Macrophage Function Research Articles

Alpha-synuclein shapes monocyte and macrophage cell biology and functions by bridging alterations of autophagy and inflammatory pathways.

Introduction: Abnormal spreading of alpha-synuclein (αS), a hallmark of Parkinson's disease, is known to promote peripheral inflammation, which occurs in part via functional alterations in monocytes/macrophages. However, underlying intracellular mechanisms remain unclear. Methods: Herein we investigate the subcellular, molecular, and functional effects of excess αS in human THP-1 monocytic cell line, THP-1-derived macrophages, and at least preliminarily, in primary monocyte-derived macrophages (MDMs). In cells cultured w/wo recombinant αS (1μM) for 4h and 24h, by Confocal microscopy, Western Blot, RT-qPCR, Elisa, and Flow Cytometry we assessed: i) αS internalization; ii) cytokine/chemokine expression/secretion, and C-C motif chemokine receptor 2 (CCR2) levels; iii) autophagy (LC3II/I, LAMP1/LysoTracker, p62, pS6/total S6); and iv) lipid droplets (LDs) accumulation, and cholesterol pathway gene expression. Transwell migration assay was employed to measure THP-1 cell migration/chemotaxis, while FITC-IgG-bead assay was used to analyze phagocytic capacity, and the fate of phagocytosed cargo in THP-1-derived macrophages. Results: Extracellular αS was internalized by THP-1 cells, THP-1-derived macrophages, and MDMs. In THP1 cells, αS induced a general pro-inflammatory profile and conditioned media from αS-exposed THP-1 cells potently attracted unstimulated cells. However, CCL2 secretion peaked at 4h αS, consistent with early internalization of its receptor CCR2, while this was blunted at 24h αS exposure, when CCR2 recycled back to the plasma membrane. Again, 4h αS-exposed THP-1 cells showed increased spontaneous migration, while 24h αS-exposed cells showed reduced chemotaxis. This occurred in the absence of cell toxicity and was associated with upregulation of autophagy/lysosomal markers, suggesting a pro-survival/tolerance mechanism against stress-related inflammation. Instead, in THP-1-derived macrophages, αS time-dependently potentiated the intracellular accumulation, and release of pro-inflammatory mediators. This was accompanied by mild toxicity, reduced autophagy-lysosomal markers, defective LDs formation, as well as impaired phagocytosis, and the appearance of stagnant lysosomes engulfed with phagocytosed cargo, suggesting a status of macrophage exhaustion reminiscent of hypophagia. Discussion: In summary, despite an apparently similar pro-inflammatory phenotype, monocytes and macrophages respond differently to intracellular αS accumulation in terms of cell survival, metabolism, and functions. Our results suggest that in periphery, αS exerts cell- and context-specific biological effects bridging alterations of autophagy, lipid dynamics, and inflammatory pathways.

Efferocytosis dysfunction in CXCL4-induced M4 macrophages: phenotypic insights in systemic sclerosis in vitro and in vivo.

Systemic sclerosis (SSc) is an autoimmune disease characterized by antinuclear antibody production, which has been linked to an excess of apoptotic cells, normally eliminated by macrophages through efferocytosis. Additionally, circulating levels of CXCL4, a novel SSc biomarker, correlate with more severe fibrotic manifestations of the disease. Considering the defective efferocytosis of macrophages in SSc and the CXCL4-related M4 macrophage phenotype, we hypothesized that CXCL4 could be involved in the alteration of phagocytic functions of macrophages in SSc, including LC3-associated phagocytosis (LAP), another phagocytic process requiring autophagy proteins and contributing to immune silencing. In this study, CXCL4 levels were measured by ELISA in vitro in the serum of SSc patients, and also in vivo in the serum and lungs of C57BL/6J SSc mice induced by intradermal injections of hypochloric acid (HOCl) or Bleomycin (BLM), with evaluation of M4 markers. Circulating monocytes from healthy donors were also differentiated in vitro into M4 monocytes-derived macrophages (MDMs) in the presence of recombinant CXCL4. In M4-MDMs, phagocytosis of fluorescent beads and expression level of efferocytic receptors were evaluated by flow cytometry in vitro, while efferocytosis of pHrodo-stained apoptotic Jurkat cells was evaluated by real-time fluorescence microscopy. LAP quantification was made by fluorescence microscopy in M4-MDMs exposed to IgG-coated beads as well as apoptotic Jurkat cells. Our results demonstrated that efferocytosis was significantly reduced in M0-MDMs from healthy donors exposed to the CXCL4-rich plasma of SSc patients. In vivo, CXCL4 expression was increased in the lungs of both SSc-mouse models, along with elevated M4 markers, while efferocytosis of BLM-mice alveolar macrophages was decreased. In vitro, M4-MDMs exhibited reduced efferocytosis compared to M0-MDMs, notably attributable to lower CD36 receptor expression and impaired phagocytosis capacities, despite enhanced LAP. Autophagic gene expression was increased both in vitro in SSc MDMs and in vivo in BLM mice, thus acting as a potential compensatory mechanism. Altogether, our results support the role of CXCL4 on the impaired efferocytosis capacities of human macrophages from SSc patients and in SSc mice.

Aberrant Expression of SLC7A11 Impairs the Antimicrobial Activities of Macrophages in Staphylococcus Aureus Osteomyelitis in Mice.

Staphylococcus aureus (S. aureus) persistence in macrophages, potentially a reservoir for recurrence of chronic osteomyelitis, contributes to resistance and failure in treatment. As the mechanisms underlying survival of S. aureus in macrophages remain largely unknown, there has been no treatment approved. Here, in a mouse model of S. aureus osteomyelitis, we identified significantly up-regulated expression of SLC7A11 in both transcriptomes and translatomes of CD11b+F4/80+ macrophages, and validated a predominant distribution of SLC7A11 in F4/80+ cells around the S. aureus abscess. Importantly, pharmacological inhibition or genetic knockout of SLC7A11 promoted the bactericidal function of macrophages, reduced bacterial burden in the bone and improved bone structure in mice with S. aureus osteomyelitis. Mechanistically, aberrantly expressed SLC7A11 down-regulated the level of intracellular ROS and reduced lipid peroxidation, contributing to the impaired bactericidal function of macrophages. Interestingly, blocking SLC7A11 further activated expression of PD-L1 via the ROS-NF-κB axis, and a combination therapy of targeting both SLC7A11 and PD-L1 significantly enhanced the efficacy of clearing S. aureus in vitro and in vivo. Our findings suggest that targeting both SLC7A11 and PD-L1 is a promising therapeutic approach to reprogram the bactericidal function of macrophages and promote bacterial clearance in S. aureus osteomyelitis.

Macrophage Polarization and Osteoclast Differentiation.

Macrophages are a key player to regulate rheumatoid arthritis pathogenesis from onset to remission. They can alter innate functions under microenvironmental conditions. To understand heterogeneous functions of macrophages in rheumatoid arthritis, several activated statuses of macrophages should be mimicked in vitro. Here, we describe basic protocols for macrophage polarization and osteoclast differentiation.

Aryl hydrocarbon receptor confers protection against macrophage pyroptosis and intestinal inflammation through regulating polyamine biosynthesis.

Rationale: The aryl hydrocarbon receptor (AhR) functions in the regulation of intestinal inflammation, but knowledge of the underlying mechanisms in innate immune cells is limited. Here, we investigated the role of AhR in modulating the functions of macrophages in inflammatory bowel disease pathogenesis. Methods: The cellular composition of intestinal lamina propria CD45+ leukocytes in a dextran sulfate sodium (DSS)-induced mouse colitis model was determined by single-cell RNA sequencing. Macrophage pyroptosis was quantified by analysis of lactate dehydrogenase release, propidium iodide staining, enzyme-linked immunosorbent assay, western blot, and flow cytometry. Differentially expressed genes were confirmed by RNA-seq, RT-qPCR, luciferase assay, chromatin immunoprecipitation, and immunofluorescence staining. Results: AhR deficiency mediated dynamic remodeling of the cellular composition of intestinal lamina propria (LP) CD45+ immune cells in a colitis model, with a significant increase in monocyte-macrophage lineage. Mice with AhR deficiency in myeloid cells developed more severe dextran sulfate sodium induced colitis, with concomitant increased macrophage pyroptosis. Dietary supplementation with an AhR pre-ligand, indole-3-carbinol, conferred protection against colitis while protection failed in mice lacking AhR in myeloid cells. Mechanistically, AhR signaling inhibited macrophage pyroptosis by promoting ornithine decarboxylase 1 (Odc1) transcription, to enhance polyamine biosynthesis. The increased polyamine, particularly spermine, inhibited NLRP3 inflammasome assembly and subsequent pyroptosis by suppressing K+ efflux. AHR expression was positively correlated with ODC1 in intestinal mucosal biopsies from patients with ulcerative colitis. Conclusions: These findings suggest a functional role for the AhR/ODC1/polyamine axis in maintaining intestinal homeostasis, providing potential targets for treatment of inflammatory bowel disease.

Unlocking Macrophage Secrets: Histone Deacetylases in Chronic Transplant Rejection.

Solid organ transplantation (SOT) offers life-saving therapy for patients with organ failure, yet chronic rejection remains a significant challenge despite advances in immunosuppression. Macrophages are central to chronic rejection, orchestrating fibrosis, and tissue damage. Since it became clear that histone deacetylases (HDACs), a family of epigenetic regulators, modulate macrophage function and polarization and eventually affect fibrosis progression, the HDACs modulation has gained great importance. This review explores the role of HDACs in chronic rejection, focusing on their impact on macrophage polarization and fibrosis. While some HDACs promote M2 polarization and fibrosis, others inhibit these processes, highlighting the complexity of HDAC function. Targeting HDACs holds promise as a therapeutic strategy for chronic rejection, offering a potential approach for intervention in transplant recipients. However, further research is needed to elucidate the specific roles of individual HDAC isoforms and their inhibition in chronic rejection.

Engineering macrophage membrane-camouflaged nanoplatforms with enhanced macrophage function for mediating sonodynamic therapy of ovarian cancer.

Cancer immunotherapy has demonstrated remarkable efficacy in the treatment of cancer, and it has been successfully applied in the treatment of various solid tumors. However, the response rates to immunotherapy in patients with ovarian cancer remain modest because of the immunosuppressive tumor microenvironment (TME). Tumor-associated macrophages (TAMs) represent the predominant myeloid cell population within the TME, which adopt the protumorigenic M2 phenotype and are blinded by the "don't eat me" signals from tumor cells. These characteristics of TAMs result in insufficient phagocytic activation. In this study, we constructed a SIM@TR-NP-mediated combination therapy of sonodynamic and immunotherapy. SIM@TR-NPs were modified by engineered macrophage membranes with overexpressed sialic acid-binding Ig-like lectin 10 (Siglec-10), and were internally loaded with sonosensitizer 4,4',4'',4'''-(porphine-5,10,15,20-tetrayl)tetrakis(benzoic acid) and immune adjuvant resiquimod. SIM@TR-NPs can block "don't eat me" signals to enhance macrophage phagocytosis and trigger the polarization of TAMs toward the M1 phenotype, thereby improving the immunosuppressive TME. Simultaneously, upon ultrasound irradiation, SIM@TR-NP-mediated sonodynamic therapy (SDT) triggered immunogenic cell death in tumor cells, in combination with TAM-based immunotherapy, transforming the "immune cold tumor" into an "immune hot tumor". SIM@TR-NP-mediated sonodynamic immunotherapy exhibited potent antitumor efficacy in ovarian cancer and exhibited substantial potential for improving the immunosuppressive TME. This study presents an emerging therapeutic regimen for ovarian cancer that synergizes TAM-based antitumor immunotherapy and SDT.

RBM15 activates glycolysis in M1-type macrophages to promote the progression of aortic aneurysm and dissection.

Aortic aneurysm and dissection (AD) represent a critical cardiovascular emergency with an alarmingly high mortality rate. Recent research has spotlighted the overexpression of genes associated with the m6A modification in AD patients, linking them to the presence of inflammatory M1-type macrophages. Moreover, glycolysis is widely recognized as a key feature of inflammatory M1-type macrophages, but biomarkers linking glycolysis and macrophage function to promote disease progression in AD have not been reported. We conducted an analysis of aortic immune cell infiltration, macrophages, and m6A-related biomarkers in AD patients using bioinformatics techniques. Subsequently, we employed a combination of RT-PCR, WB, and immunofluorescence assays to elucidate the alterations in the expression of M1- and M2-type macrophages, as well as markers of glycolysis, following the overexpression of key biomarkers. These findings were further validated in vivo through the creation of a rat model of AD with knockdown of the aforementioned key biomarkers. The findings revealed that the m6A-modified related gene RBM15 exhibited heightened expression in AD samples and was correlated with macrophage polarization. Upon overexpression of RBM15 in macrophages, there was an observed increase in the expression of M1-type macrophage markers CXCL9 and CXCL10, alongside a decrease in the expression of M2-type macrophage markers CCL13 and MRC1. Furthermore, there was an elevation in the expression of glycolytic enzymes GLUT1 and Hexokinase, as well as HIF1α, GAPDH, and PFKFB3 after RBM15 overexpression. Moreover, in vivo knockdown of RBM15 led to an amelioration of aortic aneurysm in the rat AD model. This knockdown also resulted in a reduction of the M1-type macrophage marker iNOS, while significantly increasing the expression of the M2-type macrophage marker CD206. In conclusion, our findings demonstrate that RBM15 upregulates glycolysis in macrophages, thus contributing to the progression of AD through the promotion of M1-type macrophage polarization. Conversely, downregulation of RBM15 suppresses M1-type macrophage polarization, thereby decelerating the advancement of AD. These results unveil potential novel targets for the treatment of AD.

Repurposing of multiple sclerosis (MS) and graft versus host disease (GVHD) therapeutics to inhibit chronic rejection of transplanted organs

Chronic rejection of transplanted organs is an incurable event in transplantation. Macrophages infiltrating allograft induce vessel occlusion and tissue fibrosis, which over the months-years post-transplantation cause organ failure. Patients require re-transplantation, which is extremely problematic because of the permanent lack of organ donors. Our studies showed that chronic rejection depends on the function of macrophages and the RhoA/ROCK pathway. Genetic (RhoA knockout) or pharmacologic (ROCK inhibitors) interference with the RhoA pathway inhibits macrophage functions and prevents chronic rejection in the rodent transplantation model. Most commercially available RhoA pathway inhibitors are not approved for clinical use. However, we found two compounds: fingolimod (Gilenya, FTY720) and belumosudil (Rezurock), which are clinically approved for relapsing multiple sclerosis (MS) and chronic graft versus host disease (cGVHD), respectively, which also inhibit the RhoA pathway. We tested these two drugs in the rodent transplantation model, and both inhibited chronic rejection. Thus, we proposed to repurpose these drugs for organ transplantation. A clinical trial on the effect of fingolimod in kidney transplant patients is ongoing, and the belumosudil trial is pending.

Taenia solium excretory secretory proteins (ESPs) suppresses TLR4/AKT mediated ROS formation in human macrophages via hsa-miR-125.

Helminth infections are a global health menace affecting 24% of the world population. They continue to increase global disease burden as their unclear pathology imposes serious challenges to patient management. Neurocysticercosis is classified as neglected tropical disease and is caused by larvae of helminthic cestode Taenia solium. The larvae infect humans and localize in central nervous system and cause NCC; a leading etiological agent of acquired epilepsy in the developing world. The parasite has an intricate antigenic make-up and causes active immune suppression in the residing host. It communicates with the host via its secretome which is complex mixture of proteins also called excretory secretory products (ESPs). Understanding the ESPs interaction with host can identify therapeutic intervention hot spots. In our research, we studied the effect of T. solium ESPs on human macrophages and investigated the post-translation switch involved in its immunopathogenesis. T. solium cysts were cultured in vitro to get ESPs and used for treating human macrophages. These macrophages were studied for cellular signaling and miR expression and quantification at transcript and protein level. We found that T. solium cyst ESPs treatment to human macrophages leads to activation of Th2 immune response. A complex cytokine expression by macrophages was also observed with both Th1 and Th2 cytokines in milieu. But, at the same time ESPs modulated the macrophage function by altering the host miR expression as seen with altered ROS activity, apoptosis and phagocytosis. This leads to activated yet compromised functional macrophages, which provides a niche to support parasite survival. Thus T. solium secretome induces Th2 phenomenon in macrophages which may promote parasite's survival and delay their recognition by host immune system.

Anemoside B4, a new pyruvate carboxylase inhibitor, alleviates colitis by reprogramming macrophage function.

Colitis is a global disease usually accompanied by intestinal epithelial damage and intestinal inflammation, and an increasing number of studies have found natural products to be highly effective in treating colitis. Anemoside B4 (AB4), an abundant saponin isolated from Pulsatilla chinensis (Bunge), whichwas found to have strong anti-inflammatory activity. However, the exact molecular mechanisms and direct targets of AB4 in the treatment of colitis remain to be discovered. The anti-inflammatory activities of AB4 were verified in LPS-induced cell models and 2, 4, 6-trinitrobenzene sulfonic (TNBS) or dextran sulfate sodium (DSS)-induced colitis mice and rat models. The molecular target of AB4 was identified by affinity chromatography analysis using chemical probes derived from AB4. Experiments including proteomics, molecular docking, biotin pull-down, surface plasmon resonance (SPR), and cellular thermal shift assay (CETSA) were used to confirm the binding of AB4 to itsmolecular target. Overexpression of pyruvate carboxylase (PC) and PC agonist were used to study the effects of PC on the anti-inflammatory and metabolic regulation of AB4 in vitro and in vivo. AB4 not only significantly inhibited LPS-induced NF-κB activation and increased ROS levels in THP-1 cells, but also suppressed TNBS/DSS-induced colonic inflammation in mice and rats. The molecular target of AB4 was identified as PC, a key enzyme related to fatty acid, amino acid and tricarboxylic acid (TCA) cycle. We next demonstrated that AB4 specifically bound to the His879 site of PC and altered the protein's spatial conformation, thereby affecting the enzymatic activity of PC. LPS activated NF-κB pathway and increased PC activity, which caused metabolic reprogramming, while AB4 reversed this phenomenon by inhibiting the PC activity. In vivo studies showed that diisopropylamine dichloroacetate (DADA),a PC agonist, eliminated the therapeutic effects of AB4 by changing the metabolic rearrangement of intestinal tissues in colitis mice. We identified PC as a direct cellular target of AB4 in the modulation of inflammation, especially colitis. Moreover, PC/pyruvate metabolism/NF-κB is crucial for LPS-driven inflammation andoxidative stress. These findings shed more light on thepossibilities ofPC as a potential new target for treating colitis.

Fecal Supernatants from Patients with Crohn's Disease Induce Inflammatory Alterations in M2 Macrophages and Fibroblasts.

Intestinal macrophages and fibroblasts act as microenvironmental sentinels mediating inflammation and disease progression in Crohn's disease (CD). We aimed to establish the effects of fecal supernatants (FSs) from patients with CD on macrophage and fibroblast phenotype and function. FS were obtained by ultracentrifugation, and the metabolites were analyzed. Monocyte-derived M2 macrophages and fibroblasts were conditioned with FS, and secreted proteins, surface proteins and gene expression were analyzed. M2 macrophage efferocytosis was evaluated. Patients with CD (n = 15) had a skewed fecal metabolite profile compared to healthy subjects (HS, n = 10). FS from CD patients (CD-FS) induced an anti-inflammatory response in M2 macrophages with higher expression of IL-10, IL1RA and CD206 as compared to healthy FS (HS-FS) while the efferocytotic capacity was unaltered. CD-FS did not affect extracellular matrix production from fibroblasts, but increased expression of the pro-inflammatory proteins IL-6 and MCP-1. Conditioned media from M2 macrophages treated with CD-FS modulated gene expression in fibroblasts for TGFβ superfamily members and reduced IL-4 expression compared to HS-FS. We show that M2 macrophages and fibroblasts react abnormally to the fecal microenvironment of CD patients, resulting in altered protein expression related to inflammation but not fibrosis. This implies that the gut microbiota and its metabolites have an important role in the generation and/or perpetuation of inflammation in CD.

Developmental role of macrophages modeled in human pluripotent stem cell-derived intestinal tissue

Macrophages populate the embryo early in gestation, but their role in development is not well defined. In particular, specification and function of macrophages in intestinal development remain little explored. To study this event in the human developmental context, we derived and combined human intestinal organoid and macrophages from pluripotent stem cells. Macrophages migrate into the organoid, proliferate, and occupy the emerging microanatomical niches of epithelial crypts and ganglia. They also acquire a transcriptomic profile similar to that of fetal intestinal macrophages and display tissue macrophage behaviors, such as recruitment to tissue injury. Using this model, we show that macrophages reduce glycolysis in mesenchymal cells and limit tissue growth without affecting tissue architecture, in contrast to the pro-growth effect of enteric neurons. In short, we engineered an intestinal tissue model populated with macrophages, and we suggest that resident macrophages contribute to the regulation of metabolism and growth of the developing intestine.

Phosphorylation of RGS16 at Tyr168 promote HBeAg-mediated macrophage activation by ERK pathway to accelerate liver injury.

Liver injury is closely associated with macrophage activation following HBV infection. Our previous study showed that only HBeAg, but not HBsAg and HBcAg, stably enhances inflammatory cytokine production in macrophages. And we also indicated that HBeAg could induce macrophage activation via TLR2 and thus aggravate the progression of liver fibrosis. However, the specific molecular mechanism of HBeAg in macrophage activation is not clear. We screened significantly overexpressed RGS16 from RNASeq results of HBeAg-stimulated macrophages and validated them with cellular assays, GSE83148 microarray dataset, and in clinical samples. Meanwhile, small interference, plasmid, and lentivirus transfection assays were used to establish cell models for knockdown and overexpression of RGS16, and q-PCR, ELISA, Transwell, and CCK-8 assays were used to analyze the role of RGS16 in HBeAg-induced macrophage activation. In addition, the upstream and downstream mechanisms of RGS16 in HBeAg-treated macrophage activation were explored using inhibitors, phostag gels, and RGS16 phosphorylation mutant plasmids. Finally, the effect of RGS16 on hepatic inflammation in murine tissues was evaluated by H&E staining, liver enzyme assay and immunofluorescence. RGS16 was significantly upregulated in HBeAg-induced macrophage activation, and its expression was enhanced with increasing HBeAg content and treatment time. Functional experiments showed that overexpression of RGS16 promoted the production of inflammatory factors TNF-α and IL-6 and boosted macrophage proliferation and migration, while knockdown of RGS16 exhibited the opposite effect. Mechanistically, we discovered that RGS16 is regulated by the TLR2/P38/STAT5 signaling pathway. Meanwhile, RGS16 enhanced ERK phosphorylation via its own Tyr168 phosphorylation to contribute to macrophage activation, thereby accelerating liver injury. Finally, in mice, overexpression of RGS16 markedly strengthened liver inflammation. HBeAg upregulates RGS16 expression through the TLR2-P38-STAT5 axis, and the upregulated expression of RGS16 enhances macrophage activation and accelerates liver injury by promoting ERK phosphorylation. In this process, phosphorylation of Tyr168 is necessary for RGS16 to function. KEY MESSAGES: RGS16 boosted HBeAg-induced macrophage inflammation, proliferation, and migration. Tyr168 phosphorylation of RGS16 affected by ERK promoted macrophage activation. HBeAg upregulated the expression of RGS16 through TLR2/P38/STAT5 signal pathway. RGS16 promoted liver injury by regulating macrophage functions in mouse model.

NLRX1 Prevents M2 Macrophage Polarization and Excessive Renal Fibrosis in Chronic Obstructive Nephropathy.

Chronic kidney disease often leads to kidney dysfunction due to renal fibrosis, regardless of the initial cause of kidney damage. Macrophages are crucial players in the progression of renal fibrosis as they stimulate inflammation, activate fibroblasts, and contribute to extracellular matrix deposition, influenced by their metabolic state. Nucleotide-binding domain and LRR-containing protein X (NLRX1) is an innate immune receptor independent of inflammasomes and is found in mitochondria, and it plays a role in immune responses and cell metabolism. The specific impact of NLRX1 on macrophages and its involvement in renal fibrosis is not fully understood. To explore the specific role of NLRX1 in macrophages, bone-marrow-derived macrophages (BMDMs) extracted from wild-type (WT) and NLRX1 knockout (KO) mice were stimulated with pro-inflammatory and pro-fibrotic factors to induce M1 and M2 polarization in vitro. The expression levels of macrophage polarization markers (Nos2, Mgl1, Arg1, and Mrc1), as well as the secretion of transforming growth factor β (TGFβ), were measured using RT-PCR and ELISA. Seahorse-based bioenergetics analysis was used to assess mitochondrial respiration in naïve and polarized BMDMs obtained from WT and NLRX1 KO mice. In vivo, WT and NLRX1 KO mice were subjected to unilateral ureter obstruction (UUO) surgery to induce renal fibrosis. Kidney injury, macrophage phenotypic profile, and fibrosis markers were assessed using RT-PCR. Histological staining (PASD and Sirius red) was used to quantify kidney injury and fibrosis. Compared to the WT group, an increased gene expression of M2 markers-including Mgl1 and Mrc1-and enhanced TGFβ secretion were found in naïve BMDMs extracted from NLRX1 KO mice, indicating functional polarization towards the pro-fibrotic M2 subtype. NLRX1 KO naïve macrophages also showed a significantly enhanced oxygen consumption rate compared to WT cells and increased basal respiration and maximal respiration capacities that equal the level of M2-polarized macrophages. In vivo, we found that NLRX1 KO mice presented enhanced M2 polarization markers together with enhanced tubular injury and fibrosis demonstrated by augmented TGFβ levels, fibronectin, and collagen accumulation. Our findings highlight the unique role of NLRX1 in regulating the metabolism and function of macrophages, ultimately protecting against excessive renal injury and fibrosis in UUO.

Pulmonary tuberculosis and diabetes mellitus: Epidemiology, pathogenesis and therapeutic management (Review).

The dual burden of pulmonary tuberculosis (PTB) and diabetes mellitus (DM) is a major global public health concern. There is increasing evidence to indicate an association between PTB and DM. DM is associated with immune dysfunction and altered immune components. Hyperglycemia weakens the innate immune response by affecting the function of macrophages, dendritic cells, neutrophils, and natural killer cells, and also disrupts the adaptive immune response, thus promoting the susceptibility of PTB in patients with DM. Antituberculosis drugs often cause the impairment of liver and kidney function in patients with PTB, and the infection with Mycobacterium tuberculosis weaken pancreatic endocrine function by causing islet cell amyloidosis, which disrupts glucose metabolism and thus increases the risk of developing DM in patients with PTB. The present review discusses the association between PTB and DM from the perspective of epidemiology, pathogenesis, and treatment management. The present review aims to provide information for the rational formulation of treatment strategies for patients with PTB-DM.

SP-R210 isoforms of Myosin18A modulate endosomal sorting and recognition of influenza A virus infection in macrophages

Influenza A virus (IAV) infection causes acute and often lethal inflammation in the lung. The role of macrophages in this adverse inflammation is partially understood. The surfactant protein A receptor 210 (SP-R210) consists of two isoforms, a long (L) SP-R210L and a short (S) SP-R210S isoform encoded by alternative splicing of the myosin 18A gene. We reported that disruption of SP-R210L enhances cytosolic and endosomal antiviral response pathways. Here, we report that SP-R210L antagonizes type I interferon β (IFNβ), as depletion of SP-R210L potentiates IFNβ secretion. SP-R210 antibodies enhance and attenuate IFNβ secretion in SP-R210L replete and deficient macrophages, respectively, indicating that SP-R210 isoform stoichiometry alters macrophage function intrinsically. This reciprocal response is coupled to unopposed and restricted expression of viral genes in control and SP-R210L-deficient macrophages, respectively. Human monocytic cells with sub-stoichiometric expression of SP-R210L resist IAV infection, whereas alveolar macrophages with increased abundance of SP-R210L permit viral gene expression similar to murine macrophages. Uptake and membrane binding studies show that lack of SP-R210 isoforms does not impair IAV binding and internalization. Lack of SP-R210L, however, results in macropinocytic retention of the virus that depends on both SP-R210S and interferon-inducible transmembrane protein-3 (IFITM3). Mass spectrometry and Western blot analyses indicate that SP-R210 isoforms modulate differential recruitment of the Rho-family GTPase RAC1 and guanine nucleotide exchange factors. Our study suggests that SP-R210 isoforms modulate RAC-dependent macropinosomal sorting of IAV to discrete endosomal and lysosomal compartments that either permit or prevent endolysosomal escape and inflammatory sensing of viral genomes in macrophages.

From hepatitis B virus infection to acute-on-chronic liver failure: The dynamic role of hepatic macrophages.

Acute-on-chronic liver failure (ACLF) is a progressive disease that is associated with rapid worsening of clinical symptoms and high mortality. A multicentre prospective study from China demonstrated that patients with hepatitis B virus-related ACLF (HBV-ACLF) exhibited worse clinical characteristics and higher mortality rates compared to non-HBV-ACLF patients. Immune dysregulation is closely linked to the potential mechanisms of initiation and progression of ACLF. Innate immune response, which is represented by monocytes/macrophages, is up-regulated across ACLF development. This suggests that monocytes/macrophages play an essential role in maintaining the immune homeostasis of ACLF. Information that has been published in recent years shows that the immune status and function of monocytes/macrophages vary in ACLF precipitated by different chronic liver diseases. Monocytes/macrophages have an immune activation effect in hepatitis B-precipitated-ACLF, but they exhibit an immune suppression in cirrhosis-precipitated-ACLF. Therefore, this review aims to explain whether this difference affects the clinical outcome in HBV-ACLF patients as well as the mechanisms involved. We summarize the novel findings that highlight the dynamic polarization phenotype and functional status of hepatic macrophages from the stage of HBV infection to ACLF development. Moreover, we discuss how different HBV-related liver disease tissue microenvironments affect the phenotype and function of hepatic macrophages. In summary, increasing developments in understanding the differences in immune phenotype and functional status of hepatic macrophages in ACLF patients will provide new perspectives towards the effective restoration of ACLF immune homeostasis.

Polysaccharide of Asparagus cochinchinensis (Lour.) Merr regulates macrophage immune response and epigenetic memory through TLR4-JNK/p38/ERK signaling pathway and histone modification

BackgroundInnate immune memory of macrophages is closely linked to histone modifications. While various studies have demonstrated that the polysaccharide of Asparagus cochinchinensis (Lour.) Merr (ACMP), extracted through alcohol-alkali extraction, enhances macrophages' non-specific immune function; no literature currently addresses whether ACMP's regulatory effect is related to innate immune memory and histone modification. PurposeThis study aims to investigate if ACMP induces innate immune memory emergence in macrophages via pattern recognition receptor (PRR). Study designAfter co-incubating different doses of ACMP with RAW264.7 cells and BMDM cells, we observed changes in signaling pathways related to PRR and assessed the presence of innate immune memory phenomenon in the cells. MethodsWe observed the morphological characteristics of the ACMP using a scanning electron microscope, infrared spectrum, and HPLC pre-column derivatization method. We used q-PCR, Western blot, RNA-seq, and CUT&Tag-seq methods to examine ACMP's regulation of macrophage immune response and innate immune memory and explored its specific mechanism. ResultsACMP, primarily composed of Man, GlcN, Rha, Fuc, GalA, Xyl, Glc, Gal, Ara, and, exhibited a molar ratio of each monosaccharide (1.41: 0.35: 0.49: 0.18: 1.00: 97.12: 0.36: 3.58: 1.14). ACMP regulated immunological function in macrophages through the TLR4-MAPK-JNK/p38/ERK pathway. ACMP induced elevated levels of chromosomal H3K4me1, enhancing TNF-α, IL-1β, and other genes' responsiveness, allowing macrophages to develop innate immune memory to ACMP stimulation. ConclusionThis study first time demonstrates that ACMP regulates immunological function through the TLR4-MAPK-JNK/ERK/p38 signaling pathway, distinct from prior reports. ACMP induces innate immune memory in macrophages in response to its immune stimulation by promoting increased H3K4me1 on chromosomes. This mechanism may be crucial in how plant polysaccharides regulate macrophages and the body's immune function.

Luteolin-7-O-glucoside promotes macrophage release of IFN-β by maintaining mitochondrial function and corrects the disorder of glucose metabolism during RSV infection

Respiratory syncytial virus (RSV) pneumonia is the main cause of acute bronchiolitis in infants. Luteolin-7-O-glucoside (LUT-7G) is a natural flavonoid, which exists in a variety of plants and has the potential to treat viral pneumonia. We established RSV pneumonia mouse models and RSV-infected cell models. Clodronate liposomes were used to deplete macrophages. We used HE staining and immunohistochemistry to determine inflammatory damage and virus replication. We detected the expression levels of inflammatory factors and IFN-β through qPCR and ELISA. JC-1 kit was used for detecting the cell mitochondrial Membrane potential (MMP). ROS, SOD, and MDA kits were used for detecting intracellular oxidative stress damage. Metabolites of TCA in lung tissue and serum of mice were detected by GC-MS. Pharmacodynamic studies have shown that intervention with LUT-7G can alleviate lung tissue damage caused by RSV infection, inhibit RSV replication, and downregulate TNF-α, IL-1β, and IL-6 mRNA expression. LUT-7G upregulated the IFN-β content and the expression of IFN-β, ISG15, and OAS1 mRNA. In vitro, LUT-7G inhibited RSV-induced cell death, reversed the RSV-induced decrease of MMP and decreased intracellular oxidative stress. Target metabonomics showed that RSV infection upregulated the levels of glycolysis and TCA metabolites in lung tissue and serum, while LUT-7G could improve the disorder of glucose metabolism. The results indicate that LUT-7G can promote the release of IFN-β in the lung, alleviate inflammatory damage, and inhibit RSV replication during RSV infection. These effects may be achieved by protecting the mitochondrial function of alveolar macrophages and correcting the disorder of glucose metabolism.