Stimulation Of Macrophages Research Articles

TAK1 deficiency promotes liver injury and tumorigenesis via ferroptosis and macrophage cGAS-STING signalling

Oxidative stress-mediated ferroptosis and macrophage-related inflammation play an important role in various liver diseases. Here, we explored if and how hepatocyte ferroptosis regulates macrophage stimulator of interferon genes (STING) activation in the development of spontaneous liver damage, fibrosis, and tumorigenesis. We used a transforming growth factor-beta-activated kinase 1 (TAK1) deficiency-induced model of spontaneous liver damage, fibrosis, and tumorigenesis to investigate hepatocyte ferroptosis and its impact on macrophage STING signalling. Primary hepatocytes and macrophages were used for invitro experiments. Significant liver injury and increased numbers of intrahepatic M1 macrophages were found in hepatocyte-specific TAK1-deficient (TAK1ΔHEP) mice, peaking at 4 weeks and gradually decreasing at 8 and 12 weeks. Meanwhile, activation of STING signalling was observed in livers from TAK1ΔHEP mice at 4 weeks and had decreased at 8 and 12 weeks. Treatment with a STING inhibitor promoted macrophage M2 polarisation and alleviated liver injury, fibrosis, and tumour burden. TAK1 deficiency exacerbated liver iron metabolism in mice with a high-iron diet. Moreover, consistent with the results from single-cell RNA-Seq dataset, TAK1ΔHEP mice demonstrated an increased oxidative response and hepatocellular ferroptosis, which could be inhibited by reactive oxygen species scavenging. Suppression of ferroptosis by ferrostatin-1 inhibited the activation of macrophage STING signalling, leading to attenuated liver injury and fibrosis and a reduced tumour burden. Mechanistically, increased intrahepatic and serum levels of 8-hydroxydeoxyguanosine were detected in TAK1ΔHEP mice, which was suppressed by ferroptosis inhibition. Treatment with 8-hydroxydeoxyguanosine antibody inhibited macrophage STING activation in TAK1ΔHEP mice. Hepatocellular ferroptosis-derived oxidative DNA damage promotes macrophage STING activation to facilitate the development of liver injury, fibrosis, and tumorigenesis. Inhibition of macrophage STING may represent a novel therapeutic approach for the prevention of chronic liver disease. The precise mechanism by which hepatocyte ferroptosis regulates macrophage STING activation in the progression of liver damage, fibrosis, and tumorigenesis remains unclear. Herein, we show that deletion of TAK1 in hepatocytes caused oxidative stress-mediated ferroptosis and macrophage-related inflammation in the development of spontaneous liver injury, fibrosis, and hepatocellular carcinoma.

Stimulation of the farnesoid X receptor promotes M2 macrophage polarization.

FXR is a key molecule that modulates anti-inflammatory activity in the intestinal-liver axis. Although FXR has pleiotropic functions including regulation of liver inflammation and activation of macrophages, it remains unclear whether it is involved in macrophage polarization. In this paper we demonstrated that stimulation of macrophages derived from the bone marrow using an FXR agonist activated polarization toward M2 but not M1 macrophages. The treatment of mice with chitin skewed macrophage polarization towards M2 macrophages, while co-treatment with an FXR agonist further promoted the polarization toward M2 macrophages in vivo. This skewed polarization towards M2 macrophages by an FXR agonist was accompanied by increased expression of signaling molecules related to the retinoic acid receptor. Inhibition of the retinoic acid receptor suppressed FXR agonist-mediated M2 macrophage polarization, indicating that this polarization was, at least, partly dependent on the retinoic acid receptor pathway. These data demonstrate that FXR has a role in polarization toward M2 macrophages and suggest a possible therapeutic potential of FXR agonists in M2 macrophage-related conditions.

NUCLEOLIN PROTECTS CARDIOMYOCYTES BY UPREGULATING PGC-1α AND PROMOTING MITOCHONDRIAL BIOGENESIS IN LPS-INDUCED MYOCARDIAL INJURY.

Background: Lipopolysaccride-induced myocardial injury was characterized by frequent mitochondrial dysfunction. Our previous studies found that nucleolin (NCL) played important protective roles in myocardial ischemia-reperfusion injury. Recently, it has been found that NCL has a protective effect on LPS-induced myocardial injury in vivo . However, the exact underlying mechanisms that how NCL protects myocardium against the LPS-induced myocardial injury remains unclear. Objective: The aim of the study is to investigate the protective role of NCL in LPS-induced myocardial injury from the aspect of mitochondrial biogenesis. Methods: The cardiac-specific NCL-knockout (NCL -/- ) or NCL f/f mice were injected with LPS (10 mg/kg) to induce LPS-induced myocardial injury. The supernatant generated after LPS stimulation of macrophages was used as the conditioned medium to stimulate H9C2 and established the injured cell model. Analysis of mRNA stability, RNA-binding protein immunoprecipitation assay, and luciferase reporter assay were performed to detect the mechanism by which NCL regulated the expression of PGC-1α. Results: The expression of NCL and PGC-1α was elevated in cardiac tissue and cardiomyocytes during LPS-induced myocardial injury. The cardiac-specific NCL-knockout decreased PGC-1α expression, inhibited mitochondrial biogenesis, and increased cardiomyocytes death during LPS-induced myocardial injury in vitro and in vivo . In contrast, the overexpression of NCL could improve mitochondrial biogenesis in H9C2 cells. Moreover, the analysis of mRNA stability and luciferase reporter assay revealed that the interaction between NCL and PGC-1α significantly promoted the stability of PGC-1α mRNA, thereby upregulating the expression of PGC-1α and exerting a cardioprotective effect. In addition, the activation of PGC-1α diminished the detrimental effects of NCL knockdown on mitochondrial biogenesis in vitro and in vivo . Conclusions: Nucleolin upregulated the gene expression of PGC-1α by directly binding to the 5'-UTR of PGC-1α mRNA and increasing its mRNA stabilities, then promoted mitochondrial biogenesis, and played protective effect on cardiomyocytes during LPS-induced myocardial injury. Taken together, all these data showed that NCL activated PGC-1α to rescue cardiomyocytes from LPS-induced myocardial injury insult, suggesting that the cardioprotective role of NCL might be a promising prospect for clinical treatment of patients with endotoxemia.

Von Willebrand factor: from figurant to main character in the scene of inflammation

von Willebrand factor: from figurant to main character in the scene of inflammation

Echinochrome Prevents Sulfide Catabolism-Associated Chronic Heart Failure after Myocardial Infarction in Mice.

Abnormal sulfide catabolism, especially the accumulation of hydrogen sulfide (H2S) during hypoxic or inflammatory stresses, is a major cause of redox imbalance-associated cardiac dysfunction. Polyhydroxynaphtoquinone echinochrome A (Ech-A), a natural pigment of marine origin found in the shells and needles of many species of sea urchins, is a potent antioxidant and inhibits acute myocardial ferroptosis after ischemia/reperfusion, but the chronic effect of Ech-A on heart failure is unknown. Reactive sulfur species (RSS), which include catenated sulfur atoms, have been revealed as true biomolecules with high redox reactivity required for intracellular energy metabolism and signal transduction. Here, we report that continuous intraperitoneal administration of Ech-A (2.0 mg/kg/day) prevents RSS catabolism-associated chronic heart failure after myocardial infarction (MI) in mice. Ech-A prevented left ventricular (LV) systolic dysfunction and structural remodeling after MI. Fluorescence imaging revealed that intracellular RSS level was reduced after MI, while H2S/HS- level was increased in LV myocardium, which was attenuated by Ech-A. This result indicates that Ech-A suppresses RSS catabolism to H2S/HS- in LV myocardium after MI. In addition, Ech-A reduced oxidative stress formation by MI. Ech-A suppressed RSS catabolism caused by hypoxia in neonatal rat cardiomyocytes and human iPS cell-derived cardiomyocytes. Ech-A also suppressed RSS catabolism caused by lipopolysaccharide stimulation in macrophages. Thus, Ech-A has the potential to improve chronic heart failure after MI, in part by preventing sulfide catabolism.

Probiotic activity of ropy Lactiplantibacillus plantarum NA isolated from Chinese northeast sauerkraut and comparative evaluation of its live and heat-killed cells on antioxidant activity and RAW 264.7 macrophage stimulation.

Lactic acid bacteria are known to have a positive impact on health and considered as functional supplements and additives. This study aimed to evaluate the probiotic properties of ropy Latilactobacillus isolated from Chinese northeast sauerkraut and to determine the antioxidant and immunoregulatory activities of its heat-killed cells compared to its live strains to assess its functional activity. After the analysis of the 16r DNA and phylogenetic tree, it was identified as a Lactiplantibacillus plantarum and named L. plantarum NA. L. plantarum NA was resistant to simulated gastrointestinal conditions in vitro. In addition, L. plantarum NA exhibited cholesterol degradation, antibiotic susceptibility, and antibacterial activity. Heat-killed L. plantarum NA exhibited antioxidant and immune-stimulating activities similar to live cells, which may be associated with the undamaged overall structure after heating. The results of antioxidant activity analysis suggested that both live and heat-killed L. plantarum NA possessed capacity for scavenging free radicals, including 2,2-diphenyl-1-picryl-hydrazyl (DPPH), 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), hydroxyl radicals and superoxide radicals. The ABTS scavenging activity of heat-killed cells (38%) was significantly higher than that of viable (19%) L. plantarum NA. Furthermore, RAW 264.7 macrophages treated with L. plantarum NA induced the production of nitric oxide, cytokines (IL-6 and TNF-α), and reactive oxygen species (ROS). The NO/iNOS signaling pathway could be activated by L. plantarum NA and promoted NO production. Both live and heat-killed cells had a potential impact on the immunomodulatory activity, with different dosages. These results suggest that the novel L. plantarum NA isolated from Chinese northeast sauerkraut could be useful as a probiotic strain and applied to functional food processing and pharmaceutical fields as a potential immunomodulator.

Neutrophil-dependent Mitochondrial DNA Release Associated With Extracellular Trap Formation in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is associated with increased circulating damage-associated molecular patterns, in particular, the highly pro-inflammatory mitochondrial DNA (mtDNA). Here, we study the importance of blood neutrophils in mtDNA release via neutrophil extracellular trap (NET) formation and mitochondrial NETosis, where neutrophils specifically expulse mtDNA as potential targetable biological pathways. We investigated the roles of A23187 (a known NET stimulant), granulocyte macrophage stimulating factor, lipopolysaccharide (LPS), and human IBD plasma in their ability to induce NET formation, mitochondrial NETosis, mtDNA, and total DNA release from human blood neutrophils; and the evidence for increased NET formation in IBD. We demonstrated that NET formation resulted in significant DNA (P < .0001) and mtDNA release (P < .0001) with long DNA fragments (>1000 base pairs) with NETs containing high levels of mtDNA. Using previously described invitro conditions for mitochondrial NETosis, granulocyte macrophage stimulating factor+ LPS triggered neutrophil mtDNA release at lower levels but not NETosis. LPS alone can trigger neutrophilic DNA release without NET formation. Heterologous coculture with plasma from patients with active IBD (vs remission [n= 6/group]) were not associated with significantly higher levels of NETs and mtDNA release. During coculture with active IBD plasma (vs remission), citrullinated histone 3 (CitH3) (a NETs biomarker) levels were significantly lower (P < .001). Similarly, CitH3 levels were lower in stool supernatants of patients with active IBD vs remission (n= 19/12, P= .0001). Stool CitH3 negatively correlates with stool calprotectin, a biomarker for gut inflammation (r=-0.47, P= .03). Hence, although blood neutrophils remain an important source of circulating mtDNA with defined mechanisms for release via NET formation and during neutrophil activation, our data do not support excessive systemic NET formation as a dominant underpinning pathobiological process in IBD.

The Role of Immune Mechanisms, Inflammatory Pathways, and Macrophage Activation Syndrome in the Pathogenesis of Hemophagocytic Lymphohistiocytosis.

This review article describes the pathophysiology of hemophagocytic lymphohistiocytosis (HLH). The condition is characterized by excessive stimulation of inflammatory cytokines, lymphocytes, and macrophages, leading to hyperinflammatory disorder with immune dysfunction. The main clinical and diagnostic features include fever ≥38.5°C, splenomegaly, hyperferritinemia, cytopenia, hypofibrinogenemia, hemophagocytosis on the bone marrow, low or absent of natural killer (NK) cell activity, and elevated soluble CD25. Various immunological and inflammatory mechanisms are involved in the pathogenesis of HLH. Moreover, the condition can result in multisystem organ failure, contributing to the high mortality rate in hospital settings. A thorough literature search was conducted by collecting data from multiple articles published on PubMed, Medline, and Google Scholar. The article discusses the cellular and molecular pathways that lead to HLH. Due to the high rate of morbidity and mortality, early diagnosis needs to be established. More research pertaining to molecular biology, immunology, and the genetics of HLH is needed to explore the effective management and treatment of this rare disorder.

On-Off Phagocytosis and Switchable Macrophage Activation Stimulated with NIR for Infected Percutaneous Tissue Repair of Polypyrrole-Coated Sulfonated PEEK.

Intelligent control of the immune response is essential for obtaining percutaneous implants with good sterilization and tissue repair abilities. In this study, polypyrrole (Ppy) nanoparticles enveloping a 3D frame of sulfonated polyether ether ketone (SP) surface are constructed, which enhance the surface modulus and hardness of the sulfonated layer by forming a cooperative structure of simulated reinforced concrete and exhibit a superior photothermal effect. Ppy-coated SP could quickly accumulate heat on the surface by responding to 808nm near-infrared (NIR) light, thereby killing bacteria, and destroying biofilms. Under NIR stimulation, the phagocytosis and M1 activation of macrophages cultured on Ppy-coated SP are enhanced by activating complement 3 and its receptor, CD11b. Phagocytosis and M1 activation are impaired along with abolishment of NIR stimulation in the Ppy-coated SP group, which is favorable for tissue repair. Ppy-coated SP promotes Collagen-I, vascular endothelial growth factor, connective tissue growth factor, and α-actin (Acta2) expression by inducing M2 polarization owing to its higher surface modulus. Overall, Ppy-coated SP with enhanced mechanical properties could be a good candidate for clinical percutaneous implants through on-off phagocytosis and switchable macrophage activation stimulated with NIR.

Vitamin D serves as a modulator of immune organs in grass carp (Ctenopharyngodon idella) infected with Aeromonas hydrophila

Vitamin D regulates biological processes via vitamin D receptors (VDRs) and exerts a pleiotropic effect in the prevention of pathogen infections. In various fish, the existence of two unique VDR isoforms (VDRa and VDRb) has been reported; however, the effects of vitamin D and VDR isoforms on the function of immune-related organs (e.g., the head kidney and spleen) remain poorly understood. In the present study, diets containing different vitamin D concentrations (0, 364.3, 782.5, 1167.9, 1573.8, and 1980.1 IU/kg) were provided to on-growing grass carp for 70 d. Subsequently, each group was subjected to a challenge trial by infection with Aeromonas hydrophila for 14 d. Dietary vitamin D decreased the oxidative biomarker content and upregulated the transcriptional responses of antioxidant enzymes potentially involved in Nrf2 signaling; alleviated cell apoptosis by down-regulating caspase expression, potentially in relation to the inhibition of p38MAPK molecules; enhanced immune component activity; downregulated the transcriptional levels of pro-inflammatory cytokines; and upregulated those of anti-inflammatory cytokines. Furthermore, the optimal vitamin D dose decreased the expression of nuclear factor kappa B p65, c-Rel, IKKα, IKKβ, and IKKγ and increased the transcriptional levels of iκBα, VDRb (but not VDRa), macrophage stimulating 1 (MST1), and signal transducers and activators of transcription (STAT) 3b1 and 3b2. Moreover, the protein levels of VDR, p-STAT3Tyr705, and p-MST1Thr108 were elevated both in the head kidney and spleen after treatment. In summary, vitamin D protected the immune organs from oxidative stress, apoptosis, and immunological injury. Based on lysozyme activity in the head kidney and spleen, the vitamin D requirements of on-growing grass carp (256.89–1129.67 g) were estimated to be 1150.71 and 1355.37 IU/kg, respectively. In conclusion, our findings confirmed that vitamin D can regulate molecular pathways related to immune organ function and pathogen defense in an aquaculture setting, supporting the feasibility of vitamin D supplementation to support immunological functions under infection conditions.

Antifungal and Anti-Inflammatory Activities of PS1-2 Peptide against Fluconazole-Resistant Candida albicans.

Clinically, fungal pneumonia rarely occurs in adults, and invasive fungal infections can cause substantial morbidity, and mortality due to sepsis and septic shock. In the present study, we have designed peptides that exhibit potent antifungal activities against fluconazole-resistant Candida albicans in physiological monovalent, and divalent ionic buffers, with minimal fungicidal concentrations ranging from 16 to 32 µM. None of these tested peptides resulted in the development of drug resistance similar to fluconazole. Among them, the PS1-2 peptide did not induce stimulation of macrophages by C. albicans, and it exerted antifungal and anti-inflammatory effects against C. albicans-induced intratracheal infection, in an acute lung injury mouse model. PS1-2 is likely a novel therapeutic agent for the control, and prevention of drug-resistant C. albicans infection, and our findings may be useful for designing antimicrobial peptides to combat fungal infection.

Abstract A50: IFNλ1 is a marker of DNA damage-triggered STING-signaling in lung cancer that induces immune activation through macrophage stimulation

Abstract Immunotherapy is a fundamental pillar in the treatment of lung cancer but remains inefficient in half of the treated patients. Recent clinical trials showed synergistic effects of combining chemotherapy and immunotherapy for lung cancer, suggesting that increased DNA damage imposed by chemotherapy modulates the tumor microenvironment to aid the efficacy of immunotherapy. But the immunological mechanisms related to this remains to be fully elucidated. The cGAS-STING pathway senses cytosolic DNA introduced by DNA-damaging agents such as chemotherapy. Activation of this pathway and induction of inflammatory cytokines may contribute to better immunotherapy efficacy. In particular, the release of type I IFN is a hallmark for STING pathway activation. However, we also know that IFNλ – a type III IFN – can be induced by STING-signaling. Despite the relevance of IFNλ in controlling inflammatory responses, its contribution in cancer remains unclear. Here, we investigated cGAS-STING expression and signaling in a panel of lung cancer cells. We identified IFNλ1 as an alternative hallmark for cGAS-STING DNA-sensing with higher expression after DNA-stimulation than IFNb in 6 out of 8 cell lines with one cell line having no IFNb expression at all. We then tested IFN induction after chemotherapeutic treatment in three different NSCLC cell lines and saw significant increase in both IFNb and IFNλ1 expression across four different chemotherapeutics, but with a higher fold change for IFNλ1. The induction of both IFNb and IFNλ1 was abolished when STING KO cell lines were treated with doxorubicin pointing to a STING-dependent response. To access the effect of IFNλ1 on macrophages, a prevalent cell type in the tumor microenvironment expressing IFNLR1, we performed RNA-sequencing on four primary human macrophage donors stimulated with IFNλ1. This stimulation led to a significant increase in well-known interferon-stimulated genes including the chemokines CXCL10 and CXCL11. We also explored whether IFNλ1 stimulation led to increased macrophage-dependent activation of autologous CD8+ T-cells. Interestingly, we found increased expression of both IFNg (p = 0.0215, n = 6) and Granzyme B (p = 0.0033, n = 6) measured by flow cytometry and ELISA (p &amp;gt; 0.0001, n = 6 for both). The addition of IFNλ1 during direct co-culture of macrophages and lung tumor organoids furthermore dampened the induction of the signal regulatory protein-a (SIRPa), a phagocytosis-inhibiting receptor, (p = 0.0041, n = 4) otherwise induced by the tumor organoids, but not matched healthy organoids. In support of this, there was an increased activation of CD8+ T-cells measured by IFNg production in an organoid-macrophage-CD8+ T-cell co-culture model. To summarize, we demonstrate that IFNλ1 may be a strong and broad marker of STING activation induced by chemotherapy in NSCLC and that IFNλ1 has the ability to prime a wider immune response by targeting macrophages. Hence, we propose that an IFNλ1 induced immune response has the potential to support and boost the response induced by existing immunotherapies. Citation Format: Kristine R Gammelgaard, Stine H Godsk, Albert G Olivier, Maria Riedel, Silke D Nielsen, Allard van Renterghem, Trine V Larsen, Anders Etzerodt, Emile Voest, Martin R Jakobsen. IFNλ1 is a marker of DNA damage-triggered STING-signaling in lung cancer that induces immune activation through macrophage stimulation [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A50.

OR2AT4 and OR1A2 counterregulate molecular pathophysiological processes of steroid-resistant inflammatory lung diseases in human alveolar macrophages

BackgroundTherapeutic options for steroid-resistant non-type 2 inflammation in obstructive lung diseases are lacking. Alveolar macrophages are central in the progression of these diseases by releasing proinflammatory cytokines, making them promising targets for new therapeutic approaches. Extra nasal expressed olfactory receptors (ORs) mediate various cellular processes, but clinical data are lacking. This work investigates whether ORs in human primary alveolar macrophages could impact pathophysiological processes and could be considered as therapeutic targets.MethodsHuman primary alveolar macrophages were isolated from bronchoalveolar lavages of 50 patients with pulmonary diseases. The expression of ORs was validated using RT-PCR, immunocytochemical staining, and Western blot. Changes in intracellular calcium levels were analyzed in real-time by calcium imaging. A luminescent assay was used to measure the cAMP concentration after OR stimulation. Cytokine secretion was measured in cell supernatants 24 h after stimulation by ELISA. Phagocytic ability was measured by the uptake of fluorescent-labeled beads by flow cytometry.ResultsWe demonstrated the expression of functional OR2AT4 and OR1A2 on mRNA and protein levels. Both ORs were primarily located in the plasma membrane. Stimulation with Sandalore, the ligand of OR2AT4, and Citronellal, the ligand of OR1A2, triggered a transient increase of intracellular calcium and cAMP. In the case of Sandalore, this calcium increase was based on a cAMP-dependent signaling pathway. Stimulation of alveolar macrophages with Sandalore and Citronellal reduced phagocytic capacity and release of proinflammatory cytokines.ConclusionThese are the first indications for utilizing olfactory receptors as therapeutic target molecules in treating steroid-resistant lung diseases with non-type 2 inflammation.

FABP5 Deficiency Impaired Macrophage Inflammation by Regulating AMPK/NF-κB Signaling Pathway.

Fatty acid binding protein 5 (FABP5) is mainly involved in the uptake, transport, and metabolism of fatty acid in the cytoplasm, and its role in immune cells has been recognized in recent years. However, the role of FABP5 in macrophage inflammation and its underlying mechanisms were not fully addressed. In our study, the acute liver injury and sepsis mouse models were induced by i.p. injection of LPS and cecal contents, respectively. Oleic acid (0.6 g/kg) was injected four times by intragastric administration every week, and this lasted for 1 wk before the LPS or cecal content challenge. We found that myeloid-specific deletion of FABP5 mitigated LPS-induced acute liver injury with reduced mortality of mice, histological liver damage, alanine aminotransferase, and proinflammatory factor levels. Metabolic analysis showed that FABP5 deletion increased the intracellular unsaturated fatty acids, especially oleic acid, in LPS-induced macrophages. The addition of oleic acid also decreased LPS-stimulated macrophage inflammation in vitro and reduced acute liver injury in LPS-induced or cecal content-induced sepsis mice. RNA-sequencing and molecular mechanism studies showed that FABP5 deletion or oleic acid supplementation increased the AMP/ATP ratio and AMP-activated protein kinase (AMPK) activation and inhibited the NF-κB pathway during the inflammatory response to LPS stimulation of macrophages. Inhibiting AMPK activation or expression by chemical or genetic approaches significantly rescued the decreased NF-κB signaling pathway and inflammatory response in LPS-treated FABP5-knockout macrophages. Our present study indicated that inhibiting FABP5 or supplementation of oleic acid might be used for the treatment of sepsis-caused acute liver injury.

Modulation of porcine alphacoronavirus pathogenesis by antibodies to SARS-CoV-2

BACKGROUND: A variety of antibodies against the SARS-CoV-2 with different functional activities can lead to immune pathology and/or modulation of infection due to antibody-dependent enhancement. An important role in the development of ADE, as exemplified by Dengue fever, is played by low-affinity antibodies, which interacting with the virus, do not neutralize it, but on the contrary forms the virus-antibody complex to immune cells (monocytes/macrophages, B-cells). There are conflicting data on the antibody-dependent enhancement presence in COVID-19 coronavirus infection. Some studies indicate a relationship between the severity of infection in patients infected by SARS-СoV-2 and the level of antibodies to closely related coronaviruses.&#x0D; AIM: The aim of the study was to understand the possibility of interaction between antibodies to SARS-CoV-2 with closely related porcine coronavirus (alphacoronavirus) and their ability to modulate the development of infection in vitro.&#x0D; MATERIALS AND METHODS: In the study serums and leukocytes from patients, who are convalescent after COVID-19 infection in 2020, was used. Antibody titers (IgG/IgM) was checked by commercial kit (VektorBest, Russia), based on ELISA method. In the eukaryotic expression system Chinese hamster ovary cells, monoclonal antibody to SARS-CoV-2 spike protein and its Fab version without Fc domain were obtained. As a model, swine alphacoronavirus- transmissible gastroenteritis virus (TGEV) was used. Replication activity of TGEV was studied in porcine leukocyte cell culture. The ability of this virusto induce the interferon gamma synthesis in leukocytes from convalescents was assessed TigraTest SARS-CoV-2 Enzyme-Linked SPOT analysis (Russia). Virus titration and neutralization assay with sera and monoclonal antibodies were carried out in porcine kidney PK-15 cell line.&#x0D; RESULTS: Serum samples from 43 donors (59% female, 41% male) aged from 21 to 56 was studied for the presence of class M and G antibodies to SARS-CoV-2 by ELISA method. Class M antibodies were found in only one donor. The highest titers of class G antibodies (1:400 to 1:1600) were detected among donors 4050 years old. For four donors with the highest antibody titers, HLA-II type was determined. The DQA1 allele in 3 patients was found to have the *0501 variant, which, according to literature data, is associated with autoimmune thyroid diseases. For the DQB1 allele, two convalescents had exactly the same variants (*0602-8). Among HLA DRB1, all patients had different allele variants (*09, *11, *01,*03,*13,*15,*08,*13).&#x0D; Among the samples, studied by ELISPOT method, in 45,5% of samples a positive T-cell response was noted after stimulation of macrophages with the S peptides of the SARS-CoV-2 protein and in 22.7% of samples in response to the pool of N peptides of the SARS-CoV-2 protein. Interestingly, infection of macrophages from convalescents with the transmissible gastroenteritis virus caused interferon-gamma expression in 31,8% of cases.&#x0D; The monoclonal antibody to the Spike protein SARS-CoV-2 and its Fab variant were not able to neutralize the TGEV. Interestingly, serum samples from 16 donors convalescent after COVID-19 caused virus neutralization in PK-15 cell culture at dilutions of 1:4-1:8.&#x0D; CONCLUSIONS: We have shown that porcine alphacoronavirus induces (in 31.8% of cases) the synthesis of interferon gamma in macrophages of recovered afterCOVID-19 donors, which may indicate cross-recognition of the antigen of a closely related coronavirus. However, monoclonal antibodies against the Spike protein SARS-CoV-2 did not demonstrate to TGEV neutralization. In turn, the neutralization of the TGEV by sera from recovered COVID-19 donors suggests that not only Spike protein, but also other coronavirus antigens can play a significant role in antigenic imprinting and antibody-dependent enhancement of COVID-19.

Antinociceptive effect of ultra-low dose naltrexone in a pre-clinical model of postoperative orofacial pain

Painmanagement after oral surgeries is essential to enhance recovery, reduce negative outcomes and improve the experience of the patient. Naltrexone (NTX) is a non-selective opioid receptor antagonist that has been shown to modulate neuro-inflammation when employed in low to ultra-low doses. In addition, ultra-low dose naltrexone (ULDN) has been shown to potentiate opioids’ analgesia and to have opioid-sparing effects. Herein it was investigated the effect of ULDN in a model of postoperative orofacial pain in rats, and it was tested the hypothesis that blockade of TLR4-signalling pathway contributes to its antinociceptive effect. Systemic NTX reduced heat hyperalgesia in female rats and heat and mechanical hyperalgesia in male rats after incision surgery. Combined treatment with NTX and morphine, both at ineffective doses, resulted in a significant reduction of heat hyperalgesia in male rats. NTX injection at the incision site failed to change heat hyperalgesia, but injection at the trigeminal ganglion (TG) or subnucleus caudalis (Sp5C) caused a significant reduction in heat hyperalgesia. At these sites, blockade of TLR4 impeded NTX effect. Lipopolysaccharide (LPS) injection in the intraoral mucosa resulted in facial heat hyperalgesia an increase in IL-1β levels in the TG, which were reduced by systemic NTX. Stimulation of macrophages with LPS resulted in increase of nitric oxide, IL-1β and CXCL-2 levels which were reduced by NTX. Altogether, these results provide evidence for an antinociceptive effect of ULDN in postoperative orofacial pain and suggest that blockade of TLR4 and downstream signaling pathway contribute to its effect.

Particulate matter promotes cancer metastasis through increased HBEGF expression in macrophages

Although many cohort studies have reported that long-term exposure to particulate matter (PM) can cause lung cancer, the molecular mechanisms underlying the PM-induced increase in cancer metastasis remain unclear. To determine whether PM contributes to cancer metastasis, cancer cells were cultured with conditioned medium from PM-treated THP1 cells, and the migration ability of the treated cancer cells was assessed. The key molecules involved were identified using RNA-seq analysis. In addition, metastatic ability was analyzed in vivo by injection of cancer cells into the tail vein and intratracheal injection of PM into the lungs of C57BL/6 mice. We found that PM enhances the expression of heparin-binding EGF-like growth factor (HBEGF) in macrophages, which induces epithelial-to-mesenchymal transition (EMT) in cancer cells, thereby increasing metastasis. Macrophage stimulation by PM results in activation and subsequent nuclear translocation of the aryl hydrocarbon receptor and upregulation of HBEGF. Secreted HBEGF activates EGFR on the cancer cell surface to induce EMT, resulting in increased migration and invasion in vitro and increased metastasis in vivo. Therefore, our study reveals a critical PM-macrophage-cancer cell signaling axis mediating EMT and metastasis and provides an effective therapeutic approach for PM-induced malignancy.

Effects of Oleacein, a New Epinutraceutical Bioproduct from Extra Virgin Olive Oil, in LPS-Activated Murine Immune Cells.

The present study was designed to evaluate the immunomodulatory effects of the secoiridoid from extra virgin olive oil, oleacein (OLA), deepening into the possible signaling pathways involved in LPS-activated murine peritoneal macrophages. Moreover, we have explored OLA-induced epigenetic changes in histone markers and related cytokine production in murine LPS-stimulated murine splenocytes. Murine cells were treated with OLA in the presence or absence of LPS (5 μg/mL) for 18 or 24 h. OLA modulated the oxidative stress and the inflammatory response produced by LPS stimulation in murine peritoneal macrophages, by the inhibition of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, IFN-γ, IL-17 and IL-18) and ROS production and the expression of pro-inflammatory enzymes such as iNOS, COX-2 and m-PGES1. These protective effects could be due to the activation of the Nrf-2/HO-1 axis and the inhibition of JAK/STAT, ERK and P38 MAPKs and inflammasome canonical and non-canonical signaling pathways. Moreover, OLA modulated epigenetic modifications throughout histone methylation deacetylation (H3K18ac) and (H3K9me3 and H3K27me) in LPS-activated spleen cells. In conclusion, our data present OLA as an interesting anti-inflammatory and antioxidant natural compound that is able to regulate histone epigenetic markers. Nevertheless, additional in vivo studies are required to further investigate the beneficial effects of this EVOO secoiridoid, which might be a promising epinutraceutical bioproduct for the management of immune-related inflammatory diseases.

The Impact of Sphingosine Kinases on Inflammation-Induced Cytokine Release and Vascular Endothelial Barrier Integrity.

Sphingosine kinases type 1 and 2 (SphK1/2) are required for the production of the immune modulator sphingosine 1-phosphate (S1P). SphK1 deficient mice (SphK1-/-) revealed 50% reduced S1P in plasma, while SphK2-/- mice demonstrated 2-3 times increased S1P levels in plasma. Since plasma S1P is a potent inducer of vascular endothelial cell (VEC) barrier stability, we hypothesized that higher and lower levels of S1P in SphK2-/- and SphK1-/- mice, respectively, compared to wild type (wt) mice should translate into decreased and increased severity of induced systemic inflammation due to improved or damaged VEC barrier maintenance. To our surprise, both SphK1-/- and SphK2-/- mice showed improved survival rate and earlier recovery from inflammation-induced weight loss compared to wt mice. While no difference was observed in VEC barrier stability by monitoring Evans blue leakage into peripheral tissues, SphK1-/- mice demonstrated a distinct delay and SphK2-/- mice an improved resolution of early pro-inflammatory cytokine release in plasma. Ex vivo cell culture experiments demonstrated that bone marrow-derived dendritic cells (BMDC) generated from SphK1-/- and SphK2-/- mice responded with decreased interferon-γ (IFN-γ) production upon stimulation with lipopolysaccharides (LPS) compared to wt BMDC, while activation-induced cytokine expression of lymphocytes and macrophages was not majorly altered. Ex vivo stimulation of macrophages with IFN-γ resulted in increased cytokine release. These results suggest that SphK1/2 are involved in production and secretion of IFN-γ by DC. DC-derived IFN-γ subsequently stimulates the production and secretion of a large panel of inflammatory cytokines by macrophages, which belong to the main cytokine-releasing cells of the early innate immune response. Inhibitors of SphK1/2 may therefore be attractive targets to dampen the early cytokine response of macrophages as part of the innate immune response.

Lacticaseibacillus casei Strain Shirota Modulates Macrophage-Intestinal Epithelial Cell Co-Culture Barrier Integrity, Bacterial Sensing and Inflammatory Cytokines.

Probiotic bacteria modulate macrophage immune inflammatory responses, with functional cytokine responses determined by macrophage subset polarisation, stimulation and probiotic strain. Mucosal macrophages exhibit subset functional heterogeneity but are organised in a 3-dimensional tissue, over-laid by barrier epithelial cells. This study aimed to investigate the effects of the probiotic Lacticaseibacillus casei strain Shirota (LcS) on macrophage-epithelial cell cytokine responses, pattern recognition receptor (PRR) expression and LPS responses and the impacts on barrier integrity. THP-1-derived M1 and M2 subset macrophages were co-cultured in a transwell system with differentiated Caco-2 epithelial cells in the presence or absence of enteropathogenic LPS. Both Caco-2 cells in monoculture and macrophage co-culture were assayed for cytokines, PRR expression and barrier integrity (TEER and ZO-1) by RT-PCR, ELISA, IHC and electrical resistance. Caco-2 monocultures expressed distinct cytokine profiles (IL-6, IL-8, TNFα, endogenous IL-10), PRRs and barrier integrity, determined by inflammatory context (TNFα or IL-1β). In co-culture, LcS rescued ZO-1 and TEER in M2/Caco-2, but not M1/Caco-2. LcS suppressed TLR2, TLR4, MD2 expression in both co-cultures and differentially regulated NOD2, TLR9, Tollip and cytokine secretion. In conclusion, LcS selectively modulates epithelial barrier integrity, pathogen sensing and inflammatory cytokine profile; determined by macrophage subset and activation status.