced in patients treated with intravitreal injections

PurposeTo investigate cytokine levels in the tear fluid of patients receiving serial intravitreal injections (IVI) with anti-vascular endothelial growth factor (anti-VEGF) for neovascular age-related macular degeneration (nAMD). MethodsConcentrations of six cytokines (IFN-γ, IL-1β, IL-6, IL-8, TNF and VEGF) in tears of patients receiving anti-VEGF in one eye were assayed using multiplex cytometric bead array. The fellow untreated eye served as control. Tear sampling was performed on a single occasion at a minimum of four weeks after IVI. Patients underwent a pre-IVI antisepsis protocol with povidone-iodine. ResultsTear fluid from thirty patients with a mean age of 78.8 years (range 58–90) was assayed. Subjects received a median of 43.5 (range 22–106) IVI in one eye. The median level of IFN-γ was 0.33 (interquartile range (IQR) 0.22–0.52) pg/mg of total protein in injected eyes versus 0.41 (IQR 0.21–1.05) pg/mg in fellow eyes (p = 0.017). For TNF, a median level of 0.12 (IQR 0.08–0.18) pg/mg of total protein was found in injected eyes versus 0.14 (IQR 0.07–0.33) pg/mg of total protein in fellow eyes (p = 0.019). There were no differences between injected and fellow eyes regarding the levels of IL-1β, IL-6, IL-8 and VEGF. ConclusionTear fluid in eyes receiving serial IVI with anti-VEGF and preoperative povidone-iodine antisepsis constitutes lower levels of the pro-inflammatory cytokines IFN-γ and TNF compared to fellow eyes. This provides biochemical support of previous findings of reduced signs of inflammation and healthier tear film parameters in patients treated with serial IVI.

Protection Activity of 1,4-Naphthoquinones in Rotenone-Induced Models of Neurotoxicity.

The MTS cell viability test was used to screen a mini library of natural and synthetic 1,4-naphthoquinone derivatives (1,4-NQs) from marine sources. This screening identified two highly effective compounds, U-443 and U-573, which showed potential in protecting Neuro-2a neuroblastoma cells from the toxic effects of rotenone in an in vitro model of neurotoxicity. The selected 1,4-NQs demonstrated the capability to reduce oxidative stress by decreasing the levels of reactive oxygen species (ROS) and nitric oxide (NO) in Neuro-2a neuroblastoma cells and RAW 264.7 macrophage cells and displayed significant antioxidant properties in mouse brain homogenate. Normal mitochondrial function was restored and the mitochondrial membrane potential was also regained by 1,4-NQs after exposure to neurotoxins. Furthermore, at low concentrations, these compounds were found to significantly reduce levels of proinflammatory cytokines TNF and IL-1β and notably inhibit the activity of cyclooxygenase-2 (COX-2) in RAW 264.7 macrophages. The results of docking studies showed that the 1,4-NQs were bound to the active site of COX-2, analogically to a known inhibitor of this enzyme, SC-558. Both substances significantly improved the behavioral changes in female CD1 mice with rotenone-induced early stage of Parkinson's disease (PD) in vivo. It is proposed that the 1,4-NQs, U-443 and U-573, can protect neurons and microglia through their potent anti-ROS and anti-inflammatory activities.

Triclosan and its alternatives, especially chlorhexidine, modulate macrophage immune response with distinct modes of action

Since European regulators restricted the use of bacteriocidic triclosan (TCS), alternatives for TCS are emerging. Recently, TCS has been shown to reprogram immune metabolism, trigger the NLRP3 inflammasome, and subsequently the release of IL-1β in human macrophages, but data on substitutes is scarce. Hence, we aimed to examine the effects of TCS compared to its alternatives at the molecular level in human macrophages.LPS-stimulated THP-1 macrophages were exposed to TCS or its substitutes, including benzalkonium chloride, benzethonium chloride, chloroxylenol, chlorhexidine (CHX) and cetylpyridinium chloride, with the inhibitory concentration (IC10-value) of cell viability to decipher their mode of action. TCS induced the release of the pro-inflammatory cytokine TNF and high level of IL-1β, suggesting the activation of the NLRP3-inflammasome, which was confirmed by non-apparent IL-1β under the NLRP3-inhibitor MCC950 treatment d. While IL-6 release was reduced in all treatments, the alternative CHX completely abolished the release of all investigated cytokines.To unravel the underlying molecular mechanisms, we used untargeted LC-MS/MS-based proteomics. TCS and CHX showed the strongest cellular response at the protein and signalling pathway level, whereby pathways related to metabolism, translation, cellular stress and migration were mainly affected but to different proposed modes of action. TCS inhibited mitochondrial electron transfer and affected phagocytosis. In contrast, in CHX-treated cells, the translation was arrested due to stress conditions, resulting in the formation of stress granules. Mitochondrial (e.g. ATP5F1D, ATP5PB, UQCRQ) and ribosomal (e.g. RPL10, RPL35, RPS23) proteins were revealed as putative key drivers. Furthermore, we have demonstrated the formation of podosomes by CHX, potentially involved in ECM degradation.Our results exhibit modulation of the immune response in macrophages by TCS and its substitutes and illuminated underlying molecular effects. These results illustrate critical processes involved in the modulation of macrophages' immune response by TCS and its alternatives, providing information essential for hazard assessment.

The Battle within: AML´s p53 Strategies to Evade T-Cell Attack

Bispecific T-cell recruiting antibody constructs (BsAb) have shown clinical efficacy with Blinatumomab, a CD3xCD19 BiTE ® construct used for the treatment of relapsed or refractory B-cell precursor ALL. Despite the success of BsAbs in B-cell malignancies, the translation to a myeloid setting has been unsuccessful, independently of the chosen target antigen. For example, targeting CD33 has failed to provide long-term benefits. Several reports have demonstrated that the diverse genetic makeup of AML confers resistance to T-cell based immunotherapy. Mutations of TP53 occur in 5-10% of patients with de novo AML and up to 50% in older patients with therapy related AML, thus highlighting its role as a potential resistance mechanism. We hypothesize, that genetic aberrations of TP53 in AML contribute to cell intrinsic and cell extrinsic resistance against T-cell based immunotherapy approaches. To study the impact of TP53 aberrations on T-cell based immunotherapy, MV4-11 p53 WT or MV4-11 p53 KD were cocultured with healthy donor T cells at an effector to target ratio of 1:6 with or without BsAbs. BsAb-mediated cytotoxicity was analyzed by specific lysis of the CD33 + target cells and T-cell proliferation was assessed by FarRed ® staining or CD2 + T-cell fold change calculated to day 0. Cytokine secretion was monitored in the coculture supernatants by cytometric beads arrays. To differentiate between secretome vs surfacome mediated differences, transwell assays were performed. To identify the T-cell immunosuppressive factor, secretome analysis were performed using the Olink® proteomics platform. To further dissect the effect of the p53 KD MV-411 on T cells, we performed RNA-Seq analysis of T cells after coculture with MV4-11 p53 KD vs p53 WT. As expected, BsAb-mediated cytotoxicity was reduced against p53 KD vs p53 WT MV4-11 (% specific lysis: p53 KD=47.86±7.37 vs p53 WT=76.42±3.82; p=0,0001). However, BsAb-mediated T-cell proliferation was also significantly decreased in cocultures with p53 KD vs p53 WT (% proliferated: 30.26±2.92 vs 44.25±4.08; p=0.002). In line with these findings, we observed a significant decrease in secretion of the proinflammatory cytokines IFNγ (p53 KD=2985,01±896,69 vs p53 WT=4009.15±764.97 pg/ml; p=0.068), TNF (29,38±8.16 vs 110.21±37.61 pg/ml; p=0.044) and IL-2 (1391.64±375.93 vs 2253.88±604.76 pg/ml; p=0.018). RNA-Seq analysis of T cells after coculture with MV4-11 p53 KD vs p53 WT revealed a decrease in the expression of genes associated with mitosis (such as E2F targets and mitotic spindle related genes). A lower mitotic rate provides a potential explanation for the reduction in T-cell proliferation after coculture with p53 KD. To dissect if the observed effects were due to the AML surfacome or the secretome, we performed transwell assays. Indeed, the negative impact of p53 KD AML cells on T-cell function was also observed without direct cell-cell contact. To gain further insights in the secretome of the AML cells, coculture supernatant was analysed using the Olink ® platform and demonstrated significantly higher secretion of IL-18 and LAP TGF-β1 (TGF-β1 latency associated protein) in the coculture with MV4-11 p53 KD vs MV4-11 p53 WT (normalized protein expression for IL-18: p53 KD=1.86±0.22 vs p53 WT=0.96±0.052; p=0.0153; for LAP TGF-β1: 6.52±0.18 vs 5.84±0.14; p=0.0394). The relevance of TGF-β on T-cell fitness was confirmed by the analysis of BsAb-mediated cytotoxicity with or without the addition of TGF-β. Addition of 3 ng/µl TGF-β to the coculture with p53 WT decreased the specific lysis significantly and was now comparable to the lysis of p53 KD. Confirmatory studies in primary AML samples with or without p53 aberrations further validated our findings with a clear reduction of BsAb-mediated cytotoxicity (% specific lysis: p53 KD=35.82±5.51 vs p53 WT=61.32±8.27; p=0.015), T-cell proliferation (CD2 + fold change: 3.17±0,61 vs 7.1±2.8; p=0.111) and secretion of proinflammatory cytokines. Taken together, we demonstrated that AML cells carrying a p53 aberration are less susceptible to BsAb-mediated cytotoxicity. Surprisingly, immune evasion was mediated by the AML secretome leading to a decrease in T-cell proliferation and cytokine secretion. Secretome analyses indicated TGF-β as a prominent cytokine leading to an impairment of T-cell function. Further studies are needed to understand the influence of p53 on the TGF-β pathway in AML cells.

Therapeutic Effects of Zymomonas mobilis on Experimental DSS-Induced Colitis Mouse Model.

Zymomonas mobilis, a Gram-negative bacteria observed in some popular beverages, is considered safe and has been studied for its potential therapeutic benefits. In this study, we explored its effects on the inflammatory process, tissue integrity, differential gene expression, and microbiota composition in an experimental dextran sulfate sodium (DSS)-induced colitis model in mice. As a result, Z. mobilis alleviated the symptoms caused by DSS administration, as indicated by reduced weight loss, disease activity index, a significant reduction in the colon weight/length ratio, and histopathological improvement. Also, Z. mobilis could restore the mucosal barrier as well as increase the expression of Muc3 and Ocln genes. An analysis of 16S rRNA sequences showed that Z. mobilis alters gut microbiota, increasing Akkermansia muciniphila abundance and decreasing Escherichia coli. Furthermore, Z. mobilis seems to be involved in potentiating a regulatory phenotype by inducing immunomodulatory genes like Tgfb, Il5, Il10, and Foxp3 and reducing the relative mRNA expression of proinflammatory cytokines TNF, Il6, and Il17. Our data suggest that Z. mobilis could alleviate disease progression and be considered a possible probiotic adjuvant for pathologies of the bowel.

Biochemical and gene expression studies reveal the potential of Aspergillus oryzae-fermented broken rice and brewers' rice water extracts as anti-photoageing agents.

In this study, UVA- and UVB-irradiated human fibroblasts were used to investigate the anti-photoaging efficacy of two aqueous extracts from Aspergillus oryzae-fermented broken rice (FBR) and brewers' rice (FBrR). As UVA and UVB can damage the dermal and epidermal layers, respectively, two UV radiation approaches were utilised: i) direct UVA irradiation on fibroblasts, and ii) UVB-irradiated keratinocytes indirectly co-cultured with fibroblasts to observe their epithelial-mesenchymal interaction during UVB-induced photoaging. The anti-photoaging properties were tested utilising biochemical tests and quantitative polymerase chain reaction (qPCR). The treatment of UV-irradiated human fibroblasts with FBR and FBrR dramatically downregulates MMP-1 and SFE gene expression. Nonetheless, MMP-1 secretion was inhibited by FBR and FBrR, with more substantial decreases in UVB-treated co-cultures, ranging from 0.76- to 1.89-fold relative to the untreated control. In UVA-treated fibroblasts, however, the elastase-inhibiting activity of FBR and FBrR is up to 1.63-fold and 2.13-fold more potent, respectively. In addition, post-UV irradiation treatment with FBR and FBrR was able to repair and enhance collagen formation in UVA-irradiated fibroblasts. Both FBR and FBrR were able to upregulate elastin gene expression in fibroblasts under both culture conditions, especially at 50 µg/mL. The pro-inflammatory cytokines TNF-, IL-1ß, and IL-6 were likewise lowered by FBR and FBrR, which may have contributed to the anti-photoaging effect of the UVB-treated co-culture. These results reveal that FBR and FBrR inhibit photoaging in human fibroblasts under both UV induction conditions. In conclusion, FBR and FBrR may be attractive bio-ingredients for usage in the cosmetic sector as cosmeceuticals.

CKLF induces microglial activation via triggering defective mitophagy and mitochondrial dysfunction

ABSTRACT Although microglial activation is induced by an increase in chemokines, the role of mitophagy in this process remains unclear. This study aimed to elucidate the role of microglial mitophagy in CKLF/CKLF1 (chemokine-like factor 1)-induced microglial activation and neuroinflammation, as well as the underlying molecular mechanisms following CKLF treatment. This study determined that CKLF, an inducible chemokine in the brain, leads to an increase in mitophagy markers, such as DNM1L, PINK1 (PTEN induced putative kinase 1), PRKN, and OPTN, along with a simultaneous increase in autophagosome formation, as evidenced by elevated levels of BECN1 and MAP1LC3B (microtubule-associated protein 1 light chain 3 beta)-II. However, SQSTM1, a substrate of autophagy, was also accumulated by CKLF treatment, suggesting that mitophagy flux was reduced and mitophagosomes accumulated. These findings were confirmed by transmission electron microscopy and confocal microscopy. The defective mitophagy observed in our study was caused by impaired lysosomal function, including mitophagosome-lysosome fusion, lysosome generation, and acidification, resulting in the accumulation of damaged mitochondria in microglial cells. Further analysis revealed that pharmacological blocking or gene-silencing of mitophagy inhibited CKLF-mediated microglial activation, as evidenced by the expression of the microglial marker AIF1 (allograft inflammatory factor 1) and the mRNA of proinflammatory cytokines (Tnf and Il6). Ultimately, defective mitophagy induced by CKLF results in microglial activation, as observed in the brains of adult mice. In summary, CKLF induces defective mitophagy, microglial activation, and inflammation, providing a potential approach for treating neuroinflammatory diseases. Abbreviation: 3-MA: 3-methyladenine; AIF1: allograft inflammatory factor 1; ANOVA: analysis of variance; BAF: bafilomycin A1; BSA: bovine serum albumin; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; cGAMP: cyclic GMP-AMP; CGAS: cyclic GMP-AMP synthase; CKLF/CKLF1: chemokine-like factor 1; CNS: central nervous system; DMEM: Dulbecco’s Modified Eagle Medium; DNM1L: dynamin 1 like; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescence protein; IRF3: interferon regulatory factor 3; IgG: immunoglobulin G; LAMP1: lysosomal-associated membrane protein 1; LAPTM4A: lysosomal-associated protein transmembrane 4A; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; Mdivi-1: mitochondrial division inhibitor 1; mRFP: monomeric red fluorescent protein; mtDNA: mitochondrial DNA; MTORC1: mechanistic target of rapamycin kinase complex 1; OPTN: optineurin; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PINK1: PTEN induced putative kinase 1; PLL: poly-L-lysine; PRKN: parkin RBR E3 ubiquitin protein ligase; qPCR: quantitative polymerase chain reaction; ROS: reactive oxygen species; SQSTM1: sequestosome 1; TBK1: TANK-binding kinase 1; TFEB: transcription factor EB; VDAC: voltage-dependent anion channel

Myocardial injury causes changes in the transcriptional and regulatory landscape of adaptive immune cells in the heart

Abstract Background Ischaemic heart disease is a leading cause of heart failure (HF) and despite advanced therapeutic options, morbidity and mortality rates remain high. Acute inflammation after myocardial infarction (MI) has been thoroughly studied in recent years and persistent immune-mediated damage has been suggested as a factor contributing to the development of HF. T and B cells are critical mediators of adaptive immune responses after MI, however a thorough analysis of their transcriptomic phenotypes in the heart at baseline and over time after injury are missing. Purpose Here, we aim to understand the transcriptional activity of B and T cells in the post-MI heart, in order to characterize their role in maintaining post-MI autoreactivity. Methods We obtained single cell RNA sequencing data of mouse and human hearts from Forte et al (2020), Martini et al (2019) and Litviňuková et al (2020) and performed differential expression analysis, gene regulatory inferences and trajectory-based differential expression analysis. Results Myocardial T cells subclustered into CD8+, CD4+, CD20+ and NK lineages with naïve, memory and effector functions (Fig. 1). The B cell population was divided into immature, plasma, memory, collagen-producing and B1 cell subgroups (Fig. 2). At baseline, despite a striking presence of effector populations, both B and T cells are governed by homeostatic and tolerance-promoting transcription factors (Deaf1, Ikzf1 and Nr1h3 for T cells and Spi-B as well as Bcl11 for B cells). Post-MI, the activity of transcription factors concerned with differentiation and proliferation increased. T cells express transcripts for proinflammatory chemokines and cytokines (Ccr7, Ccl5, Ifng and Tnf) as well as granzymes and perforin up until 28 days post-MI (Gzma and Prf1). Surprisingly, B cells upregulate collagen transcript levels 5-7 days post-MI (Col1a, Col5a, and Col4a) and regulon analysis supports that B cells increase the activity of transcription factors necessary for collagen production (Ets-1 and Krox20). Both B and T cells remain persistently activated post MI. 28 days post-MI T cells have increased activity of proinflammatory (Nfkb1) and cytokine producing (Crem and Rel) transcription factors, whereas B cells have increase maturation (Nfkb1), autoantibody production (Stat3 and Rel) and memory (Stat5b) transcription factor activity. Conclusion Collectively, our data shows that there are effector immune cell populations present at baseline, which are governed by homeostatic transcription factors. Post-MI, effector transcription factors are upregulated in both T cells and B cells, which promote proliferation, differentiation, cytokine production and in the case of B cells, collagen production. Even after resolution of acute inflammation, both the T and B cell population do not return fully to baseline but remain at a state of persistent activation, as effector transcription factors remain upregulated.

Gasdermin D promotes hyperinflammation and immunopathology during severe influenza A virus infection

Excessive inflammation and tissue damage during severe influenza A virus (IAV) infection can lead to the development of fatal pulmonary disease. Pyroptosis is a lytic and pro-inflammatory form of cell death executed by the pore-forming protein gasdermin D (GSDMD). In this study, we investigated a potential role for GSDMD in promoting the development of severe IAV disease. IAV infection resulted in cleavage of GSDMD in vivo and in vitro in lung epithelial cells. Mice genetically deficient in GSDMD (Gsdmd−/−) developed less severe IAV disease than wildtype mice and displayed improved survival outcomes. GSDMD deficiency significantly reduced neutrophil infiltration into the airways as well as the levels of pro-inflammatory cytokines TNF, IL-6, MCP-1, and IL-1α and neutrophil-attracting chemokines CXCL1 and CXCL2. In contrast, IL-1β and IL-18 responses were not largely impacted by GSDMD deficiency. In addition, Gsdmd−/− mice displayed significantly improved influenza disease resistance with reduced viral burden and less severe pulmonary pathology, including decreased epithelial damage and cell death. These findings indicate a major role for GSDMD in promoting damaging inflammation and the development of severe IAV disease.

Presence of MDSC associates with impaired antigen-specific T cell reactivity following COVID-19 vaccination in cirrhotic patients.

Cirrhosis entails high risk of serious infections and abated efficiency of vaccination, but the underlying mechanisms are only partially understood. This study aimed at characterizing innate and adaptive immune functions, including antigen-specific T cell responses to COVID-19 vaccination, in patients with compensated and decompensated cirrhosis. Immune phenotype and function in peripheral blood from 42 cirrhotic patients and 44 age-matched healthy controls were analysed after two doses of the mRNA-based COVID-19 vaccines [BNT162b2 (Pfizer BioNTech) or mRNA-1273 (Moderna)]. Cirrhotic patients showed significantly reduced blood counts of antigen-presenting dendritic cells (DC) and high counts of monocytic myeloid-derived suppressor cells (M-MDSC) as compared to healthy controls. In addition, monocytic cells recovered from cirrhotic patients showed impaired expression of the antigen-presenting molecule HLA-DR and the co-stimulatory molecule CD86 upon Toll-like receptor (TLR) stimulation. These features were more prominent in patients with decompensated cirrhosis (Child-Pugh classes B & C). Interestingly, while patients with compensated cirrhosis (Child-Pugh class A) showed an inflammatory profile with myeloid cells producing the proinflammatory cytokines IL-6 and TNF, decompensated patients produced reduced levels of these cytokines. Cirrhotic patients, in particular those with more advanced end-stage liver disease, mounted reduced antigen-specific T cell reactivity to COVID-19 vaccination. Vaccine efficiency inversely correlated with levels of M-MDSC. These results implicate MDSC as mediators of immunosuppression, with ensuing deficiency of vaccine-specific T cell responses, in cirrhosis.