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Atlantic wakame (AW) and sea lettuce (SL) are edible seaweeds commonly produced in Asia and Europe, typically consumed as fresh or dried biomass. Because of the short shelf life, developing new post-harvest treatments, such as the extraction of bioactive compounds, is essential for establishing reliable macroalgae supply chains for food applications. This study aims to develop a sustainable and functional seaweed-based food ingredient rich in bioactive compounds for functional food applications. The results indicated that ultrasound-assisted extraction using 70% ethanol for 10 min was optimal for both SL (410±23 mg of gallic acid equivalent [GAE] per kg) and AW (666±63 mg GAE/kg). Both extracts exhibited chemical antioxidant activity and inhibited the protection of human plasma against chemically induced oxidation. SL extract did not exhibit free radical scavenging activity (DPPH). Importantly, no cytotoxicity (IC50>250 µg/mL) was observed in human-derived THP-1, HUVEC, HT-29, and HepG2 cell lines, ensuring the relative safety of these extracts for potential food technology applications. AW and SL extracts (100 µg/mL) reduced intracellular reactive oxygen species generation in lipopolysaccharide (LPS)-challenged THP-1 cells by 66% and 30%, respectively, highlighting the higher antioxidant activity of AW extract. AW and SL extracts did not modulate TNF-α and IL-6 secretion to show anti-inflammatory activity. Gummies infused with AW and SL extracts had a higher total phenolic content than commercial products, but their antioxidant activity was lower. Sensory evaluation revealed that gummies containing seaweed extracts received lower scores for colour, taste, overall impression, and purchase intention than those without extracts.

ABSTRACT Mycobacterium tuberculosis (Mtb) as well as nontuberculous mycobacteria are intracellular pathogens whose treatment is extensive and increasingly impaired due to the rise of mycobacterial drug resistance. The loss of antibiotic efficacy has raised interest in the identification of host-directed therapeutics (HDT) to develop novel treatment strategies for mycobacterial infections. In this study, we identified amiodarone as a potential HDT candidate that inhibited both intracellular Mtb and Mycobacterium avium in primary human macrophages without directly impairing bacterial growth, thereby confirming that amiodarone acts in a host-mediated manner. Moreover, amiodarone induced the formation of (auto)phagosomes and enhanced autophagic targeting of mycobacteria in macrophages. The induction of autophagy by amiodarone is likely due to enhanced transcriptional regulation, as the nuclear intensity of the transcription factor EB, the master regulator of autophagy and lysosomal biogenesis, was strongly increased. Furthermore, blocking lysosomal degradation with bafilomycin impaired the host-beneficial effect of amiodarone. Finally, amiodarone induced autophagy and reduced bacterial burden in a zebrafish embryo model of tuberculosis, thereby confirming the HDT activity of amiodarone in vivo. In conclusion, we have identified amiodarone as an autophagy-inducing antimycobacterial HDT that improves host control of mycobacterial infections.IMPORTANCEDue to the global rise in antibiotic resistance, there is a strong need for alternative treatment strategies against intracellular bacterial infections, including Mycobacterium tuberculosis (Mtb) and non-tuberculous mycobacteria. Stimulating host defense mechanisms by host‐directed therapy (HDT) is a promising approach for treating mycobacterial infections. This study identified amiodarone, an antiarrhythmic agent, as a potential HDT candidate that inhibits the survival of Mtb and Mycobacterium avium in primary human macrophages. The antimycobacterial effect of amiodarone was confirmed in an in vivo tuberculosis model based on Mycobacterium marinum infection of zebrafish embryos. Furthermore, amiodarone induced autophagy and inhibition of the autophagic flux effectively impaired the host-protective effect of amiodarone, supporting that activation of the host (auto)phagolysosomal pathway is essential for the mechanism of action of amiodarone. In conclusion, we have identified amiodarone as an autophagy-inducing HDT that improves host control of a wide range of mycobacteria.

The NLRP3 inflammasome is an intracellular multiprotein complex described to be involved in both an effective host response to infectious agents and various diseases. Investigation into the NLRP3 inflammasome has been extensive in the past two decades, and often revolves around the analysis of a few specific readouts, including ASC-speck formation, caspase-1 cleavage or activation, and cleavage and release of IL-1β and/or IL-18. Quantification of these readouts is commonly undertaken as an endpoint analysis, where the presence of each positive outcome is assessed independently of the others. In this study, we apply time-resolved analysis of a human macrophage model (differentiated THP-1-ASC-GFP cells) to commonly accessible methods. This approach yields the additional quantifiable metrics time-resolved absolute change and acceleration, allowing comparisons between readouts. Using this methodological approach, we reveal (potential) discrepancies between inflammasome-related readouts that otherwise might go undiscovered. The study highlights the importance of time-resolved data in general and may be further extended as well as incorporated into other areas of research.

Abstract Background The purinergic receptor P2X7 plays a crucial role in infection, inflammation, and cell death. It is thought that P2X7 receptor stimulation triggers processing and release of the pro-inflammatory cytokine interleukin (IL)-1β by activation of the NLRP3 inflammasome; however, the underlying mechanisms remain poorly understood. Methods Modulation of IL-1β secretion was studied in THP-1 macrophages. Adenosine 5’-triphosphate (ATP), BzATP, nigericin and pharmacological inhibitors of P2X receptors, inflammatory caspases and the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome were used to characterize signaling. Results In primed macrophages, IL-1β release was increased after P2X7 receptor activation by ATP and 2,3-O-(4-benzoylbenzoyl)-ATP (BzATP). Pharmacological inhibition or genetic knockout of NLRP3 does not completely inhibit IL-1β release in TLR2/1-primed macrophages. Increase in extracellular K+ as well as inhibition of caspase-1 or serine proteases maintained IL-1β release in macrophages stimulated with P2X7 receptor agonists at 50%. Conclusions Our findings suggest a previously unrecognized mechanism of P2X7 receptor mediated IL-1β release and highlight the existence of an NLRP3-independent pathway in human macrophages. Video Abstract

Metabolic fluxes are at the heart of metabolism and growth in any living system. During tuberculosis (TB) infection, the pathogenic Mycobacterium tuberculosis (Mtb) adapts its nutritional behaviour and metabolic fluxes to survive in human macrophages and cause infection. The infected host cells also undergo metabolic changes. However, our knowledge of the infected host metabolism and identification of the reprogrammed metabolic flux nodes remains limited. In this study, we applied systems-based 13C-metabolic flux analysis (MFA) to measure intracellular carbon metabolic fluxes in Mtb-infected human THP-1 macrophages. We provide a flux map for infected macrophages that quantified significantly increased fluxes through glycolytic fluxes towards pyruvate synthesis and reduced pentose phosphate pathway fluxes when compared to uninfected macrophages. The tri carboxylic acid (TCA) cycle fluxes were relatively low, and amino acid fluxes were reprogrammed upon Mtb infection. The knowledge of host metabolic flux profiles derived from our work expands on how the host cell adapts its carbon metabolism in response to Mtb infection and highlights important nodes that may provide targets for developing new therapeutics to improve TB treatment. [ABSTRACT FROM AUTHOR]

Background: The purinergic receptor P2X7 plays a crucial role in infection, inflammation, and cell death. It is thought that P2X7 receptor stimulation triggers processing and release of the pro-inflammatory cytokine interleukin (IL)-1β by activation of the NLRP3 inflammasome; however, the underlying mechanisms remain poorly understood. Methods: Modulation of IL-1β secretion was studied in THP-1 macrophages. Adenosine 5'-triphosphate (ATP), BzATP, nigericin and pharmacological inhibitors of P2X receptors, inflammatory caspases and the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome were used to characterize signaling. Results: In primed macrophages, IL-1β release was increased after P2X7 receptor activation by ATP and 2,3-O-(4-benzoylbenzoyl)-ATP (BzATP). Pharmacological inhibition or genetic knockout of NLRP3 does not completely inhibit IL-1β release in TLR2/1-primed macrophages. Increase in extracellular K+ as well as inhibition of caspase-1 or serine proteases maintained IL-1β release in macrophages stimulated with P2X7 receptor agonists at 50%. Conclusions: Our findings suggest a previously unrecognized mechanism of P2X7 receptor mediated IL-1β release and highlight the existence of an NLRP3-independent pathway in human macrophages. 4h-1gv1RJyWYx97JbdiPpX Video Abstract [ABSTRACT FROM AUTHOR]

Metabolic fluxes are at the heart of metabolism and growth in any living system. During tuberculosis (TB) infection, the pathogenic Mycobacterium tuberculosis (Mtb) adapts its nutritional behaviour and metabolic fluxes to survive in human macrophages and cause infection. The infected host cells also undergo metabolic changes. However, our knowledge of the infected host metabolism and identification of the reprogrammed metabolic flux nodes remains limited. In this study, we applied systems-based 13C-metabolic flux analysis (MFA) to measure intracellular carbon metabolic fluxes in Mtb-infected human THP-1 macrophages. We provide a flux map for infected macrophages that quantified significantly increased fluxes through glycolytic fluxes towards pyruvate synthesis and reduced pentose phosphate pathway fluxes when compared to uninfected macrophages. The tri carboxylic acid (TCA) cycle fluxes were relatively low, and amino acid fluxes were reprogrammed upon Mtb infection. The knowledge of host metabolic flux profiles derived from our work expands on how the host cell adapts its carbon metabolism in response to Mtb infection and highlights important nodes that may provide targets for developing new therapeutics to improve TB treatment.

Human macrophages are innate immune cells with diverse, functionally distinct phenotypes, namely, pro‐inflammatory M1 and anti‐inflammatory M2 macrophages. Both are involved in multiple physiological and pathological processes, including would healing, infection, and cancer. However, the metabolic differences between these phenotypes are largely unexplored at single‐cell resolution. To address this knowledge gap, an untargeted live single‐cell mass spectrometry‐based metabolomic profiling coupled with a machine‐learning data analysis approach was developed to investigate the metabolic profile of each phenotype at the single‐cell level. Results show that M1 and M2 macrophages have distinct metabolic profiles, with differential levels of fatty acyls, glycerophospholipids, and sterol lipids, which are important components of plasma membrane and involved in multiple biological processes. Furthermore, we could discern several putatively annotated molecules that contribute to inflammatory response of macrophages. The combination of random forest and live single‐cell metabolomics provided an in‐depth profile of the metabolome of primary human M1 and M2 macrophages at the single‐cell level for the first time, which will pave the way for future studies targeting the differentiation of other immune cells. [ABSTRACT FROM AUTHOR]

Ambient PM2.5 was collected during the winter season from Taiyuan, Shanxi, China; Jinan, Shandong, China; and Sacramento, California, USA, and used to create PMSX, PMSD, and PMCA extracts, respectively. Time-lag experiments were performed to explore the in vivo and in vitro toxicity of the PM extracts. In vivo inflammatory lung responses were assessed in BALB/c mice using a single oropharyngeal aspiration (OPA) of PM extract or vehicle (CTRL) on Day 0. Necropsies were performed on Days 1, 2, and 4 post-OPA, and pulmonary effects were determined using bronchoalveolar lavage (BAL) and histopathology. On Day 1, BAL neutrophils were significantly elevated in all PM- versus CTRL-exposed mice, with PMCA producing the strongest response. However, histopathological scoring showed greater alveolar and perivascular effects in PMSX-exposed mice compared to all three other groups. By Day 4, BAL neutrophilia and tissue inflammation were resolved, similar across all groups. In vitro effects were examined in human HepG2 hepatocytes, and U937 cells following 6, 24, or 48 h of exposure to PM extract or DMSO (control). Luciferase reporter and quantitative polymerase chain reaction assays were used to determine in vitro effects on aryl hydrocarbon receptor (AhR) activation and gene transcription, respectively. Though all three PM extracts activated AhR, PMSX produced the greatest increases in AhR activation, and mRNA levels of cyclooxygenase-2, cytochrome P450, interleukin (IL)-8, and interleukin (IL)-1β. These effects were assumed to result from a greater abundance of polycyclic aromatic hydrocarbons (PAHs) in PMSX compared to PMSD and PMCA.

Macrophage responses following the implantation of orthopaedic implants are essential for successful implant integration in the body, partly through intimate crosstalk with human marrow stromal cells (hMSCs) in the process of new bone formation. Additive manufacturing (AM) and plasma electrolytic oxidation (PEO) in the presence of silver nanoparticles (AgNPs) are promising techniques to achieve multifunctional titanium implants. Their osteoimmunomodulatory properties are, however, not yet fully investigated. Here, we studied the effects of implants with AgNPs on human macrophages and the crosstalk between hMSCs and human macrophages when co-cultured in vitro with biofunctionalised AM Ti6Al4V implants. A concentration of 0.3 g/L AgNPs in the PEO electrolyte was found to be optimal for both macrophage viability and inhibition of bacteria growth. These specimens also caused a decrease of the macrophage tissue repair related factor C-C Motif Chemokine Ligand 18 (CCL18). Nevertheless, co-cultured hMSCs could osteogenically differentiate without any adverse effects caused by the presence of macrophages that were previously exposed to the PEO (±AgNPs) surfaces. Further evaluation of these promising implants in a bony in vivo environment with and without infection is highly recommended to prove their potential for clinical use.

Macrophages are the preeminent phagocytic cells which control multiple infections. Tuberculosis a leading cause of death in mankind and the causative organism Mycobacterium tuberculosis (MTB) infects and persists in macrophages. Macrophages use reactive oxygen and nitrogen species (ROS/RNS) and autophagy to kill and degrade microbes including MTB. Glucose metabolism regulates the macrophage-mediated antimicrobial mechanisms. Whereas glucose is essential for the growth of cells in immune cells, glucose metabolism and its downsteam metabolic pathways generate key mediators which are essential co-substrates for post-translational modifications of histone proteins, which in turn, epigenetically regulate gene expression. Herein, we describe the role of sirtuins which are NAD+-dependent histone histone/protein deacetylases during the epigenetic regulation of autophagy, the production of ROS/RNS, acetyl-CoA, NAD+, and Sadenosine methionine (SAM), and illustrate the cross-talk between immunometabolism and epigenetics on macrophage activation. We highlight sirtuins as emerging therapeutic targets for modifying immunometabolism to alter macrophage phenotype and antimicrobial function. [ABSTRACT FROM AUTHOR]

In the pathogenesis of influenza virus infection, lymphocyte apoptosis as a part of the infection and/or the immune response to the virus can be somewhat puzzling. The percentage of human T lymphocytes within the peripheral blood mononuclear cell population that becomes apoptotic greatly exceeds the percentage that are infected after exposure to the virus, consistent with substantial apoptosis of bystander T lymphocytes. Studies reveal an important role of viral neuraminidase expression by co-cultured monocyte/macrophages in induction of apoptosis, including that of uninfected bystander lymphocytes. Despite these observations, it is a reasonable perspective to recognize that the development of lymphocyte apoptosis during the response to infection does not preclude a successful immune response and recovery of the infected host in the great majority of cases. Further investigation is clearly warranted to understand its role in the pathogenesis of influenza virus infection for human subjects. [ABSTRACT FROM AUTHOR]

Oxylipins derived from the cyclooxygenase (COX) and lipoxygenase (LOX) pathways of the arachidonic acid (ARA) cascade are essential for the regulation of the inflammatory response and many other physiological functions. Comprehensive analytical methods comprised of oxylipin and protein abundance analysis are required to fully understand mechanisms leading to changes within these pathways. Here, we describe the development of a quantitative multi-omics approach combining liquid chromatography tandem mass spectrometry–based targeted oxylipin metabolomics and proteomics. As the first targeted proteomics method to cover these pathways, it enables the quantitative analysis of all human COX (COX-1 and COX-2) and relevant LOX pathway enzymes (5-LOX, 12-LOX, 15-LOX, 15-LOX-2, and FLAP) in parallel to the analysis of 239 oxylipins with our targeted oxylipin metabolomics method from a single sample. The detailed comparison between MRM3 and classical MRM-based detection in proteomics showed increased selectivity for MRM3, while MRM performed better in terms of sensitivity (LLOQ, 16–122 pM vs. 75–840 pM for the same peptides), linear range (up to 1.5–7.4 μM vs. 4–368 nM), and multiplexing capacities. Thus, the MRM mode was more favorable for this pathway analysis. With this sensitive multi-omics approach, we comprehensively characterized oxylipin and protein patterns in the human monocytic cell line THP-1 and differently polarized primary macrophages. Finally, the quantification of changes in protein and oxylipin levels induced by lipopolysaccharide stimulation and pharmaceutical treatment demonstrates its usefulness to study molecular modes of action involved in the modulation of the ARA cascade. [ABSTRACT FROM AUTHOR]

Silver nanoparticles (AgNP) are the most widely produced type of nanoparticles due to their antimicrobial and preservative properties. However, their systemic bioavailability may be considered a potential hazard. When AgNP reach the bloodstream, they interact with the immune cells, contributing to the onset and development of an inflammatory response. Monocytes and macrophages play a pivotal role in our defense system, but the interaction of AgNP with these cells is still not clear. Therefore, the main objective of this work was to assess the cytotoxic and pro-inflammatory effects induced by 5, 10, and 50 nm AgNP coated with polyvinylpyrrolidone (PVP) and citrate, in concentrations that could be attained in vivo (0–25 μg/mL), in human monocytes isolated from human blood and human macrophages derived from a monocytic cell line (THP-1). The effects of PVP and citrate-coated AgNP on cell viability, mitochondrial membrane potential, and cytokines release were evaluated. The results evidenced that AgNP exert strong harmful effects in both monocytes and macrophages, through the establishment of a strong pro-inflammatory response that culminates in cell death. The observed effects were dependent on the AgNP concentration, size and coating, being observed more pronounced cytotoxic effects with smaller PVP coated AgNP. The results showed that human monocytes seem to be more sensitive to AgNP exposure than human macrophages. Considering the increased daily use of AgNP, it is imperative to further explore the adverse outcomes and mechanistic pathways leading to AgNP-induced pro-inflammatory effects to deep insight into the molecular mechanism involved in this effect. [ABSTRACT FROM AUTHOR]

ABSTRACT The global burden of tuberculosis (TB) is aggravated by the continuously increasing emergence of drug resistance, highlighting the need for innovative therapeutic options. The concept of host-directed therapy (HDT) as adjunctive to classical antibacterial therapy with antibiotics represents a novel and promising approach for treating TB. Here, we have focused on repurposing the clinically used anticancer drug tamoxifen, which was identified as a molecule with strong host-directed activity against intracellular Mycobacterium tuberculosis (Mtb). Using a primary human macrophage Mtb infection model, we demonstrate the potential of tamoxifen against drug-sensitive as well as drug-resistant Mtb bacteria. The therapeutic effect of tamoxifen was confirmed in an in vivo TB model based on Mycobacterium marinum infection of zebrafish larvae. Tamoxifen had no direct antimicrobial effects at the concentrations used, confirming that tamoxifen acted as an HDT drug. Furthermore, we demonstrate that the antimycobacterial effect of tamoxifen is independent of its well-known target the estrogen receptor (ER) pathway, but instead acts by modulating autophagy, in particular the lysosomal pathway. Through RNA sequencing and microscopic colocalization studies, we show that tamoxifen stimulates lysosomal activation and increases the localization of mycobacteria in lysosomes both in vitro and in vivo, while inhibition of lysosomal activity during tamoxifen treatment partly restores mycobacterial survival. Thus, our work highlights the HDT potential of tamoxifen and proposes it as a repurposed molecule for the treatment of TB. IMPORTANCE Tuberculosis (TB) is the world's most lethal infectious disease caused by a bacterial pathogen, Mycobacterium tuberculosis. This pathogen evades the immune defenses of its host and grows intracellularly in immune cells, particularly inside macrophages. There is an urgent need for novel therapeutic strategies because treatment of TB patients is increasingly complicated by rising antibiotic resistance. In this study, we explored a breast cancer drug, tamoxifen, as a potential anti-TB drug. We show that tamoxifen acts as a so-called host-directed therapeutic, which means that it does not act directly on the bacteria but helps the host macrophages combat the infection more effectively. We confirmed the antimycobacterial effect of tamoxifen in a zebrafish model for TB and showed that it functions by promoting the delivery of mycobacteria to digestive organelles, the lysosomes. These results support the high potential of tamoxifen to be repurposed to fight antibiotic-resistant TB infections by host-directed therapy.

Acute respiratory distress syndrome (ARDS) is characterized by severe hypoxemia and high-permeability pulmonary edema. A hallmark of the disease is the presence of lung inflammation with features of diffuse alveolar damage. The molecular pathogenetic mechanisms of COVID-19-associated ARDS (CARDS), secondary to SARS-CoV-2 infection, are still not fully understood. Here, we investigate the effects of a cytokine-enriched conditioned medium from Spike S1-activated macrophage on alveolar epithelial A549 cells in terms of cell proliferation, induction of autophagy, and expression of genes related to protein degradation. The protective effect of baricitinib, employed as an inhibitor of JAK-STAT, has been also tested. The results obtained indicate that A549 exhibits profound changes in cell morphology associated to a proliferative arrest in the G0/G1 phase. Other alterations occur, such as a blockade of protein synthesis and the activation of autophagy, along with an increase of the intracellular amino acids content, which is likely ascribable to the activation of protein degradation. These changes correlate to the induction of IFN-regulatory factor 1 (IRF-1) due to an increased secretion of IFN-γ in the conditioned medium from S1-activated macrophages. The addition of baricitinib prevents the observed effects. In conclusion, our findings suggest that the IFN-γ-IRF-1 signaling pathway may play a role in the alveolar epithelial damage observed in COVID-19-related ARDS. [ABSTRACT FROM AUTHOR]

Background: The Janus kinase (JAK) 1/2 inhibitor ruxolitinib has been approved in an indication of myelofibrosis and is a candidate for the treatment of a number of inflammatory or autoimmune diseases. We assessed the effects of ruxolitinib on lipopolysaccharide (LPS)- and poly (I:C)-induced cytokine production by human lung macrophages (LMs) and on the LMs’ phagocytic activity.Methods: Human LMs were isolated from patients operated on for lung carcinoma. The LMs were cultured with ruxolitinib (0.5 × 10−7 M to 10–5 M) or budesonide (10–11 to 10–8 M) and then stimulated with LPS (10 ng·ml−1) or poly (I:C) (10 μg·ml−1) for 24 h. Cytokines released by the LMs into the supernatants were measured using ELISAs. The phagocytosis of labelled bioparticles was assessed using flow cytometry. Results: Ruxolitinib inhibited both the LPS- and poly (I:C)-stimulated production of tumor necrosis factor alpha, interleukin (IL)-6, IL-10, chemokines CCL2, and CXCL10 in a concentration-dependent manner. Ruxolitinib also inhibited the poly (I:C)- induced (but not the LPS-induced) production of IL-1ß. Budesonide inhibited cytokine production more strongly than ruxolitinib but failed to mitigate the production of CXCL10. The LMs’ phagocytic activity was not impaired by the highest tested concentration (10–5 M) of ruxolitinib. Conclusion: Clinically relevant concentrations of ruxolitinib inhibited the LPS- and poly (I:C)-stimulated production of cytokines by human LMs but did not impair their phagocytic activity. Overall, ruxolitinib’s anti-inflammatory activities are less intense than (but somewhat different from) those of budesonide—particularly with regard to the production of the corticosteroid-resistant chemokine CXCL-10. Our results indicate that treatment with a JAK inhibitor might be a valuable anti-inflammatory strategy in chronic obstructive pulmonary disease, Th1-high asthma, and both viral and non-viral acute respiratory distress syndromes (including coronavirus disease 2019). [ABSTRACT FROM AUTHOR]

Introduction: The inflammatory potential of SARS-CoV-2 Spike S1 (Spike) has never been tested in human primary macrophages (MΦ). Different recombinant Spikes might display different effects in vitro, according to protein length and glycosylation, and endotoxin (lipopolysaccharide, LPS) contamination. Objectives: To assess (1) the effects of different Spikes on human primary MΦ inflammation; (2) whether LPS contamination of recombinant Spike is (con)cause in vitro of increased MΦ inflammation. Methods: Human primary MΦ were incubated in the presence/absence of several different Spikes (10 nM) or graded concentrations of LPS. Pro-inflammatory marker expression (qPCR and ELISA) and supernatant endotoxin contamination (LAL test) were the main readouts. Results: LPS-free, glycosylated Spike (the form expressed in infected humans) caused no inflammation in human primary MΦ. Two (out of five) Spikes were contaminated with endotoxins ≥ 3 EU/ml and triggered inflammation. A non-contaminated non-glycosylated Spike produced in E. coli induced MΦ inflammation. Conclusions: Glycosylated Spike per se is not pro-inflammatory for human MΦ, a feature which may be crucial to evade the host innate immunity. In vitro studies with commercially available Spike should be conducted with excruciating attention to potential LPS contamination. [ABSTRACT FROM AUTHOR]

Macrophages serve an active role in the pathophysiology of chronic obstructive pulmonary disease (COPD). Erythromycin (EM) has been verified as an effective treatment for COPD. However, there are few studies on the effect of EM on the ultrastructure of macrophages exposed to cigarette smoke extract (CSE). In the present study, human macrophages were randomly divided into three groups: The control, CSE and the CSE+EM group, using electron microscopy, the effect of EM was evaluated by comparing the ultrastructural changes between these groups. The macrophages were additionally divided into a further four groups: The control, CSE, CSE+EM 24 h and CSE+EM 48 h groups. The generation of reactive oxygen species (ROS) in each group was evaluated by detecting fluorescence intensity. It was observed that the cellular ultrastructure of the CSE group exhibited abnormal changes, though this effect was reversed back to the level of the control in the CSE+EM group. Compared with the control group, the ROS expression level was significantly increased in the CSE group (P <.05); however, compared with the CSE group, the ROS concentration was decreased in the CSE+EM 24 h (P <.05) and CSE+EM 48 h groups (P <.05), though this was more apparent in the EM 48 h group. It was concluded that EM protects human macrophages against CSE. Moreover, it was hypothesized that EM may reduce the symptoms of patients with COPD by protecting the macrophage ultrastructure from the effects of CSE, resulting in the decreased generation of ROS, inhibiting autophagy and reducing endoplasmic reticulum stress. [ABSTRACT FROM AUTHOR]

Toll-like receptor 8 (TLR8) recognizes single-stranded RNA of viral and bacterial origin as well as mediates the secretion of pro-inflammatory cytokines and type I interferons by human monocytes and macrophages. TLR8, as other endosomal TLRs, utilizes the MyD88 adaptor protein for initiation of signaling from endosomes. Here, we addressed the potential role of the Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) in the regulation of TLR8 signaling in human primary monocyte-derived macrophages (MDMs). To accomplish this, we performed TIRAP gene silencing, followed by the stimulation of cells with synthetic ligands or live bacteria. Cytokine-gene expression and secretion were analyzed by quantitative PCR or Bioplex assays, respectively, while nuclear translocation of transcription factors was addressed by immunofluorescence and imaging, as well as by cell fractionation and immunoblotting. Immunoprecipitation and Akt inhibitors were also used to dissect the signaling mechanisms. Overall, we show that TIRAP is recruited to the TLR8 Myddosome signaling complex, where TIRAP contributes to Akt-kinase activation and the nuclear translocation of interferon regulatory factor 5 (IRF5). Recruitment of TIRAP to the TLR8 signaling complex promotes the expression and secretion of the IRF5-dependent cytokines IFNβ and IL-12p70 as well as, to a lesser degree, TNF. These findings reveal a new and unconventional role of TIRAP in innate immune defense. [ABSTRACT FROM AUTHOR]

Background: The Janus kinase (JAK) 1/2 inhibitor ruxolitinib has been approved in an indication of myelofibrosis and is a candidate for the treatment of a number of inflammatory or autoimmune diseases. We assessed the effects of ruxolitinib on lipopolysaccharide (LPS)- and poly (I:C)-induced cytokine production by human lung macrophages (LMs) and on the LMs’ phagocytic activity.Methods: Human LMs were isolated from patients operated on for lung carcinoma. The LMs were cultured with ruxolitinib (0.5 × 10−7 M to 10–5 M) or budesonide (10–11 to 10–8 M) and then stimulated with LPS (10 ng·ml−1) or poly (I:C) (10 μg·ml−1) for 24 h. Cytokines released by the LMs into the supernatants were measured using ELISAs. The phagocytosis of labelled bioparticles was assessed using flow cytometry.Results: Ruxolitinib inhibited both the LPS- and poly (I:C)-stimulated production of tumor necrosis factor alpha, interleukin (IL)-6, IL-10, chemokines CCL2, and CXCL10 in a concentration-dependent manner. Ruxolitinib also inhibited the poly (I:C)- induced (but not the LPS-induced) production of IL-1ß. Budesonide inhibited cytokine production more strongly than ruxolitinib but failed to mitigate the production of CXCL10. The LMs’ phagocytic activity was not impaired by the highest tested concentration (10–5 M) of ruxolitinib.Conclusion: Clinically relevant concentrations of ruxolitinib inhibited the LPS- and poly (I:C)-stimulated production of cytokines by human LMs but did not impair their phagocytic activity. Overall, ruxolitinib’s anti-inflammatory activities are less intense than (but somewhat different from) those of budesonide—particularly with regard to the production of the corticosteroid-resistant chemokine CXCL-10. Our results indicate that treatment with a JAK inhibitor might be a valuable anti-inflammatory strategy in chronic obstructive pulmonary disease, Th1-high asthma, and both viral and non-viral acute respiratory distress syndromes (including coronavirus disease 2019).

Toll-like receptor 4 (TLR4)-mediated changes in macrophages reshape intracellular lipid pools to coordinate an effective innate immune response. Although this has been previously well-studied in different model systems, it remains incompletely understood in primary human macrophages. Here we report time-dependent lipidomic and transcriptomic responses to lipopolysaccharide (LPS) in primary human macrophages from healthy donors. We grouped the variation of ~200 individual lipid species measured by LC-MS/MS into eight temporal clusters. Among all other lipids, glycosphingolipids (glycoSP) and cholesteryl esters (CE) showed a sharp increase during the resolution phase (between 8h or 16h post LPS). GlycoSP, belonging to the globoside family (Gb3 and Gb4), showed the greatest inter-individual variability among all lipids quantified. Integrative network analysis between GlycoSP/CE levels and genome-wide transcripts, identified Gb4 d18:1/16:0 and CE 20:4 association with subnetworks enriched for T cell receptor signaling (PDCD1 , CD86 , PTPRC , CD247 , IFNG) and DC-SIGN signaling (RAF1 , CD209), respectively. Our findings reveal Gb3 and Gb4 globosides as sphingolipids associated with the resolution phase of inflammatory response in human macrophages. [ABSTRACT FROM AUTHOR]

To increase our understanding of factors contributing to therapeutic failure (TF) in leishmaniasis, we have studied some plasma membrane features of host THP-1 cells infected with clinical isolates of Leishmania infantum from patients with leishmaniasis and TF. The fluorescent probes DPH and TMA-DPH were used to measure changes in membrane fluidity at various depths of the plasma membranes. Steady-state fluorescence anisotropy of DPH embedded in the infected THP-1 membranes showed a significant increase, thereby suggesting a substantial decrease in plasma membrane fluidity relative to controls. Considering that cholesterol affects membrane fluidity and permeability, we determined the cholesterol content in plasma membrane fractions of human macrophages infected with these L. infantum lines and observed a significant increase in cholesterol content that correlates with the measured decrease in plasma membrane fluidity. In order to define the pathways that could explain the increase in cholesterol content, we studied the transcriptomics of the cholesterol-enriched pathways in host THP-1 cells infected with TF clinical isolates by RNA-seq. Specifically, we focused on four enriched Gene Ontology (GO) terms namely cholesterol efflux, cholesterol transport, cholesterol metabolic process and cholesterol storage. Additionally, we analyzed the genes involved in these pathways. Overall, this study shows that these clinical isolates are able to modulate the expression of specific genes in host cells, thereby modifying the cholesterol content in plasma membranes and inducing changes in plasma membrane fluidity that could be associated with the parasite's ability to survive in the host macrophages, thereby possibly contributing to immune evasion and TF. [ABSTRACT FROM AUTHOR]

Toll-like receptor 4 (TLR4)-mediated changes in macrophages reshape intracellular lipid pools to coordinate an effective innate immune response. Although this has been previously well-studied in different model systems, it remains incompletely understood in primary human macrophages. Here we report time-dependent lipidomic and transcriptomic responses to lipopolysaccharide (LPS) in primary human macrophages from healthy donors. We grouped the variation of ~200 individual lipid species measured by LC-MS/MS into eight temporal clusters. Among all other lipids, glycosphingolipids (glycoSP) and cholesteryl esters (CE) showed a sharp increase during the resolution phase (between 8h or 16h post LPS). GlycoSP, belonging to the globoside family (Gb3 and Gb4), showed the greatest inter-individual variability among all lipids quantified. Integrative network analysis between GlycoSP/CE levels and genome-wide transcripts, identified Gb4 d18:1/16:0 and CE 20:4 association with subnetworks enriched for T cell receptor signaling (PDCD1, CD86, PTPRC, CD247, IFNG) and DC-SIGN signaling (RAF1, CD209), respectively. Our findings reveal Gb3 and Gb4 globosides as sphingolipids associated with the resolution phase of inflammatory response in human macrophages.

ABSTRACT Mycobacterium tuberculosis segregates within multiple subcellular niches with different biochemical and biophysical properties that, upon treatment, may impact antibiotic distribution, accumulation, and efficacy. However, it remains unclear whether fluctuating intracellular microenvironments alter mycobacterial homeostasis and contribute to antibiotic enrichment and efficacy. Here, we describe a live dual-imaging approach to monitor host subcellular acidification and M. tuberculosis intrabacterial pH. By combining this approach with pharmacological and genetic perturbations, we show that M. tuberculosis can maintain its intracellular pH independently of the surrounding pH in human macrophages. Importantly, unlike bedaquiline (BDQ), isoniazid (INH), or rifampicin (RIF), the drug pyrazinamide (PZA) displays antibacterial efficacy by disrupting M. tuberculosis intrabacterial pH homeostasis in cellulo. By using M. tuberculosis mutants, we confirmed that intracellular acidification is a prerequisite for PZA efficacy in cellulo. We anticipate this imaging approach will be useful to identify host cellular environments that affect antibiotic efficacy against intracellular pathogens. IMPORTANCE We still do not completely understand why tuberculosis (TB) treatment requires the combination of several antibiotics for up to 6 months. M. tuberculosis is a facultative intracellular pathogen, and it is still unknown whether heterogenous and dynamic intracellular populations of bacteria in different cellular environments affect antibiotic efficacy. By developing a dual live imaging approach to monitor mycobacterial pH homeostasis, host cell environment, and antibiotic action, we show here that intracellular localization of M. tuberculosis affects the efficacy of one first-line anti-TB drug. Our observations can be applicable to the treatment of other intracellular pathogens and help to inform the development of more effective combined therapies for tuberculosis that target heterogenous bacterial populations within the host.

To increase our understanding of factors contributing to therapeutic failure (TF) in leishmaniasis, we have studied some plasma membrane features of host THP-1 cells infected with clinical isolates of Leishmania infantum from patients with leishmaniasis and TF. The fluorescent probes DPH and TMA-DPH were used to measure changes in membrane fluidity at various depths of the plasma membranes. Steady-state fluorescence anisotropy of DPH embedded in the infected THP-1 membranes showed a significant increase, thereby suggesting a substantial decrease in plasma membrane fluidity relative to controls. Considering that cholesterol affects membrane fluidity and permeability, we determined the cholesterol content in plasma membrane fractions of human macrophages infected with these L. infantum lines and observed a significant increase in cholesterol content that correlates with the measured decrease in plasma membrane fluidity. In order to define the pathways that could explain the increase in cholesterol content, we studied the transcriptomics of the cholesterol-enriched pathways in host THP-1 cells infected with TF clinical isolates by RNA-seq. Specifically, we focused on four enriched Gene Ontology (GO) terms namely cholesterol efflux, cholesterol transport, cholesterol metabolic process and cholesterol storage. Additionally, we analyzed the genes involved in these pathways. Overall, this study shows that these clinical isolates are able to modulate the expression of specific genes in host cells, thereby modifying the cholesterol content in plasma membranes and inducing changes in plasma membrane fluidity that could be associated with the parasite’s ability to survive in the host macrophages, thereby possibly contributing to immune evasion and TF.

Recently, first insights into the regulation and the role of the RNA‐binding protein IMP2 in macrophage activation have been published by Wang et al. This study addresses differences in the regulation of IMP2 between the human and murine system. While the expression of IMP2 in anti‐inflammatory macrophages is synchronous in mice and men, IMP2 expression is regulated differently in inflammatory macrophages. [ABSTRACT FROM AUTHOR]

Background. Clinical and experimental evidence point to a dysregulated immune response caused by SARS-CoV-2 as the primary mechanism of lung disease in COVID-19. However, the pathogenic mechanisms underlying COVID-19-associated ARDS (Acute Respiratory Distress Syndrome) remain incompletely understood. This study aims to explore the inflammatory responses of alveolar epithelial cells to either the spike S1 protein or to a mixture of cytokines secreted by S1-activated macrophages. Methods and Results. The exposure of alveolar A549 cells to supernatants from spike-activated macrophages caused a further release of inflammatory mediators, with IL-8 reaching massive concentrations. The investigation of the molecular pathways indicated that NF-kB is involved in the transcription of IP-10 and RANTES, while STATs drive the expression of all the cytokines/chemokines tested, with the exception of IL-8 which is regulated by AP-1. Cytokines/chemokines produced by spike-activated macrophages are also likely responsible for the observed dysfunction of barrier integrity in Human Alveolar Epithelial Lentivirus-immortalized cells (hAELVi), as demonstrated by an increased permeability of the monolayers to mannitol, a marked decrease of TEER and a disorganization of claudin-7 distribution. Conclusion. Upon exposure to supernatants from S1-activated macrophages, A549 cells act both as targets and sources of cytokines/chemokines, suggesting that alveolar epithelium along with activated macrophages may orchestrate lung inflammation and contribute to alveolar injury, a hallmark of ARDS. [ABSTRACT FROM AUTHOR]

Keywords: allergen-specific immunotherapy; allergoid-mannan conjugates; human macrophages; immune regulation; innate immunity EN allergen-specific immunotherapy allergoid-mannan conjugates human macrophages immune regulation innate immunity 320 323 4 12/31/21 20220101 NES 220101 ACKNOWLEDGEMENT None. Allergoid-mannan conjugates imprint tolerogenic features in human macrophages LPS-stimulated Mannan-GM-MØ produced significantly lower levels of the pro-inflammatory cytokines TNF- and IL-6 and significantly higher levels of anti-inflammatory IL-10 than conventional GM-MØ (Figure 2D). [Extracted from the article]

Objective: To investigate the effects of Ginkgo biloba extract (GBE) on autophagy in human macrophages stimulated by cigarette smoke extract (CSE). Methods: The human monocyte cell line U937 was cultured in vitro, and phorbol ester (PMA) was added to the cell culture medium to induce differentiation into human macrophages. CSE was prepared by traditional methods for experiments. The cells were divided into four groups: the blank group, the CSE model group, the GBE + CSE group, and the rapamycin + CSE group. Immunofluorescence was used to identify human macrophages, transmission electron microscopy was used to observe the ultrastructure of human macrophages in each group, ELISA was used to measure the amount of IL-6 and IL-10 in the supernatant from each group of cells, the mRNA levels of p62, ATG5, ATG7, and Rab7 were measured by real-time qPCR, and the protein expression levels of p62, ATG5, ATG7, and Rab7 were measured by Western blotting. Results: U937 cells were successfully differentiated into human macrophages after induction with PMA. The CSE model group had many more autophagosomes than the blank group. Compared with the CSE model group, the GBE + CSE group and the rapamycin + CSE group had significantly more autophagolysosomal. Compared with the other groups, the CSE model group had a higher level of IL-6 but a lower level of IL-10 in the supernatant (p < 0.05). Compared with the blank group, the mRNA and protein expression levels of p62 in the CSE model group were significantly decreased, while the mRNA and protein expression levels of ATG5 and ATG7 were significantly increased in the CSE model group (p < 0.05). No difference was found in the mRNA and protein expression levels of Rab7 between the blank group and the CSE model group. Compared with the CSE model group, the IL-6 level in the GBE + CSE group and the rapamycin + CSE group cell culture supernatant decreased significantly, p62 mRNA and protein expression significantly decreased, while ATG5, ATG7, and Rab7 mRNA and protein expression levels were significantly increased (p < 0.05). Moreover, increased LC3-II/LC3-I ratio were also found in the GBE + CSE group and the rapamycin + CSE group compared with the CSE model group. Conclusions: GBE could promote the fusion of autophagosomes and lysosomes in human macrophages, enhance the autophagy function of human macrophages, and reduce the damaging effect of CSE on the autophagy function of macrophages.

In the pathogenesis of influenza virus infection, lymphocyte apoptosis as a part of the infection and/or the immune response to the virus can be somewhat puzzling. The percentage of human T lymphocytes within the peripheral blood mononuclear cell population that becomes apoptotic greatly exceeds the percentage that are infected after exposure to the virus, consistent with substantial apoptosis of bystander T lymphocytes. Studies reveal an important role of viral neuraminidase expression by co-cultured monocyte/macrophages in induction of apoptosis, including that of uninfected bystander lymphocytes. Despite these observations, it is a reasonable perspective to recognize that the development of lymphocyte apoptosis during the response to infection does not preclude a successful immune response and recovery of the infected host in the great majority of cases. Further investigation is clearly warranted to understand its role in the pathogenesis of influenza virus infection for human subjects.

Mycobacterium tuberculosis ( Mtb ) as well as nontuberculous mycobacteria are intracellular pathogens whose treatment is extensive and increasingly impaired due to the rise of mycobacterial drug resistance. The loss of antibiotic efficacy has raised interest in the identification of host-directed therapeutics (HDT) to develop novel treatment strategies for mycobacterial infections. In this study, we identified amiodarone as a potential HDT candidate that inhibited both intracellular Mtb and Mycobacterium avium in primary human macrophages without directly impairing bacterial growth, thereby confirming that amiodarone acts in a host-mediated manner. Moreover, amiodarone induced the formation of (auto)phagosomes and enhanced autophagic targeting of mycobacteria in macrophages. The induction of autophagy by amiodarone is likely due to enhanced transcriptional regulation, as the nuclear intensity of the transcription factor EB, the master regulator of autophagy and lysosomal biogenesis, was strongly increased. Furthermore, blocking lysosomal degradation with bafilomycin impaired the host-beneficial effect of amiodarone. Finally, amiodarone induced autophagy and reduced bacterial burden in a zebrafish embryo model of tuberculosis, thereby confirming the HDT activity of amiodarone in vivo . In conclusion, we have identified amiodarone as an autophagy-inducing antimycobacterial HDT that improves host control of mycobacterial infections., Importance: Due to the global rise in antibiotic resistance, there is a strong need for alternative treatment strategies against intracellular bacterial infections, including Mycobacterium tuberculosis ( Mtb ) and non-tuberculous mycobacteria. Stimulating host defense mechanisms by host-directed therapy (HDT) is a promising approach for treating mycobacterial infections. This study identified amiodarone, an antiarrhythmic agent, as a potential HDT candidate that inhibits the survival of Mtb and Mycobacterium avium in primary human macrophages. The antimycobacterial effect of amiodarone was confirmed in an in vivo tuberculosis model based on Mycobacterium marinum infection of zebrafish embryos. Furthermore, amiodarone induced autophagy and inhibition of the autophagic flux effectively impaired the host-protective effect of amiodarone, supporting that activation of the host (auto)phagolysosomal pathway is essential for the mechanism of action of amiodarone. In conclusion, we have identified amiodarone as an autophagy-inducing HDT that improves host control of a wide range of mycobacteria., Competing Interests: The authors declare no conflict of interest.

Diabetes mellites (DM) is correlated with increased susceptibility to and disease progression of tuberculosis (TB), and strongly impairs effective global TB control measures. To better control the TB-DM co-epidemic, unravelling the bidirectional interactivity between DM-associated molecular processes and immune responses to Mycobacterium tuberculosis (Mtb) is urgently required. Since poly (ADP-ribose) polymerase (PARP) activation has been associated with DM and with Mtb infection in mouse models, we have investigated whether PARP inhibition by pharmacological compounds can interfere with host protection against Mtb in human macrophage subsets, the predominant target cell of Mtb. Pharmacological inhibition of PARP decreased intracellular Mtb and MDR- Mtb levels in human macrophages, identifying PARP as a potential target for host-directed therapy against Mtb. PARP inhibition was associated with modified chemokine secretion and upregulation of cell surface activation markers by human macrophages. Targeting LDH, a secondary target of the PARP inhibitor rucaparib, resulted in decreased intracellular Mtb , suggesting a metabolic role in rucaparib-induced control of Mtb. We conclude that pharmacological inhibition of PARP is a potential novel strategy in developing innovative host-directed therapies against intracellular bacterial infections. [ABSTRACT FROM AUTHOR]

In this study, the human immune response mechanisms against Sporothrix brasiliensis and Sporothrix schenckii , two causative agents of human and animal sporotrichosis, were investigated. The interaction of S. brasiliensis and S. schenckii with human monocyte-derived macrophages (hMDMs) was shown to be dependent on the thermolabile serum complement protein C3, which facilitated the phagocytosis of Sporothrix yeast cells through opsonization. The peptidorhamnomannan (PRM) component of the cell walls of these two Sporothrix yeasts was found to be one of their surfaces exposed pathogen-associated molecular pattern (PAMP), leading to activation of the complement system and deposition of C3b on the Sporothrix yeast surfaces. PRM also showed direct interaction with CD11b, the specific component of the complement receptor-3 (CR3). Furthermore, the blockade of CR3 specifically impacted the interleukin (IL)-1β secretion by hMDM in response to both S. brasiliensis and S. schenckii , suggesting that the host complement system plays an essential role in the inflammatory immune response against these Sporothrix species. Nevertheless, the structural differences in the PRMs of the two Sporothrix species, as revealed by NMR, were related to the differences observed in the host complement activation pathways. Together, this work reports a new PAMP of the cell surface of pathogenic fungi playing a role through the activation of complement system and via CR3 receptor mediating an inflammatory response to Sporothrix species. [ABSTRACT FROM AUTHOR]

In this study, the human immune response mechanisms against Sporothrix brasiliensis and Sporothrix schenckii, two causative agents of human and animal sporotrichosis, were investigated. The interaction of S. brasiliensis and S. schenckii with human monocyte-derived macrophages (hMDMs) was shown to be dependent on the thermolabile serum complement protein C3, which facilitated the phagocytosis of Sporothrix yeast cells through opsonization. The peptidorhamnomannan (PRM) component of the cell walls of these two Sporothrix yeasts was found to be one of their surfaces exposed pathogen-associated molecular pattern (PAMP), leading to activation of the complement system and deposition of C3b on the Sporothrix yeast surfaces. PRM also showed direct interaction with CD11b, the specific component of the complement receptor-3 (CR3). Furthermore, the blockade of CR3 specifically impacted the interleukin (IL)-1β secretion by hMDM in response to both S. brasiliensis and S. schenckii, suggesting that the host complement system plays an essential role in the inflammatory immune response against these Sporothrix species. Nevertheless, the structural differences in the PRMs of the two Sporothrix species, as revealed by NMR, were related to the differences observed in the host complement activation pathways. Together, this work reports a new PAMP of the cell surface of pathogenic fungi playing a role through the activation of complement system and via CR3 receptor mediating an inflammatory response to Sporothrix species.

Diabetes mellites (DM) is correlated with increased susceptibility to and disease progression of tuberculosis (TB), and strongly impairs effective global TB control measures. To better control the TB-DM co-epidemic, unravelling the bidirectional interactivity between DM-associated molecular processes and immune responses to Mycobacterium tuberculosis (Mtb) is urgently required. Since poly (ADP-ribose) polymerase (PARP) activation has been associated with DM and with Mtb infection in mouse models, we have investigated whether PARP inhibition by pharmacological compounds can interfere with host protection against Mtb in human macrophage subsets, the predominant target cell of Mtb. Pharmacological inhibition of PARP decreased intracellular Mtb and MDR-Mtb levels in human macrophages, identifying PARP as a potential target for host-directed therapy against Mtb. PARP inhibition was associated with modified chemokine secretion and upregulation of cell surface activation markers by human macrophages. Targeting LDH, a secondary target of the PARP inhibitor rucaparib, resulted in decreased intracellular Mtb, suggesting a metabolic role in rucaparib-induced control of Mtb. We conclude that pharmacological inhibition of PARP is a potential novel strategy in developing innovative host-directed therapies against intracellular bacterial infections.

Acute respiratory distress syndrome (ARDS) is characterized by severe hypoxemia and high-permeability pulmonary edema. A hallmark of the disease is the presence of lung inflammation with features of diffuse alveolar damage. The molecular pathogenetic mechanisms of COVID-19-associated ARDS (CARDS), secondary to SARS-CoV-2 infection, are still not fully understood. Here, we investigate the effects of a cytokine-enriched conditioned medium from Spike S1-activated macrophage on alveolar epithelial A549 cells in terms of cell proliferation, induction of autophagy, and expression of genes related to protein degradation. The protective effect of baricitinib, employed as an inhibitor of JAK-STAT, has been also tested. The results obtained indicate that A549 exhibits profound changes in cell morphology associated to a proliferative arrest in the G0/G1 phase. Other alterations occur, such as a blockade of protein synthesis and the activation of autophagy, along with an increase of the intracellular amino acids content, which is likely ascribable to the activation of protein degradation. These changes correlate to the induction of IFN-regulatory factor 1 (IRF-1) due to an increased secretion of IFN-γ in the conditioned medium from S1-activated macrophages. The addition of baricitinib prevents the observed effects. In conclusion, our findings suggest that the IFN-γ-IRF-1 signaling pathway may play a role in the alveolar epithelial damage observed in COVID-19-related ARDS.

Neutrophils undergo spontaneous apoptosis within 24–48 h after leaving bone marrow. Apoptotic neutrophils are subsequently phagocytosed and cleared by macrophages, thereby maintaining neutrophil homeostasis. Previous studies have demonstrated involvement of lysophosphatidylglucoside (lysoPtdGlc), a degradation product of PtdGlc, in modality-specific repulsive guidance of spinal sensory axons, via its specific receptor GPR55. In the present study, using human monocytic cell line THP-1 as a model, we demonstrated that lysoPtdGlc induces monocyte/macrophage migration with typical bell-haped curve and a peak at concentration 10−9 M. Lysophosphatidylinositol (lysoPtdIns), a known GPR55 ligand, induced migration at higher concentration (10−7 M). LysoPtdGlc-treated cells had a polarized shape, whereas lysoPtdIns-treated cells had a spherical shape. In EZ-TAXIScan (chemotaxis) assay, lysoPtdGlc induced chemotactic migration activity of THP-1 cells, while lysoPtdIns induced random migration activity. GPR55 antagonist ML193 inhibited lysoPtdGlc-induced THP-1 cell migration, whereas lysoPtdIns-induced migration was inhibited by CB 2 -receptor inverse agonist. SiRNA experiments showed that GPR55 mediated lysoPtdGlc-induced migration, while lysoPtdIns-induced migration was mediated by CB 2 receptor. Our findings, taken together, suggest that lysoPtdGlc functions as a chemotactic molecule for human monocytes/macrophages via GPR55 receptor, while lysoPtdIns induces random migration activity via CB 2 receptor. • Lysophosphatidylglucoside identified as a pure chemokine for human monocytes and macrophages. • Lysophosphatidylglucoside identified as a natural ligand for GPR55 on human monocytes and macrophages. • Lysophosphatidylglucoside-treated monocytes and macrophages showing polarized shape. [ABSTRACT FROM AUTHOR]

Background: Multidrug-resistant (MDR) tuberculosis has low treatment success rates, and new treatment strategies are needed. We explored whether treatment with active vitamin D3 (vitD) and phenylbutyrate (PBA) could improve conventional chemotherapy by enhancing immune-mediated eradication of Mycobacterium tuberculosis.Methods: A clinically relevant model was used consisting of human macrophages infected with M. tuberculosis isolates (n = 15) with different antibiotic resistance profiles. The antimicrobial effect of vitD+PBA, was tested together with rifampicin or isoniazid. Methods included colony-forming units (intracellular bacterial growth), messenger RNA expression analyses (LL-37, β-defensin, nitric oxide synthase, and dual oxidase 2), RNA interference (LL-37-silencing in primary macrophages), and Western blot analysis and confocal microscopy (LL-37 and LC3 protein expression).Results: VitD+PBA inhibited growth of clinical MDR tuberculosis strains in human macrophages and strengthened intracellular growth inhibition of rifampicin and isoniazid via induction of the antimicrobial peptide LL-37 and LC3-dependent autophagy. Gene silencing of LL-37 expression enhanced MDR tuberculosis growth in vitD+PBA-treated macrophages. The combination of vitD+PBA and isoniazid were as effective in reducing intracellular MDR tuberculosis growth as a >125-fold higher dose of isoniazid alone, suggesting potent additive effects of vitD+PBA with isoniazid.Conclusions: Immunomodulatory agents that trigger multiple immune pathways can strengthen standard MDR tuberculosis treatment and contribute to next-generation individualized treatment options for patients with difficult-to-treat pulmonary tuberculosis. [ABSTRACT FROM AUTHOR]

The innate immune and host-protective responses to viruses, such as the airway pathogen human metapneumovirus (HMPV), depend on interferons (IFNs) that is induced through TANK-binding kinase 1 (TBK1) and IFN regulatory factors (IRFs). The transcription factor IRF1 is important for host resistance against several viruses and has a key role in induction of IFN-λ at mucosal surfaces. In most cell types IRF1 is expressed at very low levels, but its mRNA is rapidly induced when the demand for IRF1 activity arises. Despite general recognition of the importance of IRF1 to antiviral responses, the molecular mechanisms by which IRF1 is regulated during viral infections are not well understood. Here we identify the serine/threonine kinase TBK1 and IFN-β as critical regulators of IRF1 mRNA and protein levels in human monocyte-derived macrophages. We find that inhibition of TBK1 activity either by the semi-selective TBK1/IKKε inhibitor BX795 or by siRNA-mediated knockdown abrogates HMPV-induced expression of IRF1. Moreover, we show that canonical NF-κB signaling is involved in IRF1 induction and that the TBK1/IKKε inhibitor BX795, but not siTBK1 treatment, impairs HMPV-induced phosphorylation of the NF-κB subunit p65. At later time-points of the infection, IRF1 expression depended heavily on IFN-β-mediated signaling via the IFNAR-STAT1 pathway. Hence, our results suggest that TBK1 activation and TBK1/IKKε-mediated phosphorylation of the NF-κB subunit p65 control transcription of IRF1. Our study identifies a novel mechanism for IRF1 induction in response to viral infection of human macrophages that could be relevant not only to defense against HMPV, but also to other viral, bacterial and fungal pathogens.

The innate immune and host-protective responses to viruses, such as the airway pathogen human metapneumovirus (HMPV), depend on interferons (IFNs) that is induced through TANK-binding kinase 1 (TBK1) and IFN regulatory factors (IRFs). The transcription factor IRF1 is important for host resistance against several viruses and has a key role in induction of IFN-λ at mucosal surfaces. In most cell types IRF1 is expressed at very low levels, but its mRNA is rapidly induced when the demand for IRF1 activity arises. Despite general recognition of the importance of IRF1 to antiviral responses, the molecular mechanisms by which IRF1 is regulated during viral infections are not well understood. Here we identify the serine/threonine kinase TBK1 and IFN-β as critical regulators of IRF1 mRNA and protein levels in human monocyte-derived macrophages. We find that inhibition of TBK1 activity either by the semi-selective TBK1/IKKε inhibitor BX795 or by siRNA-mediated knockdown abrogates HMPV-induced expression of IRF1. Moreover, we show that canonical NF-κB signaling is involved in IRF1 induction and that the TBK1/IKKε inhibitor BX795, but not siTBK1 treatment, impairs HMPV-induced phosphorylation of the NF-κB subunit p65. At later time-points of the infection, IRF1 expression depended heavily on IFN-β-mediated signaling via the IFNAR-STAT1 pathway. Hence, our results suggest that TBK1 activation and TBK1/IKKε-mediated phosphorylation of the NF-κB subunit p65 control transcription of IRF1. Our study identifies a novel mechanism for IRF1 induction in response to viral infection of human macrophages that could be relevant not only to defense against HMPV, but also to other viral, bacterial and fungal pathogens. [ABSTRACT FROM AUTHOR]

We studied the effect of conditioned media of GM-CSF-differentiated human macrophages polarized in M1(LPS), M2a(IL-4), M2c(dexamethasone), and M2(low serum) phenotypes on proliferation, differentiation, and collagen-producing activity of dermal fibroblasts. It was found that M1(LPS) and M2a(IL-4) were characterized by moderate influence on functional activity of fibroblasts. At the same time, soluble factors of M2c(dexamethasone) significantly enhanced the proliferative response of fibroblasts, but not their differentiation and type I collagen production. On the contrary, M2(low serum) generated under conditions of growth factors deficiency had a pronounced stimulating effect on the differentiation of fibroblasts and production of type I collagen by these cells, but moderately stimulated the fibroblast proliferation. Thus, the secretory activity of various functional phenotypes of macrophages is an important mechanism of fibrogenesis regulation. [ABSTRACT FROM AUTHOR]

Macrophages play the key role in the regulation of neuroregeneration. For evaluation of the neuroregenerative potential of M2 macrophages, we studied the effect of macrophages polarized with IL-4 (M2a (IL-4)) and by efferocytosis under conditions of serum deprivation (LS, Low Serum; M2(LS)) on proliferative activity and apoptosis of SH-SY5Y cells under conditions of deficiency of growth/serum factors. Conditioned media of both M2(LS) and M2a(IL-4) stimulated proliferation of SH-SY5Y cells. Moreover, soluble factors of M2(LS) and M2a(IL-4) reduced the degree of early apoptosis of SH-SY5Y cells and the protective effect of M2(LS) was observed at earlier terms of culturing. Our findings suggest that M2 macrophages have high neuroregenerative potential that is mediated through soluble factors and manifests itself both in stimulation of proliferation and inhibition of apoptosis of SH-SY5Y cells. [ABSTRACT FROM AUTHOR]

We studied the expression of arginase-1 (Arg1) and tyrosine kinase Mer (MerTK) in GMCSF-differentiated human macrophage populations М0, М1(IFNγ), М2а(IL-4), and М2(low serum) generated under conditions of growth/serum factor deficiency. The maximum relative content of Arg1+ and MerTK+ cells was found in М2 macrophage populations: М2а(IL-4) and М2(low serum). As the uptake of apoptotic cells is the key mechanism of M2 polarization during M2(low serum) generation, we performed a special series of experiments and showed that incubation with allogeneic apoptotic neutrophils significantly increased the percentages of CD206+ macrophages co-expressing Arg1 and MerTK. [ABSTRACT FROM AUTHOR]

The high- and the low-molecular weight hyaluronic acids (HMW-HA and LMW-HA, respectively) showed different biological activities in inflammation. However, the role of LMW-HA in inflammatory response is controversial. In this study, we aimed to investigate the effect of bioactive hyaluronan (B-HA) on lipopolysaccharide (LPS)-induced inflammatory responses in human macrophages and mice. B-HA was produced from HA treated with glycosylated recombinant human hyaluronidase PH20. Human THP-1 cells were induced to differentiate into macrophages. THP-1-derived macrophages were treated with B-HA, LPS, or B-HA + LPS. The mRNA expression and the production of inflammatory cytokines were determined using quantitative real-time PCR and enzyme-linked immunosorbent assay. The phosphorylation levels of proteins in the nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), and IRF-3 signaling pathways were measured using Western blot. The in vivo efficacy of B-HA was assessed in a mouse model of LPS-induced inflammation. Results showed that B-HA inhibited the expression of TNF-α, IL-6, IL-1, and IFN-β, and enhanced the expression of the antiinflammatory cytokine IL-10 in LPS-induced inflammatory responses in THP-1-derived macrophages and in vivo. B-HA significantly suppressed the phosphorylation of the TLR4 signaling pathway proteins p65, IKKα/β, IκBα, JNK1/2, ERK1/2, p38, and IRF-3. In conclusion, our results demonstrated that the B-HA attenuated the LPS-stimulated inflammatory response by inhibiting the activation of the TLR4 signaling pathway. B-HA could be a potential anti-inflammatory drug in the treatment of inflammatory disease. [ABSTRACT FROM AUTHOR]

Toll-like receptor 8 (TLR8) recognizes single-stranded RNA of viral and bacterial origin as well as mediates the secretion of pro-inflammatory cytokines and type I interferons by human monocytes and macrophages. TLR8, as other endosomal TLRs, utilizes the MyD88 adaptor protein for initiation of signaling from endosomes. Here, we addressed the potential role of the Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) in the regulation of TLR8 signaling in human primary monocyte-derived macrophages (MDMs). To accomplish this, we performed TIRAP gene silencing, followed by the stimulation of cells with synthetic ligands or live bacteria. Cytokine-gene expression and secretion were analyzed by quantitative PCR or Bioplex assays, respectively, while nuclear translocation of transcription factors was addressed by immunofluorescence and imaging, as well as by cell fractionation and immunoblotting. Immunoprecipitation and Akt inhibitors were also used to dissect the signaling mechanisms. Overall, we show that TIRAP is recruited to the TLR8 Myddosome signaling complex, where TIRAP contributes to Akt-kinase activation and the nuclear translocation of interferon regulatory factor 5 (IRF5). Recruitment of TIRAP to the TLR8 signaling complex promotes the expression and secretion of the IRF5-dependent cytokines IFNβ and IL-12p70 as well as, to a lesser degree, TNF. These findings reveal a new and unconventional role of TIRAP in innate immune defense.

Trichinella spiralis is a foodborne zoonotic nematode, which causes trichinellosis. During the infection, parasite evades the host immune responses by direct and indirect (through excretory-secretory products) contact with host immune cells. One of the main targets for immunomodulation induced by helminths are macrophages. In this study, we examined whether direct contact of different stages of T. spiralis can affect the polarization of human THP-1 macrophages. Co-culture of adult parasite stage and cells in direct contact without LPS addition had a significant impact on TNFα levels. Interestingly, in settings with the addition of LPS, the levels of IL-1β and TNFα significantly increased in adult parasite and newborn larvae (NBL) but not for muscle larvae (ML). While we tested muscle larvae ESP products to compare its effect with whole ML parasite, we detect an increase of pro-inflammatory cytokines like IL-1β and TNFα in no LPS conditions. Whereas, muscle larvae ESP significantly suppressed the inflammatory response measured by IL-1β, TNFα, and IL-6 levels and anti-inflammatory IL-10 compared to LPS control. Our findings indicate the anti-inflammatory potential of T. spiralis muscle larvae excretory-secretory products and propose signaling pathways which might be engaged in the mechanism of how muscle larvae ESP affect human macrophages. [ABSTRACT FROM AUTHOR]

The NLRP3 inflammasome is an intracellular multiprotein complex described to be involved in both an effective host response to infectious agents and various diseases. Investigation into the NLRP3 inflammasome has been extensive in the past two decades, and often revolves around the analysis of a few specific readouts, including ASC-speck formation, caspase-1 cleavage or activation, and cleavage and release of IL-1β and/or IL-18. Quantification of these readouts is commonly undertaken as an endpoint analysis, where the presence of each positive outcome is assessed independently of the others. In this study, we apply time-resolved analysis of a human macrophage model (differentiated THP-1-ASC-GFP cells) to commonly accessible methods. This approach yields the additional quantifiable metrics time-resolved absolute change and acceleration, allowing comparisons between readouts. Using this methodological approach, we reveal (potential) discrepancies between inflammasome-related readouts that otherwise might go undiscovered. The study highlights the importance of time-resolved data in general and may be further extended as well as incorporated into other areas of research., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:co-author affiliated to Nanoxis Consulting AB - R.K. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

Introduction. Staphylococcus epidermidis biofilms are one of the major causes of bloodstream infections related to the use of medical devices. The diagnosis of these infections is challenging, delaying their treatment and resulting in increased morbidity and mortality rates. As such, it is urgent to characterize the mechanisms employed by this bacterium to endure antibiotic treatments and the response of the host immune system, to develop more effective therapeutic strategies. In several bacterial species, the gene codY was shown to encode a protein that regulates the expression of genes involved in biofilm formation and immune evasion. Additionally, in a previous study, our group generated evidence indicating that codY is involved in the emergence of viable but non-culturable (VBNC) cells in S. epidermidis . Gap statement/Hypothesis. As such, we hypothesized that the gene codY has have an important role in this bacterium virulence. Aim. This study aimed to assess, for the first time, the impact of the deletion of the gene codY in S. epidermidis virulence, namely, in antibiotic susceptibility, biofilm formation, VBNC state emergence and in vitro host immune system response. Methodology. Using an allelic replacement strategy, we constructed and then characterized an S. epidermidis strain lacking codY, in regards to biofilm and VBNC cell formation, susceptibility to antibiotics as well as their role in the interaction with human blood and plasma. Additionally, we investigate whether the codY gene can impact the activation of innate immune cells by evaluating the production of both pro- and anti-inflammatory cytokines by THP-1 macrophages. Results. We demonstrated that the deletion of the gene codY by human macrophages, a stronger cytokine response was elicited by the mutant in a toll-like receptor 4-dependent manner. in vitro by human macrophages, a stronger cytokine response was elicited by the mutant in a toll-like receptor 4-dependent manner. Conclusion. Our results indicate that codY contributes to S. epidermidis virulence, which in turn may have an impact on our ability to manage the biofilm-associated infections caused by this bacterium.