Mature Macrophages Research Articles

Expression of spleen macrophages in a mouse model of alveolar bone resorption periodontitis.

It has been shown that macrophages can be endotoxin-tolerant under the stimulation of continuous endotoxin of Porphyromonas gingivalis. Macrophage transforms into M2-type which inhibits inflammation, and its pro-inflammatory cytokine secretion is reduced to avoid the tissue damaged by inflammation. This experiment established the corresponding animal model to explore the relative number, phenotypic proportion, and function of spleen macrophages in mice with chronic periodontitis. Twenty 16-week-old mice were randomly divided into a true ligation group (LFP group) and a pseudo-ligation group (LFC group). The periodontitis in the LFP group was induced by experimental ligation, and the LFC group was treated as a control. After 10 days of ligation, the maxilla was taken, IHC and HE staining were performed to observe the pathological changes of periodontal tissues, and IHC staining was performed to observe the RANKL/OPG ratio. Spleen mononuclear cells were isolated and counted. The ratio of M1 and M2 phenotypes was determined by fluorescence-activated cell sorting (FACS) in the spleen. The relative expression levels of macrophage-associated inflammatory cytokine TNF-a, IL-1β and anti-inflammatory cytokine IL-10 mRNA were detected by real-time PCR. Compared with the control group (LFC:M2/M110.04%), the M2 ratio among spleen mature macrophages in the periodontitis group (LFP: M2/M135.86%) was significantly increased (P<0.01) in the spleen. The proportion of M1 macrophages was not significantly different, and the ratio of M1/M2 was significantly decreased (P<0.05) in the spleen. But the expression level of M1-type macrophage inflammatory factor TNF-a mRNA was inclined. Chronic periodontitis can up-regulate the proportion of M2 macrophages, decrease the ratio of macrophage phenotype M1/M2, and incline the expression of pro-inflammatory factor TNF-a mRNA.

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-γ and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

Induced pluripotent and CD34+ stem cell derived myeloid cells display differential responses to particle and dust mite exposure

Myeloid cells form an essential component of initial responses to environmental hazards and toxic exposures. The ability to model these responses in vitro is central to efforts tasked with identifying hazardous materials and understanding mechanisms of injury and disease. Induced pluripotent stem cell (iPSC) derived cells have been suggested as alternatives to more established primary cell testing systems for these purposes. iPSC derived macrophage and dendritic like cells were compared to CD34+ haematopoietic stem cell derived populations using transcriptomic analysis. Using single cell sequencing-based characterisation of iPSC derived myeloid cells, we identified transitional, mature and M2 like macrophages as well as dendritic like antigen presenting cells and fibrocytes. Direct transcriptomic comparisons between iPSC and CD34+ cell derived populations revealed higher expression of myeloid differentiation genes such as MNDA, CSF1R and CSF2RB in CD34+ cells, while iPSC populations had higher fibroblastic and proliferative markers. Exposure of differentiated macrophage populations to nanoparticle alone or in combination with dust mite, resulted in differential gene expression on combination only, with responses markedly absent in iPSC compared to CD34+ derived cells. The lack of responsiveness in iPSC derived cells may be attributable to lower levels of dust mite component receptors CD14, TLR4, CLEC7A and CD36. In summary, iPSC derived myeloid cells display typical characteristics of immune cells but may lack a fully mature phenotype to adequately respond to environmental exposures.

Decreased bone resorption in Ezh2 myeloid cell conditional knockout mouse model.

Osteoclasts derived from hematopoietic stem cells control bone resorption. Identifying novel molecules that can epigenetically regulate osteoclastogenesis is important for developing novel treatments for osteoporosis and other disorders associated with bone deterioration and promoting healthy bone formation. The polycomb group (PcG) protein enhancer of zeste homolog 2 (Ezh2), a histone lysine methyltransferase, is associated with epigenetic regulation of numerous cellular processes, but its involvement in bone cell development and homeostasis is not yet clear. Here, LysM-Cre mice were crossed with Ezh2flox/flox mice to delete Ezh2 in myeloid cell lineage mature macrophages. Conditional knockout of Ezh2 (CKO) in myeloid cell line resulted in significant increases in postnatal bone growth in the first 6 months of life for both male and female mice. For female mice, optimal bone mass was seen for mice with Ezh2 deleted in both chromosomes in a pair (f/f Cre+ ; CKO). For male mice, optimal bone mass was found after deletion of Ezh2 from just one chromosome (f/- Cre+ ) with no difference in bone phenotype between f/- Cre+ and CKO male mice. In addition to the gender-specific difference in bone phenotype, Ezh2 CKO mice had significantly less macrophages (CD11b+) present in the bone marrow compared with control mice as well as significantly more mature osteoblasts and bone formation biomarkers present (P1NP, osteocalcin). Inflammatory array for protein lysed from bone tissue revealed deletion of Ezh2 decreased inflammatory milieu in both male and female mice compared with controls. Unexpectedly, myeloid cell deletion of Ezh2 also increased the number of mature osteoblast present in the bone. Deletion of Ezh2 also led to an increase in gene expression of osteoclast-suppressive genes IRF8, MafB, and Arg1 due to a decrease in the presence of the suppressive H3K27me3 epigenetic mark. These findings suggest that manipulation of Ezh2 expression may be a viable strategy to combat bone resorptive disorders such as osteoporosis or arthritis.

Leishmaniasis: Immune Cells Crosstalk in Macrophage Polarization.

Leishmaniasis is a complex infectious parasitic disease caused by protozoa of the genus Leishmania, belonging to a group of neglected tropical diseases. It establishes significant global health challenges, particularly in socio-economically disadvantaged regions. Macrophages, as innate immune cells, play a crucial role in initiating the inflammatory response against the pathogens responsible for this disease. Macrophage polarization, the process of differentiating macrophages into pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes, is essential for the immune response in leishmaniasis. The M1 phenotype is associated with resistance to Leishmania infection, while the M2 phenotype is predominant in susceptible environments. Notably, various immune cells, including T cells, play a significant role in modulating macrophage polarization by releasing cytokines that influence macrophage maturation and function. Furthermore, other immune cells can also impact macrophage polarization in a T-cell-independent manner. Therefore, this review comprehensively examines macrophage polarization's role in leishmaniasis and other immune cells' potential involvement in this intricate process.

Cell-penetrating TLR inhibitor peptide alleviates ulcerative colitis by the functional modulation of macrophages.

Toll-like receptors (TLRs) have a crucial role not only in triggering innate responses against microbes but in orchestrating an appropriate adaptive immunity. However, deregulated activation of TLR signaling leads to chronic inflammatory conditions such as inflammatory bowel disease (IBD). In this study, we evaluated the immunomodulatory potential of a TLR inhibitor in the form of a cell-penetrating peptide using an ulcerative colitis animal model. A peptide derived from the TIR domain of the TLR adaptor molecule TIRAP that was conjugated with a cell-penetrating sequence (cpTLR-i) suppressed the induction of pro-inflammatory cytokines such as TNF-α and IL-1β in macrophages. In DSS-induced colitis mice, cpTLR-i treatment ameliorated colitis symptoms, colonic tissue damage, and mucosal inflammation. Intriguingly, cpTLR-i attenuated the induction of TNF-α-expressing proinflammatory macrophages while promoting that of regulatory macrophages expressing arginase-1 and reduced type 17 helper T cell (Th17) responses in the inflamed colonic lamina propria. An in vitro study validated that cpTLR-i enhanced the differentiation of monocyte-driven macrophages into mature macrophages with a regulatory phenotype in a microbial TLR ligand-independent manner. Furthermore, the cocultivation of CD4 T cells with macrophages revealed that cpTLR-i suppressed the activation of Th17 cells through the functional modulation of macrophages. Taken together, our data show the immunomodulatory potential of the TLR inhibitor peptide and suggest cpTLR-i as a novel therapeutic candidate for the treatment of IBD.

Manipulation of Leucocytic NADPH Oxidase Maturation May Drive Macrophage Differentiation and Affect Atherogenesis

Abstract Atherosclerosis is a process of arterial intima thickening caused by lipid deposition as a consequence of insufficient efferocytosis of apoptotic cells by M2 macrophages and excessive inflammatory cytokine secretion from M1 macrophages. NADPH oxidase 2 (NOX2) is recognized as a major source of ROS for enhancing macrophage efferocytosis and functional polarization. We previously found that human macrophages contain significant amount of endoplasmic reticulum (ER)-retained gp91 phox, a major component of NOX2, which may hinder the formation of functional NOX2. To address the role of macrophage NOX2 in atherosclerosis, we established a mouse model bearing Apoe −/−and the CYBBpoint mutation (C1024T), which leads to ER-retained gp91 phoxin immune cells. We also developed a method for targeted delivery of a SERCA inhibitor with poly (lacticcoglycolic acid) (PLGA) nanoparticles (NPs), which may promote the glycosylation and maturation of ER-retained gp91 phox. We first found that oxLDL-administrated M1 macrophages expressed increased M2 marker CD163 and increased efferocytotic ability after being treated with NPs. We also found that there was an increased glycosylation of gp91 phoxin those macrophages from human and mice with CYBBpoint mutation (C1024T) after the treatment of NPs. Macrophage ATP-binding cassette transporters, which could decrease cholesterol overloading in macrophages, were upregulated in macrophages after macrophages were treated with NPs. Our results demonstrated that NPs not only can recover the maturation of ER-retained gp91 phoxin macrophages but also may facilitate the differentiation of M1 to M2-like macrophages with enhanced efferocytotic activity and cholesterol efflux.

PD-L1 blockade enhances brain resident memory T cell function and stimulates brain to lymph node crosstalk

Abstract Resident memory T cells (T RM) are a unique subset of memory T cells that persist within non-lymphoid tissues. Previous studies have identified T RMwithin the brain (bT RM) after peripheral infection and vaccination in mice, with more recent appreciation in human brain tissue. While bT RMare important for protection of the CNS against reinfection, their heterogeneity at the single cell level and overall involvement in shaping the neuroimmune landscape remains unknown. Here, we defined viral-specific bT RMheterogeneity by single cell RNA sequencing and identified unique subsets dependent on local antigen encounter. These data also confirmed previous findings that bT RMexpress markers associated with T cell exhaustion, such as PD-1. Despite this, intracranial delivery of cognate viral peptide led to robust bT RMreactivation and initiated a cascade of immune activation and accumulation within the brain, including rapid activation of microglia, NK cells and T cells, DC maturation, and infiltration of macrophages and monocyte derived DCs. In the presence of PD-L1 blockade, despite observing higher effector molecule production from reactivated bT RM, we found no apparent difference in downstream immune activation in the brain. Interestingly, however, we found a significant increase in DC and B cell maturation in the CNS draining deep cervical lymph nodes (dcLN) only in the presence of PD-L1 blockade. These data indicate a potential unique crosstalk between bT RMand APCs in the dcLN normally suppressed by PD-1/PD-L1 signaling. These studies provide insight into brain T RMfunctions and potential mechanisms for strengthening immune activation that can guide immunotherapies for immunologically cold brain tumors. Supported by grants from the NIH (K22AI148508-02, T32 AI007363)

Modelling the erythroblastic island niche of dyserythropoietic anaemia type IV patients using induced pluripotent stem cells.

Introduction: Congenital dyserythropoietic anaemia (CDA) type IV has been associated with an amino acid substitution, Glu325Lys (E325K), in the transcription factor KLF1. These patients present with a range of symptoms, including the persistence of nucleated red blood cells (RBCs) in the peripheral blood which reflects the known role for KLF1 within the erythroid cell lineage. The final stages of RBCs maturation and enucleation take place within the erythroblastic island (EBI) niche in close association with EBI macrophages. It is not known whether the detrimental effects of the E325K mutation in KLF1 are restricted to the erythroid lineage or whether deficiencies in macrophages associated with their niche also contribute to the disease pathology. Methods: To address this question, we generated an in vitro model of the human EBI niche using induced pluripotent stem cells (iPSCs) derived from one CDA type IV patient as well as two iPSC lines genetically modified to express an KLF1-E325K-ERT2 protein that could be activated with 4OH-tamoxifen. The one patient iPSC line was compared to control lines from two healthy donors and the KLF1-E325K-ERT2 iPSC line to one inducible KLF1-ERT2 line generated from the same parental iPSCS. Results: The CDA patient-derived iPSCs and iPSCs expressing the activated KLF1-E325K-ERT2 protein showed significant deficiencies in the production of erythroid cells with associated disruption of some known KLF1 target genes. Macrophages could be generated from all iPSC lines but when the E325K-ERT2 fusion protein was activated, we noted the generation of a slightly less mature macrophage population marked by CD93. A subtle trend in their reduced ability to support RBC enucleation was also associated with macrophages carrying the E325K-ERT2 transgene. Discussion: Taken together these data support the notion that the clinically significant effects of the KLF1-E325K mutation are primarily associated with deficiencies in the erythroid lineage but it is possible that deficiencies in the niche might have the potential to exacerbate the condition. The strategy we describe provides a powerful approach to assess the effects of other mutations in KLF1 as well as other factors associated with the EBI niche.

Abstract 2335: Targeting a positive feedback loop of MERTK and STAT3 during macrophage differentiation may provide anti-tumor immune function

Abstract MERTK tyrosine kinase expression is upregulated upon monocyte to macrophage differentiation. This receptor tyrosine kinase enables macrophages to efficiently clear apoptotic cells to maintain tissue homeostasis. Activation of MERTK in macrophages during efferocytosis promotes an immunosuppressive phenotype, which is hijacked by tumors to inhibit anti-tumor immunity. Further, this immunosuppressive phenotype in MERTK expressing macrophages in the tumor microenvironment is reversed using MERTK selective inhibitors, suggesting that inhibiting MERTK expression or activity in the tumor microenvironment may be of therapeutic benefit in the treatment of cancer. In an attempt to further understand the mechanism of MERTK upregulation in macrophages, we treated the monocytic leukemia cell line THP1 with PMA to differentiate the cells from a monocytic morphology to an adherent macrophage-like morphology. Following differentiation of the THP-1 cells, MERTK upregulation was confirmed by western blot and flow cytometry. In a similar fashion, treatment with of murine bone marrow derived monocytic cells with PMA induced MERTK expression. Proteomics cytokine array analysis also revealed increased levels of multiple chemokines including MCP-1 and RANTES. Treatment of THP-1 cells with a pan STAT inhibitor in the presence of PMA abrogated the induction of MERTK expression, suggesting a critical role for STAT pathways in the regulation of MERTK expression during monocyte differentiation to macrophages. Specifically, the STAT3 pathway was found to be important in MERTK regulation, as treatment with a STAT3 selective inhibitor was sufficient to abrogate MERTK expression in the presence of PMA treatment. Single cell sequencing of the immune cells in the bone marrow demonstrated Stat3 expression in monocytic lineage cells. Furthermore, both CD11C+Ly6c+CD45+ population and MERTK+CD11C+Ly6c+CD45+ populations were lower in Stat3 -/- bone marrow cells treated with PMA relative to the untreated cells. Collectively, these data suggest that MERTK expression during macrophage maturation may be mediated by STAT3 activation. Previously published data have also demonstrated that STAT3 can be activated downstream of MERTK activation. Thus, we propose that MERTK and STAT3 form a positive feedback loop during macrophage maturation. Treatment with either a MERTK and/or STAT3 inhibitor may interfere with this feedback pathway, potentially reversing an immunosuppressive phenotype in the macrophages in the tumor microenvironment. Citation Format: Dan Yan, Justus M. Huelse, Swati Sharma Bhasin, Manoj Bhasin, Deborah DeRyckere, Douglas K. Graham. Targeting a positive feedback loop of MERTK and STAT3 during macrophage differentiation may provide anti-tumor immune function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2335.

Role of GM-CSF in lung balance and disease.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor originally identified as a stimulus that induces the differentiation of bone marrow progenitor cells into granulocytes and macrophages. GM-CSF is now considered to be a multi-origin and pleiotropic cytokine. GM-CSF receptor signals activate JAK2 and induce nuclear signals through the JAK-STAT, MAPK, PI3K, and other pathways. In addition to promoting the metabolism of pulmonary surfactant and the maturation and differentiation of alveolar macrophages, GM-CSF plays a key role in interstitial lung disease, allergic lung disease, alcoholic lung disease, and pulmonary bacterial, fungal, and viral infections. This article reviews the latest knowledge on the relationship between GM-CSF and lung balance and lung disease, and indicates that there is much more to GM-CSF than its name suggests.

Abstract 5663: Discovery and characterization of anti-LILRB2 antibody SPX-104

Abstract T-cell based immunotherapies have produced durable remission in certain cancer patients, but owing at least partially to immunosuppression in the tumor microenvironment, response rates with current therapies remains challenging. Reversing the immunosuppression is a recognized pathway for enhancing the activity of existing IO therapies. LILRB2 (ILT4) is a key receptor for macrophage maturation and polarization and an emerging therapeutic target in immuno-oncology. This research aims to produce anti-LILRB2 antibodies to redirect the polarization of myeloid cells, specifically macrophages, in the tumor microenvironment. The LILRB2 antibodies were generated through the mouse hybridoma method. After humanization and maturation, potent and selective IgG4 anti-LILRB2 antibodies were obtained. Binding affinities were evaluated through ELISA and 293T-LILRB2 cell flow cytometry binding assays which demonstrated sub-nanomolar binding affinity as well as effective blocking of the interaction of LILRB2 with its ligands HLA-G and HLA-A2 for multiple antibodies. SPX-104 was shown to redirect macrophage polarization in primary cultured human PBMC and human monocyte derived macrophage (HMDM) models. SPX-104 treatment induced hTNFa expression and suppressed hIL10 expression in the LPS stimulated PBMC model. In the HMDM model, SPX-104 effectively induced pro-inflammatory cytokine hTNFα expression in M-CSF. Furthermore, an autologous mixed lymphocyte reaction (auto-MLR) assay to evaluate T cell response showed dose-dependent increases in hIFNγ and hGM-CSF expression following SPX-104 treatment. In a CD34-humanized hLILRB2 transgenic mouse efficacy model, SPX-104 in combination with an immune checkpoint inhibitor, effectively inhibited tumor growth. These results confirm enhanced T cell activity following macrophage redirected polarization. The in vitro and in vivo evaluation, of SPX-104 relative to other anti-LILRB2 antibodies will be presented. Citation Format: Anthony Haight, Qian Chen, Ernesto Rodriguez, Martin Siekierzycki, Victoria Hall, Brandon Williams, Jun Shi, Dinh-Duc Nguyen, Robert Cai, Jichun Ma, Michael White, Jingdong Qin, Jadon Shen, Chuanke Zhao, Zhuona Rong, Lin Meng, Chengchao Shou. Discovery and characterization of anti-LILRB2 antibody SPX-104. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5663.

Ginsenoside Rh1 ameliorates the asthma and allergic inflammation via inhibiting Akt, MAPK, and NF-κB signaling pathways in vitro and in vivo

AimsOverproduction of pro-inflammatory cytokines and its-mediated immune cell infiltration play a crucial role in asthma progression. In this study, we investigated the role of ginsenoside Rh1 (Rh1) in ovalbumin (OVA)/lipopolysaccharide (LPS)-induced allergic asthma both in vitro and in vivo. Materials and main methodsThe phorbol ester (PMA) and LPS were used to induce inflammation in lung airway cells and macrophage activation, respectively. Western blotting, quantitative reverse transcription-PCR, and immunofluorescence (IF) assays were performed to elucidate the underlying molecular mechanisms. To evaluating the effects of Rh1 in vivo, OVA and LPS were used to establish allergic asthma models. Key findingsRh1 significantly suppressed PMA-induced lung inflammation and macrophage activation by suppressing pro-inflammatory cytokines (TNF-α, IL-1β, MCP-1), ICMA-1, and matrix metallopeptidase 9 (MMP9) in A549 cells. Rh1 abolished the PMA-induced inflammation by suppressing MAPK, Akt, and NF-κB p65. Pretreatment with Rh1 blocked PMA-mediated translocation of NF-κB, a key marker of pro-inflammatory cytokine release, into the nucleus. Similar to PMA-induced lung inflammation, Rh1 suppressed LPS-induced macrophage activation by suppressing NF-κB p65 activation and inducible nitric oxide synthase protein and mRNA expression. Consistent with in vitro data, LPS injection enhanced the number of immune cells induced by OVA in bronchoalveolar lavage fluid, whereas 20 mg/kg Rh1 significantly decreased OVA/LPS-mediated immune cell induction. In addition, Rh1 inhibited eosinophil, macrophage, and neutrophil maturation through by IL-4 and OVA-specific IgE production. SignificanceRh1 protects against OVA/LPS-induced allergic asthma by suppressing immune cell infiltration by blocking the activation of MAPK, Akt, and NF-κB signaling pathways.

Identification of FCN1 as a novel macrophage infiltration-associated biomarker for diagnosis of pediatric inflammatory bowel diseases

BackgroundThe incidence of pediatric inflammatory bowel disease (PIBD) has been steadily increasing globally. Delayed diagnosis of PIBD increases the risk of complications and contributes to growth retardation. To improve long-term outcomes, there is a pressing need to identify novel markers for early diagnosis of PIBD.MethodsThe candidate biomarkers for PIBD were identified from the GSE117993 dataset by two machine learning algorithms, namely LASSO and mSVM-RFE, and externally validated in the GSE126124 dataset and our PIBD cohort. The role of ficolin-1 (FCN1) in PIBD and its association with macrophage infiltration was investigated using the CIBERSORT method and enrichment analysis of the single-cell dataset GSE121380, and further validated using immunoblotting, qRT-PCR, and immunostaining in colon biopsies from PIBD patients, a juvenile murine DSS-induced colitis model, and THP-1-derived macrophages.ResultsFCN1 showed great diagnostic performance for PIBD in an independent clinical cohort with the AUC of 0.986. FCN1 expression was upregulated in both colorectal biopsies and blood samples from PIBD patients. Functionally, FCN1 was associated with immune-related processes in the colonic mucosa of PIBD patients, and correlated with increased proinflammatory M1 macrophage infiltration. Furthermore, single-cell transcriptome analysis and immunostaining revealed that FCN1 was almost exclusively expressed in macrophages infiltrating the colonic mucosa of PIBD patients, and these FCN1+ macrophages were related to hyper-inflammation. Notably, proinflammatory M1 macrophages derived from THP-1 expressed high levels of FCN1 and IL-1β, and FCN1 overexpression in THP-1-derived macrophages strongly promoted LPS-induced activation of the proinflammatory cytokine IL-1β via the NLRP3-caspase-1 axis.ConclusionsFCN1 is a novel and promising diagnostic biomarker for PIBD. FCN1+ macrophages enriched in the colonic mucosa of PIBD exhibit proinflammatory phenotypes, and FCN1 promotes IL-1β maturation in macrophages via the NLRP3-caspase-1 axis.

Asprosin Enhances Cytokine Production by a Co-Culture of Fully Differentiated Mature Adipocytes and Macrophages Leading to the Exacerbation of the Condition Typical of Obesity-Related Inflammation.

Asprosin, a fasting-induced, glucogenic, and orexigenic adipokine, has gained popularity in recent years as a potential target in the fight against obesity and its complications. However, the contribution of asprosin to the development of moderate obesity-related inflammation remains still unknown. The present study aimed to evaluate the effect of asprosin on the inflammatory activation of adipocyte-macrophage co-cultures at various stages of differentiation. The study was performed on co-cultures of the murine 3T3L1 adipocyte and the RAW264.7 macrophage cell lines treated with asprosin before, during, and after 3T3L1 cell differentiation, with or without lipopolysaccharide (LPS) stimulation. Cell viability, overall cell activity, and the expression and release of key inflammatory cytokines were analyzed. In the concentration range of 50-100 nM, asprosin increased the pro-inflammatory activity in the mature co-culture and enhanced the expression and release of tumor necrosis factor α (TNF-α), high-mobility group box protein 1 (HMGB1), and interleukin 6 (IL-6). Macrophage migration was also increased, which could be related to the upregulated expression and release of monocyte chemoattractant protein-1 (MCP-1) by the adipocytes. In summary, asprosin exerted a pro-inflammatory effect on the mature adipocyte-macrophage co-culture and may contribute to the spread of moderate obesity-associated inflammation. Nevertheless, further research is needed to fully elucidate this process.

An Innate Checkpoint Determines Immune Dysregulation and Immunopathology during Pulmonary Murine Coronavirus Infection.

Hallmarks of life-threatening, coronavirus-induced disease include dysregulated antiviral immunity and immunopathological tissue injury. Nevertheless, the sampling of symptomatic patients overlooks the initial inflammatory sequela culminating in severe coronavirus-induced disease, leaving a fundamental gap in our understanding of the early mechanisms regulating anticoronavirus immunity and preservation of tissue integrity. In this study, we delineate the innate regulators controlling pulmonary infection using a natural mouse coronavirus. Within hours of infection, the cellular landscape of the lung was transcriptionally remodeled altering host metabolism, protein synthesis, and macrophage maturation. Genetic perturbation revealed that these transcriptional programs were type I IFN dependent and critically controlled both host cell survival and viral spread. Unrestricted viral replication overshooting protective IFN responses culminated in increased IL-1β and alarmin production and triggered compensatory neutrophilia, interstitial inflammation, and vascular injury. Thus, type I IFNs critically regulate early viral burden, which serves as an innate checkpoint determining the trajectory of coronavirus dissemination and immunopathology.

Apoptotic cell fragments locally activate tingible body macrophages in the germinal center

Germinal centers (GCs) that form within lymphoid follicles during antibody responses are sites of massive cell death. Tingible body macrophages (TBMs) are tasked with apoptotic cell clearance to prevent secondary necrosis and autoimmune activation by intracellular self antigens. We show by multiple redundant and complementary methods that TBMs derive from a lymph node-resident, CD169-lineage, CSF1R-blockade-resistant precursor that is prepositioned in the follicle. Non-migratory TBMs use cytoplasmic processes to chase and capture migrating dead cell fragments using a "lazy" search strategy. Follicular macrophages activated by the presence of nearby apoptotic cells can mature into TBMs in the absence of GCs. Single-cell transcriptomics identified a TBM cell cluster in immunized lymph nodes which upregulated genes involved in apoptotic cell clearance. Thus, apoptotic B cells in early GCs trigger activation and maturation of follicular macrophages into classical TBMs to clear apoptotic debris and prevent antibody-mediated autoimmune diseases.

MnOOH-Catalyzed Autoxidation of Glutathione for Reactive Oxygen Species Production and Nanocatalytic Tumor Innate Immunotherapy.

The antioxidant system, signed with reduced glutathione (GSH) overexpression, is the key weapon for tumor to resist the attack by reactive oxygen species (ROS). Counteracting the ROS depletion by GSH is an effective strategy to guarantee the antitumor efficacy of nanocatalytic therapy. However, simply reducing the concentration of GSH does not sufficiently improve tumor response to nanocatalytic therapy intervention. Herein, a well-dispersed MnOOH nanocatalyst is developed to catalyze GSH autoxidation and peroxidase-like reaction concurrently and respectively to promote GSH depletion and H2O2 decomposition to produce abundant ROS such as hydroxyl radical (·OH), thereby generating a highly effective superadditive catalytic therapeutic efficacy. Such a therapeutic strategy that transforms endogenous "antioxidant" into "oxidant" may open a new avenue for the development of antitumor nanocatalytic medicine. Moreover, the released Mn2+ can activate and sensitize the cGAS-STING pathway to the damaged intratumoral DNA double-strands induced by the produced ROS to further promote macrophage maturation and M1-polarization, which will boost the innate immunotherapeutic efficacy. Resultantly, the developed simple MnOOH nanocatalytic medicine capable of simultaneously catalyzing GSH depletion and ROS generation, and mediating innate immune activation, holds great potential in the treatment of malignant tumors.

DOP28 Aconitate decarboxylase 1 (ACOD1) represents a crucial regulator of monocyte recruitment, survival and proinflammatory status in intestinal inflammation

Abstract Background Aconitate decarboxylase 1 (ACOD1; also known as IRG-1) is the ubiquitous source of itaconate, which dampens inflammasome activation by preventing HIF1a production of IL-1b and prevents TNF production through NRF2 activation. We reasoned that expression of ACOD1 is an inhibitory pathway within monocytes to curtail their inflammatory function, which could be manipulated in IBD therapy. Methods We performed single-cell RNA sequencing of monocytes from colonoscopic biopsies of treatment naïve IBD patients, as well as complementary analysis of DSS treated C57BL/6J (WT) mice, using single-cell protein-mRNA (Primeflow) technologies for validation. To determine the role of ACOD1 in colitis, Acod1–/– mice were administered 2% DSS in drinking water for 6 days (acute colitis) or 4 days followed by 14 day recovery period (inflammation resolution) and assessed the colon myeloid compartment by multi-parameter flow cytometry. This was complemented with competitive bone marrow chimeric mice to assess the intrinsic role of ACOD1 controlling monocyte survival and proinflammatory status. Transcriptional profiling (Nanostring) was used to determine the molecular pathways influenced by ACOD1. Results We found that ACOD1/Acod1 represented an evolutionarily conserved of feature of inflammation-associated monocytes, with expression present in mucosal but not blood monocytes. Acod1–/– mice demonstrated increased susceptibility to acute intestinal injury with DSS compared to co-housed WT controls, characterised by increased weight loss and colon shortening with significant accumulation of colonic neutrophils and CD64+Ly6C+ MHC-II+/– monocytes. Consistent with Acod1 limiting inflammasome activation, Acod1–/– monocytes demonstrated significantly greater IL-1b producing capabilities than WT controls. To assess Acod1 deficient and sufficient haemopoietic cells in in the same environment, we generated competitive bone marrow chimeric mice. Acod1 deficiency conferred a significant advantage to monocyte recruitment to the colon, characterised by a 10fold increase in Ly6C+ MHC-II–colon monocytes in colitis. Transcriptional profiling of WT and Acod1–/– colonic monocytes revealed significantly greater expression of molecules required for monocyte extravasation, including Sell (CD62L), Icam1, Ccr2 and Itgal (CD11a). In addition, expression of genes indicative of macrophage maturation such as Itgax (CD11c), Cd163 and Cx3cr1 were lower in the context of Acod1 deficiency, suggesting that normal maturation of these cells may be perturbed by loss of Acod1. Conclusion ACOD1/Acod1 represents an evolutionarily conserved, monocyte specific immuno-metabolic pathway that acts to limit monocyte recruitment, fate and pro-inflammatory function within the inflamed intestine.

Lack of iron, zinc, and vitamins as a contributor to the etiology of atopic diseases.

Micronutritional deficiencies are common in atopic children suffering from atopic dermatitis, food allergy, rhinitis, and asthma. A lack of iron, in particular, may impact immune activation with prolonged deficiencies of iron, zinc, vitamin A, and vitamin D associated with a Th2 signature, maturation of macrophages and dendritic cells (DCs), and the generation of IgE antibodies. In contrast, the sufficiency of these micronutrients establishes immune resilience, promotion of regulatory cells, and tolerance induction. As micronutritional deficiencies mimic an infection, the body's innate response is to limit access to these nutrients and also impede their dietary uptake. Here, we summarize our current understanding of the physiological function of iron, zinc, and vitamins A and D in relation to immune cells and the clinical consequences of deficiencies in these important nutrients, especially in the perinatal period. Improved dietary uptake of iron is achieved by vitamin C, vitamin A, and whey compounds, whereas zinc bioavailability improves through citrates and proteins. The addition of oil is essential for the dietary uptake of beta-carotene and vitamin D. As for vitamin D, the major source comes via sun exposure and only a small amount is consumed via diet, which should be factored into clinical nutritional studies. We summarize the prevalence of micronutritional deficiencies of iron, zinc, and vitamins in the pediatric population as well as nutritional intervention studies on atopic diseases with whole food, food components, and micronutrients. Dietary uptake via the lymphatic route seems promising and is associated with a lower atopy risk and symptom amelioration. This review provides useful information for clinical studies and concludes/emphasizes that a healthy, varied diet containing dairy products, fish, nuts, fruits, and vegetables as well as supplementing foods or supplementation with micronutrients as needed is essential to combat the atopic march.