Activation Of Human Macrophages Research Articles

Widespread S-persulfidation in activated macrophages as a protective mechanism against oxidative-inflammatory stress

Acute inflammatory responses often involve the production of reactive oxygen and nitrogen species by innate immune cells, particularly macrophages. How activated macrophages protect themselves in the face of oxidative-inflammatory stress remains a long-standing question. Recent evidence implicates reactive sulfur species (RSS) in inflammatory responses; however, how endogenous RSS affect macrophage function and response to oxidative and inflammatory insults remains poorly understood. In this study, we investigated the endogenous pathways of RSS biogenesis and clearance in macrophages, with a particular focus on exploring how hydrogen sulfide (H2S)-mediated S-persulfidation influences macrophage responses to oxidative-inflammatory stress. We show that classical activation of mouse or human macrophages using lipopolysaccharide and interferon-γ (LPS/IFN-γ) triggers substantial production of H2S/RSS, leading to widespread protein persulfidation. Biochemical and proteomic analyses revealed that this surge in cellular S-persulfidation engaged ∼2% of total thiols and modified over 800 functionally diverse proteins. S-persulfidation was found to be largely dependent on the cystine importer xCT and the H2S-generating enzyme cystathionine γ-lyase and was independent of changes in the global proteome. We further investigated the role of the sulfide-oxidizing enzyme sulfide quinone oxidoreductase (SQOR), and found that it acts as a negative regulator of S-persulfidation. Elevated S-persulfidation following LPS/IFN-γ stimulation or SQOR inhibition was associated with increased resistance to oxidative stress. Upregulation of persulfides also inhibited the activation of the macrophage NLRP3 inflammasome and provided protection against inflammatory cell death. Collectively, our findings shed light on the metabolism and effects of RSS in macrophages and highlight the crucial role of persulfides in enabling macrophages to withstand and alleviate oxidative-inflammatory stress.

Effect of the polysaccharide capsule and its heptose on the resistance of Campylobacter jejuni to innate immune defenses.

Campylobacter jejuni is a commensal in many animals but causes diarrhea in humans. Its polysaccharide capsule contributes to host colonization and virulence in a strain- and model-specific manner. We investigated if the capsule and its heptose are important for interactions of strain NCTC 11168 with various hosts and their innate immune defenses. We determined that they support bacterial survival in Drosophila melanogaster and enhance virulence in Galleria mellonella. We showed that the capsule had limited antiphagocytic activity in human and chicken macrophages, decreased adherence to chicken macrophages, and decreased intracellular survival in both macrophages. In contrast, the heptose increased uptake by chicken macrophages and supported adherence to human macrophages and survival within them. While the capsule triggered nitric oxide production in chicken macrophages, the heptose mitigated this and protected against nitrosative assault. Finally, the C. jejuni strain NCTC 11168 elicited strong cytokine production in both macrophages but quenched ROS production independently from capsule and heptose, and while the capsule and heptose did not protect against oxidative assault, they favored growth in biofilms under oxidative stress. This study shows that the wild-type capsule with its heptose is optimized to resist innate defenses in strain NCTC 11168 often via antagonistic effects of the capsule and its heptose.

Cutting Edge: STING Induces ACLY Activation and Metabolic Adaptations in Human Macrophages through TBK1.

The 2'3'-cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of IFN genes (STING) pathway can sense infection and cellular stress by detecting cytosolic DNA. Upon ligand binding, cGAS produces the cyclic dinucleotide messenger cGAMP, which triggers its receptor STING. Active STING initiates gene transcription through the transcription factors IFN regulatory factor 3 (IRF3) and NF-κB and induces autophagy, but whether STING can cause changes in the metabolism of macrophages is unknown. In this study, we report that STING signaling activates ATP-citrate lyase (ACLY) by phosphorylation in human macrophages. Using genetic and pharmacologic perturbation, we show that STING targets ACLY via its prime downstream signaling effector TANK (TRAF family member-associated NF-κB activator)-binding kinase 1 (TBK1). We further identify that TBK1 alters cellular metabolism upon cGAMP treatment. Our results suggest that STING-mediated metabolic reprogramming adjusts the cellular response to DNA sensing in addition to transcription factor activation and autophagy induction.

PSGL-1 Blockade Induces Classical Activation of Human Tumor-associated Macrophages.

This work is a significant and actionable advance, as it offers a novel approach to treating patients with cancer who do not respond to T-cell checkpoint inhibitors, as well as to patients with tumors lacking T-cell infiltration. We expect that this mechanism will be applicable in multiple indications characterized by infiltration of TAMs.

AGEs induce high expression of Dll4 via endoplasmic reticulum stress PERK signaling-mediated internal ribosomal entry site mechanism in macrophages

Background and aimAdvanced glycation end products (AGEs)- exposed macrophages was characterized by Delta-like ligand 4 (Dll4) high expressed and has been shown to participate in diabetes-related atherosclerosis. This study was aimed to investigate the translational regulatory mechanism of Dll4 high expression in macrophages exposed to AGEs. MethodsHuman Dll4 5′ untranslated region (5′UTR) sequence was cloned and inserted into a bicistronic reporter plasmid. Human THP-1 macrophages transfected with the bicistronic reporter plasmids were exposed to AGEs. Dual-luciferase assay was used to detect internal ribosome entry site (IRES) activity contained in Dll4 5′UTR. Small interference RNA transfection was used to knock-down specific gene expression. Localization of protein was analyzed. ResultsAGEs exposure significantly induced IRES activity in Dll4 5′ UTR in human macrophages. Internal potential promoter and ribosome read-through mechanisms were excluded. Inhibition of endoplasmic reticulum stress and specific silencing of protein kinase R-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α) signaling pathway activation reduced IRES activity in Dll4 5′ UTR in human macrophages. Dll4 5′ UTR IRES activity was also inhibited by targeted silencing of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1). Moreover, specific inhibition of PERK/eIF2α signaling pathway led to deactivation of hnRNPA1, resulting to reduction of AGEs- induced Dll4 5’ UTR IRES activity in human macrophages. ConclusionsAGEs induced Dll4 5′ UTR IRES activity in human macrophages which was dependent on endoplasmic reticulum stress PERK/eIF2α signaling pathway. hnRNPA1 acted the role as an ITAF was also indispensable for AGEs-induced Dll4 5′UTR IRES activity in human macrophages.

Visualization of Caspase Activation in Human Monocyte-Derived Macrophages

Visualization of Caspase Activation in Human Monocyte-Derived Macrophages

Visualization of Caspase Activation in Human Monocyte-Derived Macrophages

Visualization of Caspase Activation in Human Monocyte-Derived Macrophages

Therapeutic Effect of HDAC5 Binding and Cell Penetrating Peptide for the Treatment of Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is an incurable disease that negatively influences the quality of life of patients. Current and emerging therapies target proinflammatory cytokines and/or receptors to downregulate proinflammatory responses, but insufficient remission requires other therapeutic agents. Herein, we report that the synthetic anti-inflammatory peptide 15 (SAP15) is capable of cell penetration and anti-inflammatory activity in human macrophages. SAP15 was labeled with fluorescence and administered to human leukemiamonocytic cells (THP-1) cells for cell penetration analysis. Using biolayer interferometry analysis, the binding affinity of SAP15 with histone deacetylase 5 (HDAC5) was measured. SAP15-treated THP-1 cells were analyzed by protein phosphorylation assay, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). In addition, in vivo analysis of the therapeutic effect on IBD was observed in a dextran sulfate sodium (DSS)-induced model. Samples from SAP15-treated mice were analyzed at both the macroscopic and microscopic levels using ELISA, myeloperoxidase (MPO) assays, and histological evaluations. SAP15 was internalized within the cytosol and nucleus of THP-1 cells and bound to the HDAC5 protein. SAP15-treated macrophages were assessed for protein phosphorylation and showed inhibited phosphorylation of HDAC5 and other immune-related proteins, which led to increased M2-like macrophage markers and decreased M1-like macrophage markers and tumor necrosis factor-α and interleukin-6 cytokine levels. The SAP15 treatment on IBD model showed significant recovery of colon length. Further histological analysis of colon demonstrated the therapeutic effect of SAP15 on mucosal layer. Moreover, proinflammatory cytokine levels and MPO activity from the plasma show that SAP15 is effective in reduced proinflammatory responses. These findings suggest that SAP15 is a novel peptide with a novelcell-penetratingpeptide with anti-inflammatory property that can be used as a therapeutic agent for IBD and other inflammatory diseases.

Targeting macrophagic PIM-1 alleviates osteoarthritis by inhibiting NLRP3 inflammasome activation via suppressing mitochondrial ROS/Cl− efflux signaling pathway

BackgroundOsteoarthritis (OA), in which macrophage-driven synovitis is considered closely related to cartilage destruction and could occur at any stage, is an inflammatory arthritis. However, there are no effective targets to cure the progression of OA. The NOD-, LRR-,and pyrin domain-containing protein 3 (NLRP3) inflammasome in synovial macrophages participates in the pathological inflammatory process and treatment strategies targeting it are considered to be an effective approach for OA. PIM-1 kinase, as a downstream effector of many cytokine signaling pathways, plays a pro-inflammatory role in inflammatory disease.MethodsIn this study, we evaluated the expression of the PIM-1 and the infiltration of synovial macrophages in the human OA synovium. The effects and mechanism of PIM-1 were investigated in mice and human macrophages stimulated by lipopolysaccharide (LPS) and different agonists such as nigericin, ATP, Monosodium urate (MSU), and Aluminum salt (Alum). The protective effects on chondrocytes were assessed by a modified co-culture system induced by macrophage condition medium (CM). The therapeutic effect in vivo was confirmed by the medial meniscus (DMM)-induced OA in mice.ResultsThe expression of PIM-1 was increased in the human OA synovium which was accompanied by the infiltration of synovial macrophages. In vitro experiments, suppression of PIM-1 by SMI-4a, a specific inhibitor, rapidly inhibited the NLRP3 inflammasome activation in mice and human macrophages and gasdermin-D (GSDME)-mediated pyroptosis. Furthermore, PIM-1 inhibition specifically blocked the apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization in the assembly stage. Mechanistically, PIM-1 inhibition alleviated the mitochondrial reactive oxygen species (ROS)/chloride intracellular channel proteins (CLICs)-dependent Cl− efflux signaling pathway, which eventually resulted in the blockade of the ASC oligomerization and NLRP3 inflammasome activation. Furthermore, PIM-1 suppression showed chondroprotective effects in the modified co-culture system. Finally, SMI-4a significantly suppressed the expression of PIM-1 in the synovium and reduced the synovitis scores and the Osteoarthritis Research Society International (OARSI) score in the DMM-induced OA model.ConclusionsTherefore, PIM-1 represented a new class of promising targets as a treatment of OA to target these mechanisms in macrophages and widened the road to therapeutic strategies for OA.

Lipoprotein(a) induces caspase-1 activation and IL-1 signaling in human macrophages.

Lipoprotein(a) (Lp(a)) is an LDL-like particle with an additional apolipoprotein (apo)(a) covalently attached. Elevated levels of circulating Lp(a) are a risk factor for atherosclerosis. A proinflammatory role for Lp(a) has been proposed, but its molecular details are incompletely defined. To explore the effect of Lp(a) on human macrophages we performed RNA sequencing on THP-1 macrophages treated with Lp(a) or recombinant apo(a), which showed that especially Lp(a) induces potent inflammatory responses. Thus, we stimulated THP-1 macrophages with serum containing various Lp(a) levels to investigate their correlations with cytokines highlighted by the RNAseq, showing significant correlations with caspase-1 activity and secretion of IL-1β and IL-18. We further isolated both Lp(a) and LDL particles from three donors and then compared their atheroinflammatory potentials together with recombinant apo(a) in primary and THP-1 derived macrophages. Compared with LDL, Lp(a) induced a robust and dose-dependent caspase-1 activation and release of IL-1β and IL-18 in both macrophage types. Recombinant apo(a) strongly induced caspase-1 activation and IL-1β release in THP-1 macrophages but yielded weak responses in primary macrophages. Structural analysis of these particles revealed that the Lp(a) proteome was enriched in proteins associated with complement activation and coagulation, and its lipidome was relatively deficient in polyunsaturated fatty acids and had a high n-6/n-3 ratio promoting inflammation. Our data show that Lp(a) particles induce the expression of inflammatory genes, and Lp(a) and to a lesser extent apo(a) induce caspase-1 activation and IL-1 signaling. Major differences in the molecular profiles between Lp(a) and LDL contribute to Lp(a) being more atheroinflammatory.

PKM2 promotes proinflammatory macrophage activation in ankylosing spondylitis.

Macrophages play a critical role in ankylosing spondylitis by promoting autoimmune tissue inflammation through various effector functions. The inflammatory potential of macrophages is highly influenced by their metabolic environment. Here, we demonstrate that glycolysis is linked to the proinflammatory activation of human blood monocyte-derived macrophages in ankylosing spondylitis. Specifically, ankylosing spondylitis macrophages produced excessive inflammation, including TNFα, IL1β, and IL23, and displayed an overactive status by exhibiting stronger costimulatory signals, such as CD80, CD86, and HLA-DR. Moreover, we found that patient-derived monocyte-derived M1-type macrophages (M1 macrophages) exhibited intensified glycolysis, as evidenced by a higher extracellular acidification rate. Upregulation of PKM2 and GLUT1 was observed in ankylosing spondylitis-derived monocytes and monocyte-derived macrophages, especially in M1 macrophages, indicating glucose metabolic alteration in ankylosing spondylitis macrophages. To investigate the impact of glycolysis on macrophage inflammatory ability, we treated ankylosing spondylitis M1 macrophages with 2 inhibitors: 2-deoxy-D-glucose, a glycolysis inhibitor, and shikonin, a PKM2 inhibitor. Both inhibitors reduced proinflammatory function and reversed the overactive status of ankylosing spondylitis macrophages, suggesting their potential utility in treating the disease. These data place PKM2 at the crosstalk between glucose metabolic changes and the activation of inflammatory macrophages in patients with ankylosing spondylitis.

The nuclear receptor Nurr1 regulates the expression and activity of PPARγ in human pro-inflammatory macrophages

Abstract Nurr1 is a member of the orphan nuclear receptor family NR4A that modulates inflammation in several cell lineages through inhibition of the NF-κB signaling pathway. Nurr1 was found to be able to interact with the peroxisome proliferator-activated receptor (PPAR)γ, as well as to be recruited to the PPARG promoter in LPS-activated microglial cells; however, the functional relationship between Nurr1 and PPARγ is not clearly established. Here, we aimed to investigate the role of Nurr1 on PPARγ activity in human macrophages. Blood monocytes were cultured with GM-CSF or M-CSF to generate pro- (GM-MDMs) and anti-inflammatory (M-MDMs) macrophages, respectively. GM-MDMs showed enhanced expression of both Nurr1 and PPARγ compared to M-MDMs, and they were preferentially located at the cytoplasmic (Nurr1) and nuclear (PPARγ) compartments. PPARγ activation in GM-MDMs with the agonist rosiglitazone did not modify Nurr1 expression. Conversely, Nurr1 activation with the agonist C-DIM12 increased PPARγ expression. This was concomitant with enhanced expression of two PPARγ target genes, CD36 and PLIN2, while silencing Nurr1 expression led to a decrease in the amounts of CD36 transcripts. Co-treatment of GM-MDMs with C-DIM12 and rosiglitazone synergistically enhanced the transcriptional activity of PPARγ, whereas C-DIM12 alone had no effect. PPARγ and Nurr1 agonists alone exerted anti-inflammatory effects on GM-MDMs, and also showed synergistic activity in combined form. In sum, our data suggests that Nurr1 positively regulates PPARγ activity at least partially through induction of PPARγ expression, which may represent a novel mechanism of Nurr1 targeting metabolic pathways and inflammation. Supported by grants from the Secretaría de Educación Pública-Consejo Nacional de Ciencia y Tecnología (SEP-CONACYT, #A1-S-9430)

A POLY(SBMA) ZWITTERIONIC HYDROGEL COATING OF POLYIMIDE SURFACES TO REDUCE THE FOREIGN BODY REACTION TO INVASIVE NEURAL INTERFACES

Intraneural electrodes can be harnessed to control neural prosthetic devices in human amputees. However, in chronic implants we witness a gradual loss of device functionality and electrode isolation due to a nonspecific inflammatory response to the implanted material, called foreign body reaction (FBR). FBR may eventually lead to a fibrous encapsulation of the electrode surface. Poly(ethylene glycol) (PEG) is one of the most common low-fouling materials used to coat and protect electrode surfaces. Yet, PEG can easily undergo encapsulation and oxidative damage in long-term in vivo applications. Poly(sulfobetaine methacrylate) - poly(SBMA) - zwitterionic hydrogels may represent more promising alternatives to minimize the FBR due to their ultra-low fouling features. Here, we tested and compared the poly(SBMA) zwitterionic hydrogel coating with the PEG coating in reducing adhesion and activation of pro-inflammatory and pro-fibrotic cells to polyimide surfaces, which are early hallmarks of FBR. We aimed to coat polyimide surfaces with a hydrogel thin film and analysed the release of a model drug from the hydrogel.We performed hydrogel synthesis, mechanical characterization and biocompatibility analysis. Cell adhesion, viability and morphology of human myofibroblasts cultured on PEG- and hydrogel-coated surfaces were evaluated through confocal microscopy-based high-content analysis (HCA). Reduced activation of pro-inflammatory human macrophages cultured on hydrogels was assessed as well as the hydrogel drug release profile.Because of its high hydration, biocompatibility, low stiffness and ultra-low fouling characteristics the hydrogel enabled lower adhesion and activation of pro-inflammatory and pro-fibrotic cells vs. polystyrene controls, and showed a long-term release of the anti-fibrotic drug Everolimus. Furthermore, a polyimide surface was successfully coated with a hydrogel thin film.Our soft zwitterionic hydrogel could outperform PEG as more suitable coating material of neural electrodes for mitigating the FBR. Such poly(SBMA)-based biomaterial could also be envisioned as long-term delivery system for a sustained release of anti-inflammatory and anti-fibrotic drugs in vivo.

Comparing the Effects of Rocaglates on Energy Metabolism and Immune Modulation on Cells of the Human Immune System.

A promising new approach to broad spectrum antiviral drugs is the inhibition of the eukaryotic translation initiation factor 4A (elF4A), a DEAD-box RNA helicase that effectively reduces the replication of several pathogenic virus types. Beside the antipathogenic effect, modulation of a host enzyme activity could also have an impact on the immune system. Therefore, we performed a comprehensive study on the influence of elF4A inhibition with natural and synthetic rocaglates on various immune cells. The effect of the rocaglates zotatifin, silvestrol and CR-31-B (-), as well as the nonactive enantiomer CR-31-B (+), on the expression of surface markers, release of cytokines, proliferation, inflammatory mediators and metabolic activity in primary human monocyte-derived macrophages (MdMs), monocyte-derived dendritic cells (MdDCs), T cells and B cells was assessed. The inhibition of elF4A reduced the inflammatory potential and energy metabolism of M1 MdMs, whereas in M2 MdMs, drug-specific and less target-specific effects were observed. Rocaglate treatment also reduced the inflammatory potential of activated MdDCs by altering cytokine release. In T cells, the inhibition of elF4A impaired their activation by reducing the proliferation rate, expression of CD25 and cytokine release. The inhibition of elF4A further reduced B-cell proliferation, plasma cell formation and the release of immune globulins. In conclusion, the inhibition of the elF4A RNA helicase with rocaglates suppressed the function of M1 MdMs, MdDCs, T cells and B cells. This suggests that rocaglates, while inhibiting viral replication, may also suppress bystander tissue injury by the host immune system. Thus, dosing of rocaglates would need to be adjusted to prevent excessive immune suppression without reducing their antiviral activity.

IL-27 inhibits anti- Mycobacterium tuberculosis innate immune activity of primary human macrophages

Mycobacterium tuberculosis (M. tuberculosis) is an intracellular pathogen that primarily infects macrophages. Despite a robust anti-mycobacterial response, many times macrophages are unable to control M. tuberculosis. The purpose of this study was to investigate the mechanism by which the immunoregulatory cytokine IL-27 inhibits the anti-mycobacterial activity of primary human macrophages. We found concerted production of IL-27 and anti-mycobacterial cytokines by M. tuberculosis-infected macrophages in a toll-like receptor (TLR) dependent manner. Notably, IL-27 suppressed the production of anti-mycobacterial cytokines TNFα, IL-6, IL-1β, and IL-15 by M. tuberculosis-infected macrophages. IL-27 limits the anti-mycobacterial activity of macrophages by reducing Cyp27B, cathelicidin (LL-37), LC3B lipidation, and increasing IL-10 production. Furthermore, neutralizing both IL-27 and IL-10 increased the expression of proteins involved in LC3-associated phagocytosis (LAP) pathway for bacterial clearance, namely vacuolar-ATPase, NOX2, and RUN-domain containing protein RUBCN. These results implicate IL-27 is a prominent cytokine that impedes M. tuberculosis clearance.

Differential Modulation of Human M1 and M2 Macrophage Activity by ICOS-Mediated ICOSL Triggering.

Activated T cells express the inducible T-cell co-stimulator (ICOS) that, upon binding to its ubiquitously expressed ligand (ICOSL), regulates the immune response and tissue repair. We sought to determine the effect of ICOS:ICOSL interaction on human M1 and M2 macrophages. M1 and M2 macrophages were polarized from monocyte-derived macrophages, and the effect of a soluble recombinant form of ICOS (ICOS-CH3) was assessed on cytokine production and cell migration. We show that ICOS-CH3 treatment increased the secretion of CCL3 and CCL4 in resting M1 and M2 cells. In LPS-treated M1 cells, ICOS-CH3 inhibited the secretion of TNF-α, IL-6, IL-10 and CCL4, while it increased that of IL-23. In contrast, M2 cells treated with LPS + IL4 displayed enhanced secretion of IL-6, IL-10, CCL3 and CCL4. In CCL7- or osteopontin-treated M1 cells, ICOS-CH3 boosted the migration rate of M1 cells while it decreased that of M2 cells. Finally, β-Pix expression was upregulated in M1 cells and downregulated in M2 cells by treatment with ICOS-CH3. These findings suggest that ICOSL activation modulates the activity of human M1 and M2 cells, thereby eliciting an overall anti-inflammatory effect consistent with its role in promoting tissue repair.

5-Ethynyluridine: A Bio-orthogonal Uridine Variant for mRNA-Based Therapies and Vaccines.

The identification of pseudo- and N1 -methylpseudo-uridine (Ψ and mΨ, respectively) as immunosilent uridine analogues has propelled the development of mRNA-based vaccines and therapeutics. Here, we have characterised another uridine analogue, 5-ethynyluridine (EU), which has an ethynyl moiety. We show that this uridine analogue does not cause immune activation in human macrophages, as it does not induce interleukin-6 secretion or expression of the inflammatory and antiviral genes MX1, PKR, and TAP2. Moreover, EU allows for prolonged expression, as shown with mRNA coding for yellow fluorescent protein (YFP). Side-by-side comparisons of EU with unmodified, Ψ, and mΨ revealed that EU-modified mRNA is expressed at lower levels, but confers similar stability and low immunogenicity to the other uridine analogues. Furthermore, structure analysis of modified mRNAs suggests that the observed phenotype is largely independent of RNA folding. Thus, EU is a potential candidate for RNA-based vaccines and therapeutics.

Bovine blood derived macrophages are unable to control Coxiella burnetii replication under hypoxic conditions.

Coxiella burnetii is a zoonotic pathogen, infecting humans, livestock, pets, birds and ticks. Domestic ruminants such as cattle, sheep, and goats are the main reservoir and major cause of human infection. Infected ruminants are usually asymptomatic, while in humans infection can cause significant disease. Human and bovine macrophages differ in their permissiveness for C. burnetii strains from different host species and of various genotypes and their subsequent host cell response, but the underlying mechanism(s) at the cellular level are unknown. C. burnetii infected primary human and bovine macrophages under normoxic and hypoxic conditions were analyzed for (i) bacterial replication by CFU counts and immunofluorescence; (ii) immune regulators by westernblot and qRT-PCR; cytokines by ELISA; and metabolites by gas chromatography-mass spectrometry (GC-MS). Here, we confirmed that peripheral blood-derived human macrophages prevent C. burnetii replication under oxygen-limiting conditions. In contrast, oxygen content had no influence on C. burnetii replication in bovine peripheral blood-derived macrophages. In hypoxic infected bovine macrophages, STAT3 is activated, even though HIF1α is stabilized, which otherwise prevents STAT3 activation in human macrophages. In addition, the TNFα mRNA level is higher in hypoxic than normoxic human macrophages, which correlates with increased secretion of TNFα and control of C. burnetii replication. In contrast, oxygen limitation does not impact TNFα mRNA levels in C. burnetii-infected bovine macrophages and secretion of TNFα is blocked. As TNFα is also involved in the control of C. burnetii replication in bovine macrophages, this cytokine is important for cell autonomous control and its absence is partially responsible for the ability of C. burnetii to replicate in hypoxic bovine macrophages. Further unveiling the molecular basis of macrophage-mediated control of C. burnetii replication might be the first step towards the development of host directed intervention measures to mitigate the health burden of this zoonotic agent.

Inflammasome Activation in Human Macrophages: IL-1β Cleavage Detection by Fully Automated Capillary-Based Immunoassay.

Interleukin (IL)-1β is a key mediator of inflammation and activates via pattern recognition receptors (PRR) of the inflammasome family by proteolytic maturation. Proteolysis is driven by proteases such as caspase-1 (also known as IL-1 converting enzyme, ICE) and converts the intact pro-IL-1β ~31kDa pro-peptide into a mature, ~17kDa form that can exit cells through nanomolecular pores or via microvesicles. Whereas pro-IL-1β fails to trigger IL-1 receptor (IL-1R) activation, mature IL-1β, upon release from the cell, triggers pleiotropic downstream effects, establishing an inflammatory state. Hence, being able to detect IL-1β conversion is physiologically relevant for measuring inflammation, but it cannot be easily accomplished by conventional ELISA or flow cytometry as most commercially available antibodies do not discriminate mature and pro-form. Furthermore, unlike for other cytokines, the mere induction and translation of IL1B mRNA cannot serve as a proxy of inflammasome PRR activation. Rather the cleavage of IL-1β needs to be verified. Hence, conventional immunoblotting has emerged as the gold standard for demonstrating inflammasome activation as the difference in molecular weight between pro- and mature form can easily be detected. However, conventional immunoblotting suffers from poor standardization, quantification, and reproducibility, may require sample concentration, and is also not suitable for medium to high throughput. Some of these shortcomings are prohibitive for analysis of human primary samples but can be overcome by fully automated capillary-based immunoassay as we outline here. We here provide a practical guide to quantify pro- vs mature IL-1β directly from unconcentrated supernatants of human monocyte-derived macrophages. The assay may be useful for more standardized and medium-throughput analysis in these cells or other biospecimen.

Pyruvate dehydrogenase kinase supports macrophage NLRP3 inflammasome activation during acute inflammation

Activating the macrophage NLRP3 inflammasome can promote excessive inflammation with severe cell and tissue damage and organ dysfunction. Here, we show that pharmacological or genetic inhibition of pyruvate dehydrogenase kinase (PDHK) significantly attenuates NLRP3 inflammasome activation in murine and human macrophages and septic mice by lowering caspase-1 cleavage and interleukin-1β (IL-1β) secretion. Inhibiting PDHK reverses NLRP3 inflammasome-induced metabolic reprogramming, enhances autophagy, promotes mitochondrial fusion over fission, preserves crista ultrastructure, and attenuates mitochondrial reactive oxygen species (ROS) production. The suppressive effect of PDHK inhibition on the NLRP3 inflammasome is independent of its canonical role as a pyruvate dehydrogenase regulator. Our study suggests a non-canonical role of mitochondrial PDHK in promoting mitochondrial stress and supporting NLRP3 inflammasome activation during acute inflammation.