Cytokine Production In Human Macrophages Research Articles

Deletion of codY impairs Staphylococcus epidermidis biofilm formation, generation of viable but non-culturable cells and stimulates cytokine production in human macrophages.

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 resulted in biofilms with less c.f.u. counts and fewer VBNC cells. Furthermore, we show that although WT and mutant cells were similarly internalized 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.

SARS-CoV-2 Delta spike protein enhances the viral fusogenicity and inflammatory cytokine production

The Delta variant had spread globally in 2021 and caused more serious disease than the original virus and Omicron variant. In this study, we investigated several virological features of Delta spike protein (SPDelta), including protein maturation, its impact on viral entry of pseudovirus and cell-cell fusion, and its induction of inflammatory cytokine production in human macrophages and dendritic cells. The results showed that SPΔCDelta exhibited enhanced S1/S2 cleavage in cells and pseudotyped virus-like particles (PVLPs). Further, SPΔCDelta elevated pseudovirus entry in human lung cell lines and significantly enhanced syncytia formation. Furthermore, we revealed that SPΔCDelta-PVLPs had stronger effects on stimulating NF-κB and AP-1 signaling in human monocytic THP1 cells and induced significantly higher levels of proinflammatory cytokine, such as TNF-α, IL-1β, and IL-6, released from human macrophages and dendritic cells. Overall, these studies provide evidence to support the important role of SPΔCDelta during virus infection, transmission, and pathogenesis.

Growth Arrest-Specific Factor 6 (GAS6) Is Increased in COVID-19 Patients and Predicts Clinical Outcome.

Background: Growth arrest-specific factor 6 (GAS6) and the Tyro3, AXL, and MERTK (TAM) receptors counterbalance pro-inflammatory responses. AXL is a candidate receptor for SARS-CoV-2, particularly in the respiratory system, and the GAS6/AXL axis is targeted in current clinical trials against COVID-19. However, GAS6 and TAMs have not been evaluated in COVID-19 patients at emergency admission. Methods: Plasma GAS6, AXL, and MERTK were analyzed in 132 patients consecutively admitted to the emergency ward during the first peak of COVID-19. Results: GAS6 levels were higher in the SARS-CoV-2-positive patients, increasing progressively with the severity of the disease. Patients with initial GAS6 at the highest quartile had the worst outcome, with a 3-month survival of 65%, compared to a 90% survival for the rest. Soluble AXL exhibited higher plasma concentration in deceased patients, without significant differences in MERTK among SARS-CoV-2-positive groups. GAS6 mRNA was mainly expressed in alveolar cells and AXL in airway macrophages. Remarkably, THP-1 human macrophage differentiation neatly induces AXL, and its inhibition (bemcentinib) reduced cytokine production in human macrophages after LPS challenge. Conclusions: Plasma GAS6 and AXL levels reflect COVID-19 severity and could be early markers of disease prognosis, supporting a relevant role of the GAS6/AXL system in the immune response in COVID-19.

Dynamin-related protein 1 inhibition reduces hepatic PCSK9 secretion.

Proteostasis maintains protein homeostasis and participates in regulating critical cardiometabolic disease risk factors including proprotein convertase subtilisin/kexin type 9 (PCSK9). Endoplasmic reticulum (ER) remodeling through release and incorporation of trafficking vesicles mediates protein secretion and degradation. We hypothesized that ER remodeling that drives mitochondrial fission participates in cardiometabolic proteostasis. We used in vitro and in vivo hepatocyte inhibition of a protein involved in mitochondrial fission, dynamin-related protein 1 (DRP1). Here, we show that DRP1 promotes remodeling of select ER microdomains by tethering vesicles at ER. A DRP1 inhibitor, mitochondrial division inhibitor 1 (mdivi-1) reduced ER localization of a DRP1 receptor, mitochondrial fission factor, suppressing ER remodeling-driven mitochondrial fission, autophagy, and increased mitochondrial calcium buffering and PCSK9 proteasomal degradation. DRP1 inhibition by CRISPR/Cas9 deletion or mdivi-1 alone or in combination with statin incubation in human hepatocytes and hepatocyte-specific Drp1-deficiency in mice reduced PCSK9 secretion (-78.5%). In HepG2 cells, mdivi-1 increased low-density lipoprotein receptor via c-Jun transcription and reduced PCSK9 mRNA levels via suppressed sterol regulatory binding protein-1c. Additionally, mdivi-1 reduced macrophage burden, oxidative stress, and advanced calcified atherosclerotic plaque in aortic roots of diabetic Apoe-deficient mice and inflammatory cytokine production in human macrophages. We propose a novel tethering function of DRP1 beyond its established fission function, with DRP1-mediated ER remodeling likely contributing to ER constriction of mitochondria that drives mitochondrial fission. We report that DRP1-driven remodeling of select ER micro-domains may critically regulate hepatic proteostasis and identify mdivi-1 as a novel small molecule PCSK9 inhibitor.

Human milk oligosaccharides and its acid hydrolysate LNT2 show immunomodulatory effects via TLRs in a dose and structure-dependent way

Abstract Human milk oligosaccharides (HMOs) have beneficial immune effects but the mechanisms of action are not well-understood. Here we study Toll-like receptor (TLR) signaling of the HMOs 2′-FL, 3-FL, 6′-SL, LNT2, and LNnT and their effect on cytokine production in human macrophages. 3-FL activated TLR2 and LNT2 activated all TLRs in a dose-dependent way. In an inhibition assay, 2′-FL, 6′-SL, and LNnT inhibited TLR5 and 7, while 3-FL inhibited TLR5, 7, and 8. 6′-SL showed a synergistic effect on ssRNA40-induced TLR8 activation. In addition, we measured HMO-induced cytokine production in THP1 macrophages. IL-10 and TNFα were induced by LNT2, and the effects were NF-κB dependent, while the other HMOs had minor effects. The potent effects of LNT2 might be explained by the unique N-acetylglucosamine end that binds to leucine-rich repeats on TLRs. Our data suggest that the effects of HMOs on TLR signaling and immunomodulation of macrophages are HMO-structure dependent.

Abatacept blocks anti-citrullinated protein antibody and rheumatoid factor\xa0mediated cytokine production in human macrophages in IDO-dependent manner

BackgroundThe anti-inflammatory effect of abatacept is most pronounced in patients with high-titer autoantibodies (including anticitrullinated protein antibodies [ACPA] and rheumatoid factor [RF]). Considering that autoantibodies trigger inflammatory cytokine production by monocytes and that abatacept binds to monocytes, influencing their functional state, we hypothesized that abatacept may effectively inhibit the production of several different cytokines by ACPA- or RF-challenged monocytes.MethodsPeripheral blood CD68+ monocytes stimulated with macrophage colony-stimulating factor for 24 h were exposed to random immunoglobulin G alone (negative control), purified ACPA, purified RF, or lipopolysaccharide (positive control) in cell culture plates coated with citrullinated vimentin (to allow ACPA immune complex formation). Stimulations were done in the presence or absence of abatacept or tumor necrosis factor (TNF) antibody (adalimumab) with or without indoleamine 2,3-dioxygenase (IDO) inhibitor 1-methyl-d-tryptophan. Supernatants were analyzed for key proinflammatory cytokines TNF-α, interleukin (IL)-1β, IL-6, IL-8, and chemokine (C-C motif) ligand 2 (CCL2) after 24 h.ResultsExposure to ACPA or RF significantly induced the production of TNF-α (20-fold and 27-fold, respectively), IL-1β (each 4-fold), IL-6 (12-fold and 11-fold, respectively), IL-8 (43-fold and 30-fold, respectively), and CCL2 (each 4-fold) in human monocytes. Abatacept inhibited this autoantibody-mediated upregulation of cytokines, reducing TNF-α by > 75%, IL-1β by > 65%, IL-6 and IL-8 by > 80%, and CCL2 by > 60%. In contrast, a TNF inhibitor did not influence autoantibody-induced proinflammatory cytokine production. IDO inhibition reversed the effect of abatacept and again permitted the induction of cytokine production by ACPA and RF.ConclusionsThese data show that abatacept interferes with autoantibody-mediated cytokine production by monocytes through induction of IDO. This inhibitory effect on the production of several effector cytokines in RA may explain the fast anti-inflammatory effect of abatacept as well as its preferential efficacy in patients with high-titer ACPA and RF.

Neurotrophic, Cytoprotective, and Anti-inflammatory Effects of St. John's Wort Extract on Differentiated Mouse Hippocampal HT-22 Neurons.

Introduction: Since ancient times Hypericum perforatum L. named St. John's wort (SJW), has been used in the management of a wide range of applications, including nervous disorders. Development of mood disorders are due to alterations in glutamate metabolism, initiation of inflammatory pathways, and changes of the neuronal plasticity. Previous studies suggest that the glutamatergic system contributes to the pathophysiology of depression. Extracts of SJW have been recommended for the treatment of depression. The aim of the present in vitro study was to evaluate the action of STW3-VI, a special SJW extract in differentiated mouse hippocampal HT-22 neurons. We evaluated the stimulation of neurogenesis, the protective effect against glutamate or N-methyl-D-aspartate receptor induced-excitotoxicity and its anti-inflammatory properties in LPS-activated human macrophages.Results: After 48 h treatment, STW3-VI stimulated the neurite formation by 25% in comparison with the control and showed protective effects against glutamate- or NMDA-induced cytotoxicity by significantly increasing the viability about +25 or +50%. In conjunction with these effects, after pretreatment with STW3-VI, the intracellular reduced glutathione content was significantly 2.3-fold increased compared with the neurons incubated with glutamate alone. Additionally, pre-treatment of human macrophages with STW3-VI showed anti-inflammatory effects after 24 or 48 h concerning inhibition of LPS-induced TNF release by −47.3 and −53.8% (24 h) or −25.0 to −64.8% (48 h).Conclusions: Our data provide new evidence that STW3-VI protects hippocampal cells from NMDA- or glutamate-induced cytotoxicity. Moreover, our results indicate a morphological remodeling by increasing neurite outgrowth and activation of the anti-inflammatory defense by inhibition of the cytokine production in human macrophages after STW3-VI treatment. These protective, neurotrophic and anti-inflammatory properties may be beneficial in the treatment of depressive disorders.

H5N1 influenza virus-specific miRNA-like small RNA increases cytokine production and mouse mortality via targeting poly(rC)-binding protein 2

Infection of H5N1 influenza virus causes the highest mortality among all influenza viruses. The mechanisms underlying such high viral pathogenicity are incompletely understood. Here, we report that the H5N1 influenza virus encodes a microRNA-like small RNA, miR-HA-3p, which is processed from a stem loop-containing viral RNA precursor by Argonaute 2, and plays a role in enhancing cytokine production during H5N1 infection. Mechanistic study shows that miR-HA-3p targets poly(rC)-binding protein 2 (PCBP2) and suppresses its expression. Consistent with PCBP2 being an important negative regulator of RIG-I/MAVS-mediated antiviral innate immunity, suppression of PCBP2 expression by miR-HA-3p promotes cytokine production in human macrophages and mice infected with H5N1 virus. We conclude that miR-HA-3p is the first identified influenza virus-encoded microRNA-like functional RNA fragment and a novel virulence factor contributing to H5N1-induced 'cytokine storm' and mortality.

Comprehensive RNAi-based screening of human and mouse TLR pathways identifies species-specific preferences in signaling protein use

Abstract Toll-like receptors (TLRs) are a major class of pattern recognition receptors, which mediate the responses of innate immune cells to microbial stimuli. To systematically determine the roles of proteins in canonical TLR signaling pathways, we conducted an RNA interference (RNAi)–based screen in human and mouse macrophages. We observed a pattern of conserved signaling module dependencies across species, but found notable species-specific requirements at the level of individual proteins. Among these, we identified unexpected differences in the involvement of members of the interleukin-1 receptor–associated kinase (IRAK) family between the human and mouse TLR pathways. Whereas TLR signaling in mouse macrophages depended primarily on IRAK4 and IRAK2, with little or no role for IRAK1, TLR signaling and pro inflammatory cytokine production in human macrophages depended on IRAK1, with knockdown of IRAK4 or IRAK2 having less of an effect. Consistent with species-specific roles for these kinases, IRAK4 orthologs failed to rescue signaling in IRAK4-deficient macrophages from the other species, and only mouse macrophages required the kinase activity of IRAK4 to mediate TLR responses. The identification of a critical role for IRAK1 in TLR signaling in humans could potentially explain the association of IRAK1 with several autoimmune diseases. Furthermore, this study demonstrated how systematic screening can be used to identify important characteristics of innate immune responses across species, which could optimize therapeutic targeting to manipulate human TLR-dependent outputs.

Comprehensive RNAi-based screening of human and mouse TLR pathways identifies species-specific preferences in signaling protein use.

Toll-like receptors (TLRs) are a major class of pattern recognition receptors, which mediate the responses of innate immune cells to microbial stimuli. To systematically determine the roles of proteins in canonical TLR signaling pathways, we conducted an RNA interference (RNAi)-based screen in human and mouse macrophages. We observed a pattern of conserved signaling module dependencies across species, but found notable species-specific requirements at the level of individual proteins. Among these, we identified unexpected differences in the involvement of members of the interleukin-1 receptor-associated kinase (IRAK) family between the human and mouse TLR pathways. Whereas TLR signaling in mouse macrophages depended primarily on IRAK4 and IRAK2, with little or no role for IRAK1, TLR signaling and proinflammatory cytokine production in human macrophages depended on IRAK1, with knockdown of IRAK4 or IRAK2 having less of an effect. Consistent with species-specific roles for these kinases, IRAK4 orthologs failed to rescue signaling in IRAK4-deficient macrophages from the other species, and only mouse macrophages required the kinase activity of IRAK4 to mediate TLR responses. The identification of a critical role for IRAK1 in TLR signaling in humans could potentially explain the association of IRAK1 with several autoimmune diseases. Furthermore, this study demonstrated how systematic screening can be used to identify important characteristics of innate immune responses across species, which could optimize therapeutic targeting to manipulate human TLR-dependent outputs.

Synergistic Activation of Inflammatory Cytokine Genes by Interferon-γ-Induced Chromatin Remodeling and Toll-like Receptor Signaling

Synergistic activation of inflammatory cytokine genes by interferon-γ (IFN-γ) and Toll-like receptor (TLR) signaling is important for innate immunity and inflammatory disease pathogenesis. Enhancement of TLR signaling, a previously proposed mechanism, is insufficient to explain strong synergistic activation of cytokine production in human macrophages. Rather, we found that IFN-γ induced sustained occupancy of transcription factors STAT1, IRF-1, and associated histone acetylation at promoters and enhancers at the TNF, IL6, and IL12B loci. This priming of chromatin did not activate transcription but greatly increased and prolonged recruitment of TLR4-induced transcription factors and RNA polymerase II to gene promoters and enhancers. Priming sensitized cytokine transcription to suppression by Jak inhibitors. Genome-wide analysis revealed pervasive priming of regulatory elements by IFN-γ and linked coordinate priming of promoters and enhancers with synergistic induction of transcription. Our results provide a synergy mechanism whereby IFN-γ creates a primed chromatin environment to augment TLR-induced gene transcription.

129 : IFN-g and TNF alter epigenetic landscapes to control TLR-induced transcriptional responses in macrophages

Macrophage activation phenotypes are determined by the interplay between cytokines such as IFN-g or TNF and acute inflammatory stimuli such as TLR ligands. Synergistic activation of inflammatory cytokine genes by IFN-g and TLR signaling is important for innate immunity and inflammatory disease pathogenesis. Enhancement of TLR signaling, a previously proposed mechanism, is insufficient to explain massive synergistic activation of cytokine production in human macrophages. We found that IFN-g induced sustained occupancy of STAT1, IRF-1 and associated histone acetylation at TSS-proximal and distal regulatory elements at the TNF , IL6 and IL12B gene loci in human macrophages. This priming or opening of chromatin did not activate transcription, but greatly increased and prolonged recruitment of TLR4-induced transcription factors and RNA polymerase II to gene promoters and enhancers. Genome-wide analysis revealed pervasive priming of regulatory elements by IFN-g and linked coordinate priming of promoters and enhancers with synergistic induction of transcription. IFN-g-mediated priming conferred sensitivity of cytokine gene transcription to inhibitors of Jak kinases or BET chromatin regulators. These results provide a synergy mechanism whereby IFN-g creates a primed chromatin environment to augment TLR-induced gene transcription, and suggest therapeutic approaches that selectively target priming mechanisms. In contrast, TNF tolerizes macrophages such that inflammatory cytokine genes are not activated in response to subsequent TLR stimulation. Combined genome-wide analysis using RNAseq and ChIPseq identified transcriptional modules that represent subsets of tolerized genes that are regulated by distinct alterations in chromatin states. Overall the results highlight the importance of chromatin-based mechanisms in regulating transcriptional responses to acute stimuli that activate canonical inflammatory signaling pathways.

Priming of regulatory DNA elements activates inflammatory cytokine genes synergistically with TLR signaling to increase transcription (P1230)

Abstract Synergistic activation of inflammatory cytokine genes by IFN-γ and Toll-like receptor signaling is important for innate immunity and inflammatory disease pathogenesis. Enhancement of Toll-like receptor signaling events, a previously proposed mechanism, is insufficient to explain massive activation of cytokine production in human macrophages. We found that IFN-γ induced sustained occupancy of STAT1 and IRF1 at proximal and distal regulatory regions of the TNF, IL6 and IL12B gene loci in human macrophages, which was associated with increased histone acetylation, an indicator of an active histone state. This opening of chromatin greatly facilitated further recruitment of other transcription factors and the assembly of the RNA polymerase II complex upon Toll-like receptor activation, which resulted in increased and prolonged transcription of these inflammatory cytokines. Genome-wide analysis revealed pervasive priming of regulatory elements by IFN-γ and linked coordinate priming of promoters and enhancers with synergistic induction of transcription. Furthermore, IFN-γ-mediated priming conferred sensitivity of cytokine gene transcription to inhibitors of Jak kinases or chromatin regulators of the bromo and extra terminal protein family. Our results provide a synergy mechanism whereby IFN-γ creates a primed chromatin environment to augment Toll-like receptor-induced gene transcription, and suggest therapeutic approaches that selectively target priming mechanisms.

Differences in Cytokine Production in Human Macrophages and in Virulence in Mice Are Attributable to the Acidic Polymerase Protein of Highly Pathogenic Influenza A Virus Subtype H5N1

The pathogenesis of influenza A virus subtype H5N1 (hereafter, "H5N1") infection in humans is not completely understood, although hypercytokinemia is thought to play a role. We previously reported that most H5N1 viruses induce high cytokine responses in human macrophages, whereas some H5N1 viruses induce only a low level of cytokine production similar to that induced by seasonal viruses. To identify the viral molecular determinants for cytokine induction of H5N1 viruses in human macrophages, we generated a series of reassortant viruses between the high cytokine inducer A/Vietnam/UT3028II/03 clone 2 (VN3028IIcl2) and the low inducer A/Indonesia/UT3006/05 (IDN3006) and evaluated cytokine expression in human macrophages. Viruses possessing the acidic polymerase (PA) gene of VN3028IIcl2 exhibited high levels of hypercytokinemia-related cytokine expression in human macrophages, compared with IDN3006, but showed no substantial differences in viral growth in these cells. Further, the PA gene of VN3028IIcl2 conferred enhanced virulence in mice. These results demonstrate that the PA gene of VN3028IIcl2 affects cytokine production in human macrophages and virulence in mice. These findings provide new insights into the cytokine-mediated pathogenesis of H5N1 infection in humans.

β-Glucans inhibit intracellular growth of Mycobacterium bovis BCG but not virulent Mycobacterium tuberculosis in human macrophages

The yeast polysaccharide, β-glucan, has been shown to promote both anti-microbial and anti-tumor activities through its interaction with macrophages. Here we analyzed the effects of an insoluble whole glucan particle (WGP), a 1,3/1,6-β-glucan from Saccharomyces cerevisiae, and a soluble poly-1-6-β-d-glucopyranosyl-1-3-β-d-glucopyranose (PGG), a hydrolytic product of WGP, on the anti-microbial response of human macrophages against mycobacterial infection. Treatment of macrophages with WGP and PGG significantly decreased cell association and intracellular growth of Mycobacterium bovis BCG, but not Mycobacterium tuberculosis (M.tb) when compared to untreated controls. We characterized the influence of β-glucans on the generation of macrophage oxidative products and pro-inflammatory cytokines, two important anti-microbial defense mechanisms. WGP but not PGG treatment enhanced the oxidative response of macrophages as determined by the 2′,7′-dichlorofluorescin (DCF) assay. WGP treatment also induced macrophages to produce pro-inflammatory cytokines. The β-glucan receptor, Dectin-1, was found to be involved in the WGP-induced macrophage oxidative burst and intracellular growth inhibition of M. bovis BCG. This report indicates that although some forms of β-glucan are able to stimulate the respiratory burst and cytokine production in human macrophages, and exhibit anti-microbial properties against M. bovis BCG, the β-glucans tested here did not inhibit growth of M.tb within human macrophages.