Wild-type Bone Marrow-derived Macrophages Research Articles

Nuclear receptor Nur77 regulates immunomechanics of macrophages

Nuclear receptor Nur77 plays a pivotal role in immune regulation across various tissues, influencing pro-inflammatory signaling pathways and cellular metabolism. While cellular mechanics have been implicated in inflammation, the contribution of Nur77 to these mechanical processes remains elusive. Macrophages exhibit remarkable plasticity in their morphology and mechanics, enabling them to adapt and execute essential inflammatory functions, such as navigating through inflamed tissue and pathogen engulfment. However, the precise regulatory mechanisms governing these dynamic changes in macrophage mechanics during inflammation remain poorly understood. To establish the potential correlation of Nur77 with cellular mechanics, we compared bone marrow-derived macrophages (BMDMs) from wild-type (WT) and Nur77-deficient (Nur77-KO) mice and employed cytoskeletal imaging, single-cell acoustic force spectroscopy (AFS), migration and phagocytosis assays, and RNA-sequencing. Our findings reveal that Nur77-KO BMDMs exhibit changes to their actin networks compared to WT BMDMs, which is associated with a stiffer and more rigid phenotype. Subsequent in vitro experiments validated our observations, showcasing that Nur77 deficiency leads to enhanced migration, reduced adhesion, and increased phagocytic activity. The transcriptomics data confirmed altered mechanics-related pathways in Nur77-deficient macrophage that are accompanied by a robust pro-inflammatory phenotype. Utilizing previously obtained ChIP-data, we revealed that Nur77 directly targets differentially expressed genes associated with cellular mechanics. In conclusion, while Nur77 is recognized for its role in reducing inflammation of macrophages by inhibiting the expression of pro-inflammatory genes, our study identifies a novel regulatory mechanism where Nur77 governs macrophage inflammation through the modulation of expression of genes involved in cellular mechanics. Our findings suggest that immune regulation by Nur77 may be partially mediated through alterations in cellular mechanics, highlighting a potential avenue for therapeutic targeting.

Transient receptor potential vanilloid 1 interacts with TLR4/CD14 signaling pathway in lipopolysaccharide-mediated inflammation in macrophages.

Transient receptor potential vanilloid 1 (TRPV1), a ligand-gated cation channel, is a receptor for vanilloids on sensory neurons and is also activated by capsaicin, heat, protons, arachidonic acid metabolites, and inflammatory mediators on neuronal or non-neuronal cells. However, the role of the TRPV1 receptor in pro-inflammatory cytokine secretion and its potential regulatory mechanisms in lipopolysaccharide (LPS)-induced inflammation has yet to be entirely understood. To investigate the role and regulatory mechanism of the TRPV1 receptor in regulating LPS-induced inflammatory responses, bone marrow-derived macrophages (BMDMs) harvested from wild-type (WT) and TRPV1 deficient (Trpv1-/-) mice were used as the cell model. In WT BMDMs, LPS induced an increase in the levels of tumor necrosis factor-α, interleukin-1β, inducible nitric oxide synthase (iNOS), and nitric oxide, which were attenuated in Trpv1-/- BMDMs. Additionally, the phosphorylation of IκBα and mitogen-activated protein kinases, as well as the translocation of NF-κB and AP-1, were all decreased in LPS-treated Trpv1-/- BMDMs. Immunoprecipitation assay revealed that LPS treatment increased the formation of TRPV1-TLR4-CD14 complex in WT BMDMs. Genetic deletion of TRPV1 in BMDMs impaired the LPS-triggered immune-complex formation of TLR4, MyD88, and IRAK, all of which are essential regulators in LPS-induced activation of the TLR4 signaling pathway. Moreover, genetic deletion of TRPV1 prevented the LPS-induced lethality and pro-inflammatory production in mice. In conclusion, the TRPV1 receptor may positively regulate the LPS-mediated inflammatory responses in macrophages by increasing the interaction with the TLR4-CD14 complex and activating the downstream signaling cascade.

Expansion and Blunted Inflammatory Responses of Jak2 V617F Cells in the Setting of Electronic Cigarette Aerosol Inhalation

Clonal hematopoiesis of intermediate potential (CHIP) increases the risk for hematological malignancies and cardiovascular disease with Jak2 being a commonly mutated gene in CHIP. Age is the strongest determinant of CHIP but environmental factors also play a role since only a fraction of the individuals with CHIP eventually present with overt clonal hematopoiesis. The popularity of electronic (E-) cigarette (E-cig) usage has increased but their health effects are not well-established. E-cigarettes have been associated with inflammation and oxidative stress and these can provide a selective pressure for the outgrowth of CHIP mutant cells. Here, we determined the impact of E-cig aerosol inhalation on the survival and/or expansion of Jak2 mutant hematopoietic cells by exposing bone marrow transplanted mice to E-cig aerosols. Competitive bone marrow transplant assays were established to determine expansion of Jak2V617F mutant cells to E-cigarette aerosols. Lethally irradiated recipient mice in the CD45.1/2 background were transplanted with equal numbers of whole bone marrow cells from wild type (WT) (CD45.1) and Jak2V617F(CD45.2) mice (WT:Jak2V617F), or WT (CD45.1) and WT (CD45.2) (WT:WT) as transplant controls. Eight weeks post-transplant, mice were exposed to E-cigarette aerosols using a nose-only inhalation exposure system for 2 hours/day, 4 days/week for 3 months. Control mice were exposed to room air using the nose-only inhalation approach. At regular intervals, peripheral blood was drawn to determine the percentage of WT and Jak2V617F cells by flow cytometry. At euthanasia, the percentage of WT and mutant cells in the bone marrow (BM) and spleen was determined. In competitive bone marrow transplants in a 1:1 ratio, this mouse model of Jak2V617F does not gain a selective advantage in relation to WT cells. Instead, we observe declining levels of Jak2V617F cells in the peripheral blood over time. The time point at which the Jak2V617F cells were at their minimum was recorded as time point 0 (T0). Mice were exposed for 12 weeks and then allowed to rest, to determine how E-cig exposure promotes Jak2V617F expansion, until termination of the experiment (Tend). Mice exposed to E-cig aerosols demonstrated significant expansion of Jak2V617F cells at the end of the experiment compared to air exposed controls (Wilcoxon paired test, p<0.05). Jak2V617F Lin-, c-Kit+, Sca-1+ (LKS) cells in the bone marrow were also slightly increased in the E-cig group compared to the air group. To assess how inflammation is altered in the setting of prior E-cig aerosol inhalation, bone marrow derived macrophages (BMDMs) from exposed mice were stimulated with lipopolysaccharide (LPS) and secretion of the pro-inflammatory cytokines, TNFα and IL-6, were quantified by ELISA. WT:WT BMDMs from the E-cig cohort produced more TNFα and IL-6 than the WT:WT BMDMs from the air cohort. However, interestingly E-cig exposed WT:Jak2V617F BMDMs produced significantly less TNFα and IL-6 than the E-cig exposed WT:WT BMDMs. This indicates that prior E-cig aerosol inhalation can sensitize WT cells to inflammatory stimuli while Jak2V617F mutant cells are more resistant to LPS mediated inflammation post E-cig exposure. This is particularly relevant in inflammation-induced hematopoietic stem cell exhaustion since a diminishing pool of WT HSCs provides selection pressure for the outgrowth of mutant HSCs. Environmental factors such as combustible cigarette smoke and E-cigarette products can play a role in the clinical consequences of CHIP. We observed that in vivo exposure to E-cigarette aerosols supports the survival and expansion of Jak2V617F cells in bone marrow transplanted mice. Our data also indicates that E-cig exposure sensitizes WT cells to inflammation more than Jak2V617F mutant cells. Future work is aimed at determining E-cig exposure mediated inflammatory and apoptotic effects on WT and mutant HSCs and subsets of mature hematopoietic cells.

Caveolin-1 forms a complex with P2X7 receptor and tunes P2X7-mediated ATP signaling in mouse bone marrow-derived macrophages.

The ionotropic purinergic P2X7 receptor responds to extracellular ATP and can trigger proinflammatory immune signaling in macrophages. Caveolin-1 (Cav-1) is known to modulate functions of macrophages and innate immunity. However, it is unknown how Cav-1 modulates P2X7 receptor activity in macrophages. We herein examined P2X7 receptor activity and macrophage functions using bone marrow-derived macrophages (BMDMs) from wild-type (WT) and Cav-1 knockout (KO) mice. ATP (1 mM) application caused biphasic increase in cytosolic [Ca2+] and sustained decrease in cytosolic [K+]. A specific P2X7 receptor blocker, A-740003, inhibited the maintained cytosolic [Ca2+] increase and cytosolic [K+] decrease. Total internal reflection fluorescent imaging and proximity ligation assays revealed a novel molecular complex formation between P2X7 receptors and Cav-1 in WT BMDMs that were stimulated with lipopolysaccharides. This molecular coupling was increased by ATP application. Specifically, the ATP-induced Ca2+ influx and K+ efflux through P2X7 receptors were increased in Cav-1 KO BMDMs, even though the total and surface protein levels of P2X7 receptors in WT and Cav-1 KO BMDMs were unchanged. Cell-impermeable dye (TO-PRO3) uptake analysis revealed that macropore formation of P2X7 receptors was enhanced in Cav-1 KO BMDMs. Cav-1 KO BMDMs increased ATP-induced IL-1β secretion, reactive oxygen species production, Gasdermin D (GSDMD) cleavage, and lactate dehydrogenase release indicating pyroptosis. A-740003 completely prevented ATP-induced pyroptosis. In combination, these datasets show that Cav-1 has a negative effect on P2X7 receptor activity in BMDMs and that Cav-1 in macrophages may contribute to finely tuned immune responses by preventing excessive IL-1β secretion and pyroptosis.NEW & NOTEWORTHY In bone marrow-derived macrophages, Cav-1 suppresses the macropore formation of P2X7 receptors through their direct or indirect interactions, resulting in reduced membrane permeability of cations (Ca2+ and K+) and large cell-impermeable dye (TO-PRO3) induced by ATP. Cav-1 also inhibits ATP-induced IL-1β secretion, ROS production, GSDMD cleavage, and pyroptosis. Cav-1 contributes to the maintenance of proper immune responses by finely tuning IL-1β secretion and cell death in macrophages.

MyD88-dependent Toll-like receptor 2 signaling modulates macrophage activation on lysate-adsorbed Teflon™ AF surfaces in an in vitro biomaterial host response model.

The adsorbed protein layer on an implanted biomaterial surface is known to mediate downstream cell-material interactions that drive the host response. While the adsorption of plasma-derived proteins has been studied extensively, the adsorption of damage-associated molecular patterns (DAMPs) derived from damaged cells and matrix surrounding the implant remains poorly understood. Previously, our group developed a DAMP-adsorption model in which 3T3 fibroblast lysates were used as a complex source of cell-derived DAMPs and we demonstrated that biomaterials with adsorbed lysate potently activated RAW-Blue macrophages via Toll-like receptor 2 (TLR2). In the present study, we characterized the response of mouse bone marrow derived macrophages (BMDM) from wildtype (WT), TLR2-/- and MyD88-/- mice on Teflon™ AF surfaces pre-adsorbed with 10% plasma or lysate-spiked plasma (10% w/w total protein from 3T3 fibroblast lysate) for 24 hours. WT BMDM cultured on adsorbates derived from 10% lysate in plasma had significantly higher gene and protein expression of IL-1β, IL-6, TNF-α, IL-10, RANTES/CCL5 and CXCL1/KC, compared to 10% plasma-adsorbed surfaces. Furthermore, the upregulation of pro-inflammatory cytokine and chemokine expression in the 10% lysate in plasma condition was attenuated in TLR2-/- and MyD88-/- BMDM. Proteomic analysis of the adsorbed protein layers showed that even this relatively small addition of lysate-derived proteins within plasma (10% w/w) caused a significant change to the adsorbed protein profile. The 10% plasma condition had fibrinogen, albumin, apolipoproteins, complement, and fibronectin among the top 25 most abundant proteins. While proteins layers generated from 10% lysate in plasma retained fibrinogen and fibronectin among the top 25 proteins, there was a disproportionate increase in intracellular proteins, including histones, tubulins, actins, and vimentin. Furthermore, we identified 7 DAMPs or DAMP-related proteins enriched in the 10% plasma condition (fibrinogen, apolipoproteins), compared to 39 DAMPs enriched in the 10% lysate in plasma condition, including high mobility group box 1 and histones. Together, these findings indicate that DAMPs and other intracellular proteins readily adsorb to biomaterial surfaces in competition with plasma proteins, and that adsorbed DAMPs induce an inflammatory response in adherent macrophages that is mediated by the MyD88-dependent TLR2 signaling pathway.

Abstract P3088: Hexokinase 3 Regulates Macrophage Polarization And Cardiac Remodeling Likely Through Serine Metabolism

Myocardial infarction (MI) is associated with activation of macrophages in the myocardium. Hexokinase 3 (HK3) is the mainly expressed hexokinase in macrophage. Along with its role as the first enzyme in glycolysis, HK3 has shown to be involved in the regulation of diverse immune system activities. In this study we studied the role of HK3 in the regulation of macrophage polarization into inflammatory phenotype (termed as M1) and anti-inflammatory and repair phenotype (termed as M2). Using CRISPR-Cas9 technology, we generated mice with deletion of exons 2 and 3 of the Hk3 gene, which are needed for the function of the protein. Bone marrow-derived macrophages (BMDM) isolated from Hk3 -/- mice displayed higher levels of M2 markers, including Arg1, Ym1, Mrc1, PD-L2, IRF4, Mgl2, and Mgl1 compared to BMDMs from wild-type mice. Also, we found that pSTAT6 protein, was higher in BMDMs from Hk3 -/- mice. Furthermore, there was a higher oxygen consumption rate and lower extracellular acidification rate in Hk3 -/- BMDMs. Steady-state metabolomics in BMDMs isolated from WT and Hk3 -/- mice showed that there was a significant increase in metabolites in the serine biosynthesis pathway (SBP), indicating that glucose metabolism is more shunted into the SBP with Hk3 deletion. The removal of serine/glycine from media resulted in a significant decrease in the M2 markers in wild-type BMDMs; However, higher M2 markers were observed in Hk3 -/- BMDMs. This result indicated that the SBP is needed for the polarization of macrophages into the M2 subtype, and HK3 mediated M2 polarization is likely through the SBP. We then subjected Hk3 -/- to LAD and assessed cardiac function after seven days using echocardiography. Result demonstrated that both male and female Hk3 -/- mice display improved cardiac function, as assessed by measurement of ejection fraction (EF), fractional shortening (FS), and interventricular septum thickness in diastole (IVSd). Our results demonstrate that HK3 is a novel regulator of macrophage polarization into the phenotype with more anti-inflammatory and tissue repair characteristic. This study is clinically important by providing a potent novel approach to the treatment of ischemic heart disease.

DCIR suppresses osteoclastic proliferation and resorption by downregulating M-CSF and RANKL signaling.

Dendritic cell immunoreceptor (DCIR) is an inhibitory C-type lectin receptor that acts as a negative regulator in the immune system and bone metabolism. We previously revealed that DCIR deficiency enhanced osteoclastogenesis and antigen presentation of dendritic cells, and that asialo-biantennary N-glycan (NA2) functions as a ligand for DCIR. NA2 binding to DCIR suppressed murine and human osteoclastogenesis that occurs in the presence of M-CSF and RANKL. The DCIR-NA2 axis, therefore, plays an important role in regulating osteoclastogenesis in both mice and humans, although the underlying mechanisms remain unclear. Here we found that Dcir -/- bone marrow-derived macrophages (BMMs) exhibited greater proliferative and differentiation responses to M-CSF and RANKL, respectively, than wild-type (WT) BMMs. Moreover, Dcir -/- osteoclasts (OCs) increased resorptive activity and cell fusion more significantly than WT OCs. DCIR deficiency affects gene expression patterns in OCs, and we found that the expression of neuraminidase 4 was increased in Dcir -/- OCs. Furthermore, DCIR-NA2 interaction in WT BMMs, but not Dcir -/- BMMs, decreased Akt phosphorylation in response to M-CSF and RANKL. These data suggest that DCIR regulates osteoclastogenesis by downregulating M-CSF and RANKL signaling, and that DCIR-mediated signaling may contribute to the terminal modification of oligosaccharides by controlling the expression of glycosylation enzymes.

Rnf144b alleviates the inflammatory responses and cardiac dysfunction in sepsis.

Sepsis is an inflammatory disease with high mortality and morbidity. Inflammation plays an essential role in sepsis, and suppressing inflammation has been shown to ameliorate sepsis. Rnf144b is an ubiquitin E3 ligation with anti-inflammation activities. Its precise roles in sepsis remain unknown. Here, we explored the function of Rnf144b in sepsis. We generated conditional knockout mice with Rnf144b deficiency in the myeloid cells. We monitored the Rnf144b expression in peripheral blood mononuclear cells from healthy donor and patients with sepsis, and in lipopolysaccharides (LPS)-treated bone marrow-derived macrophages (BMDMs). The cytokine expression between wild-type BMDMs and Rnf144b-deficient BMDMs after LPS and CpG treatments was compared. The survival rate and cardiac function were monitored. The activation of TANK binding kinase 1 and nuclear factor kappa-B was examined by Western blot and real-time PCR. Up-regulated expression of Rnf144b was observed in peripheral blood mononuclear cells from patients with sepsis. LPS induced the expression of Rnf144b in BMDMs. Rnf144b-deficient BMDMs produced more inflammatory cytokines after LPS or CpG stimulation. Septic mice with Rnf144b deficiency in myeloid cells had higher mortality and exacerbated cardiac dysfunction. Rnf144b interacted with TANK binding kinase 1 and Rnf144b deficiency resulted in impaired activation of TBK1 but enhanced activation of nuclear factor kappa-B. Rnf144b prevents inflammatory responses and cardiac dysfunction in sepsis.

Immune Dysregulation in Human Telomere Diseases

Germline variants in a diversity of genes involved in telomere maintenance result in premature telomere shortening and cause clinical conditions that are collectively called telomeropathies, which range from mucocutaneous findings (skin hypopigmentation, nail dystrophy and oral leukoplakia) to bone marrow failure, myeloid neoplasms, pulmonary fibrosis and cirrhosis. Even though pulmonary fibrosis and liver cirrhosis develop in up to a quarter of patients with telomeropathies, the exact mechanisms promoting the fibrotic pathology remain unclear. Considering that the hematopoietic compartment is particularly sensitive to telomere erosion, we aimed to investigate whether the immune response may play a role on the development of fibrotic lesions in a murine model, and to evaluate immunological biomarkers in patients diagnosed with telomere diseases. To assess the effects of telomere attrition on the development of liver fibrosis and immune response, G3 Terc-/- and Tert-/- mice (background C57BL/6J) and wild-type (WT) mice were infected with S. mansoni cercariae and euthanized after 12 weeks. Telomere length (TL) was determined by qPCR in leukocytes and liver sections and confirmed to be shorter in both knockout (KO) models in comparison to WT animals (p≤0.01). Collagen deposition in liver tissue was quantified with Picro Sirius Red staining and showed larger fibrotic areas in WT mice (14 ± 5,2%) when compared to Terc-/- (7,2 ± 4,3%; p≤0.05) and Tert-/- (6,3 ± 2,4%; p≤0.05). Macrophage infiltration was evaluated by immunostaining using antibodies against F4/80, CD206 and iNOS, and revealed less positive cells within the liver of Terc-/- and Tert-/- mice in comparison to WT (p≤0.001). We studied the ability of bone marrow-derived macrophages (BMDMs) to be polarized towards M1 and M2-like phenotypes ex vivo, upon stimulation with either LPS or IL-4, respectively. Expression of TNF-alpha, IL-6 and MMP9 demonstrated that the classical activation of Terc-/- and Tert-/- BMDMs was impaired in a similar fashion, and these cells did not exhibit the M1-like phenotype as observed in the WT BMDMs. Conversely, CD206 and ARG1 were significantly upregulated in KOs stimulated with IL-4, suggesting that telomerase-null BMDMs are skewed towards an M2 polarization. We also evaluated 18 patients with telomere diseases (median age, 33.9 years; range, 12.8-62.5 years; male/female, 9/9; marrow failure, 15; pulmonary manifestations, 5; hepatic manifestations, 5) and 10 healthy individuals (median age, 32.1; range, 13.9-56.3 years; male/female, 5/5) for their immune profile. Eight patients had very short TL (<1st percentile) and ten had short TL (<10th percentile). All healthy individuals had TL >10th percentile. We measured serum levels of cytokines, chemokines and growth factors using a customized Luminex panel to assess 32 analytes in a single assay. Spearman's correlation coefficient was used to analyze associations between TL z-scores and the variables of interest. Analysis showed significant correlations (p≤0.05) with angiopoietin-1, IL-1α, IL-1β, IL-1RA, IL-3, IL-4, IL-7, IL-23, IL-27, MCP-1, M-CSF, MIP-3α and PDGF-BB and, in all cases, Spearman's rho was positive, indicating that shorter telomeres were correlated with decreased protein serum levels. In order to study the maturation of T- and B-cells, T-cell receptor excision circles (TRECs) and kappa-deleting excision circles (KRECs) were quantified by qPCR in dried blood spot samples from ten patients with short telomeres and ten age-matched healthy individuals. Both biomarkers were significantly reduced in patients (p≤0.05), showing that production and/or maturation of T- and B-lymphocytes is impaired. TRECs were negatively correlated to age in the control group, (rs = -0.66; p = 0.043), but not in patients (p>0.05), suggesting that TRECs do not follow the same pattern of age-dependent decrease in short telomere syndromes. Altogether, the results demonstrate that telomere erosion contributes to impaired immune responses and T- and B-cell maturation, which may contribute to the pathophysiology of several clinical manifestations in telomeropathies. Further studies are necessary to deeply characterize innate and adaptative immune response in telomere syndromes and better understand how it correlates to fibrosis development. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Murine macrophages do not support the proliferation of Leishmania (Viannia) braziliensis amastigotes even in absence of nitric oxide and presence of high arginase activity.

Leishmania (Viannia) braziliensis is the main species responsible for American tegumentary leishmaniasis in Brazil. Nevertheless, the use of this parasite species to study Leishmania infection in the murine model has been less conducted when compared with other Leishmania species. The control of murine infection with Leishmania has been associated with nitric oxide (NO) produced by inducible NO synthase (iNOS) from M1 macrophages, while arginase expressed by M2 macrophages is related to Leishmania proliferation. Here we use three different strains of L. (V.) braziliensis and one strain of L. (L.) major to study a 9-day infection of macrophages in vitro. Wild-type bone marrow-derived macrophages (BMDM) supported the proliferation of L. (L) major amastigotes from the 3rd day after infection, while all strains of L. (V.) braziliensis did not proliferate even inside IL-4-treated or iNOS knockout (KO) macrophages. The arginase activity was higher in iNOS KO than IL-4-treated macrophage showing that the absence of proliferation is independent of arginase. Importantly, L. (V.) braziliensis was able to cause uncontrolled disease in iNOS KO mice in vivo demonstrating that murine macrophages present at the site of infection have additional changes beyond inhibition of NO production or stimulation of arginase activity to support parasite proliferation.

Myeloid DNA methyltransferase3b deficiency aggravates pulmonary fibrosis by enhancing profibrotic macrophage activation

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic, progressive and severe disease characterized by excessive matrix deposition in the lungs. Macrophages play crucial roles in maintaining lung homeostasis but are also central in the pathogenesis of lung diseases like pulmonary fibrosis. Especially, macrophage polarization/activation seems to play a crucial role in pathology and epigenetic reprograming is well-known to regulate macrophage polarization. DNA methylation alterations in IPF lungs have been well documented, but the role of DNA methylation in specific cell types, especially macrophages, is poorly defined.MethodsIn order to determine the role of DNA methylation in macrophages during pulmonary fibrosis, we subjected macrophage specific DNA methyltransferase (DNMT)3B, which mediates the de novo DNA methylation, deficient mice to the bleomycin-induced pulmonary fibrosis model. Macrophage polarization and fibrotic parameters were evaluated at 21 days after bleomycin administration. Dnmt3b knockout and wild type bone marrow-derived macrophages were stimulated with either interleukin (IL)4 or transforming growth factor beta 1 (TGFB1) in vitro, after which profibrotic gene expression and DNA methylation at the Arg1 promotor were determined.ResultsWe show that DNMT3B deficiency promotes alternative macrophage polarization induced by IL4 and TGFB1 in vitro and also enhances profibrotic macrophage polarization in the alveolar space during pulmonary fibrosis in vivo. Moreover, myeloid specific deletion of DNMT3B promoted the development of experimental pulmonary fibrosis.ConclusionsIn summary, these data suggest that myeloid DNMT3B represses fibrotic macrophage polarization and protects against bleomycin induced pulmonary fibrosis.

C3aR dampens intracellular cAMP to control TRIF-mediated inflammation in activated macrophages

Abstract Recent studies from our lab and others have shown that complement components can enhance cell-intrinsic inflammatory signaling pathways. Specifically, we have found that activation of complement receptor C3aR enhances TLR4 signaling in lipopolysaccharide (LPS)-treated macrophages via enhanced p-38 phosphorylation. We found C3aR−/− mice treated with systemic LPS have decreased inflammatory cytokines (IL-6 and TNF) compared to wild-type (WT) mice. Our results corroborate previous studies showing C3aR−/− mice display decreased systemic cytokines (IL-6 and TNF) and increased bacterial burden during Gram-negative (GN) infection compared to WT mice. These results suggest C3aR is a critical regulator of TLR4 signaling. TLR4-TRIF signaling leads to activation of IRF3 and expression of interferon-regulated genes. We find that primary bone marrow-derived macrophages (BMDMs) from C3aR−/− mice show significantly reduced TRIF-dependent ifnb expression and IFNb secretion following 24h LPS exposure. Additionally, WT BMDMs treated with a C3aR antagonist (C3aRi) show a similar reduction in ifnb expression after LPS-treatment. These data demonstrate C3aR is critical for TRIF-dependent signaling downstream of TLR4 activation. Here we find WT BMDMs treated with C3aRi and LPS show increased intracellular cAMP. These findings demonstrate C3aR activation leads to depleted cAMP, which is essential for TRIF-mediated signaling downstream of TLR4. We hypothesize that C3aR is working as an inhibitory GPCR to regulate inflammation via intracellular cAMP and enhancing TRIF signaling in LPS-treated macrophages. Our findings are the first to suggest C3aR reduces cAMP to control TRIF-mediated inflammation in activated macrophages.

Salmonella Enteritidis GalE Protein Inhibits LPS-Induced NLRP3 Inflammasome Activation.

Microbial infection can trigger the assembly of inflammasomes and promote secretion of cytokines, such as IL-1β and IL-18. It is well-known that Salmonella modulates the activation of NLRC4 (NLR family CARD domain-containing protein 4) and NLRP3 (NLR family pyrin domain-containing 3) inflammasomes, however the mechanisms whereby Salmonella avoids or delays inflammasome activation remain largely unknown. Therefore, we used Salmonella Enteritidis C50336ΔfliC transposon library to screen for genes involved in modulating inflammasomes activation. The screen revealed the galactose metabolism-related gene galE to be essential for inflammasome activation. Here, we found that inflammasome activation was significantly increased in J774A.1 cells or wild-type bone marrow-derived macrophages (BMDMs) during infection by ΔfliCΔgalE compared to cells infected with ΔfliC. Importantly, we found that secretion of IL-1β was Caspase-1-dependent, consistent with canonical NLRP3 inflammasome activation. Furthermore, the virulence of ΔfliCΔgalE was significantly decreased compared to ΔfliC in a mouse model. Finally, RNA-seq analysis showed that multiple signaling pathways related to the inflammasome were subject to regulation by GalE. Taken together, our results suggest that GalE plays an important role in the regulatory network of Salmonella evasion of inflammasome activation.

Osteopontin aggravates acute lung injury in influenza virus infection by promoting macrophages necroptosis

Infection with influenza A virus (IAV) can trigger pulmonary inflammation and lung damage. Osteopontin (OPN) is an essential regulator of cell death and immunity. However, the role and underlying mechanism of OPN in cell death in IAV-induced pulmonary injury remain poorly understood. Here, we demonstrated that OPN-deficient (OPN−/−) mice were insensitive to IAV, exhibiting decreased viral loads and attenuated lung injury after IAV infection compared to those in wild-type (WT) mice. Moreover, macrophage necroptosis was significantly reduced in OPN−/− mice infected with IAV compared to that in infected WT mice. OPN increased the expression of necroptosis-related genes and exacerbated macrophage necroptosis in IAV-infected THP1 cells. Notably, adoptive transfer of WT bone marrow-derived macrophages (BMDMs) or OPN−/− BMDMs into mice restored resistance to influenza infection, and the rescue effect of OPN−/− BMDMs was better than that of WT BMDMs. Collectively, these results suggest that OPN deficiency in macrophages reduces necroptosis, which leads to a decrease in viral titers and protects against IAV infection. Therefore, OPN is a potential target for the treatment of IAV infection.

Fibrinogen-like protein 2 deficiency inhibits virus-induced fulminant hepatitis through abrogating inflammatory macrophage activation.

BACKGROUNDHeterogeneous macrophages play an important role in multiple liver diseases, including viral fulminant hepatitis (VFH). Fibrinogen-like protein 2 (FGL2) is expressed on macrophages and regulates VFH pathogenesis; however, the underlying mechanism remains unclear. AIMTo explore how FGL2 regulates macrophage function and subsequent liver injury during VFH. METHODSMurine hepatitis virus strain 3 (MHV-3) was used to induce VFH in FGL2-deficient (Fgl2-/-) and wild-type (WT) mice. The dynamic constitution of hepatic macrophages was examined. Adoptive transfer of Fgl2-/- or WT bone marrow-derived macrophages (BMDMs) into WT recipients with macrophages depleted prior to infection was carried out and the consequent degree of liver damage was compared. The signaling cascades that may be regulated by FGL2 were detected in macrophages.RESULTSFollowing MHV-3 infection, hepatic macrophages were largely replenished by proinflammatory monocyte-derived macrophages (MoMFs), which expressed high levels of FGL2. In Fgl2-/- mice, the number of infiltrating inflammatory MoMFs was reduced compared with that in WT mice after viral infection. Macrophage depletion ameliorated liver damage in WT mice and further alleviated liver damage in Fgl2-/- mice. Adoptive transfer of Fgl2-/- BMDMs into macrophage-removed recipients significantly reduced the degree of liver damage. Inhibition of monocyte infiltration also significantly ameliorated liver damage. Functionally, Fgl2 deletion impaired macrophage phagocytosis and the antigen presentation potential and attenuated the proinflammatory phenotype. At the molecular level, FGL2 deficiency impaired IRF3, IRF7, and p38 phosphorylation, along with NF-κB activation in BMDMs in response to viral infection. CONCLUSIONInfiltrated MoMFs represent a major source of hepatic inflammation during VFH progression, and FGL2 expression on MoMFs maintains the proinflammatory phenotype via p38-dependent positive feedback, contributing to VFH pathogenesis.

Cytokine-Mediated Regulation of ARG1 in Macrophages and Its Impact on the Control of Salmonella enterica Serovar Typhimurium Infection.

Arginase 1 (ARG1) is a cytosolic enzyme that cleaves L-arginine, the substrate of inducible nitric oxide synthase (iNOS), and thereby impairs the control of various intracellular pathogens. Herein, we investigated the role of ARG1 during infection with Salmonella enterica serovar Typhimurium (S.tm). To study the impact of ARG1 on Salmonella infections in vitro, bone marrow-derived macrophages (BMDM) from C57BL/6N wild-type, ARG1-deficient Tie2Cre+/−ARG1fl/fl and NRAMPG169 C57BL/6N mice were infected with S.tm. In wild-type BMDM, ARG1 was induced by S.tm and further upregulated by the addition of interleukin (IL)-4, whereas interferon-γ had an inhibitory effect. Deletion of ARG1 did not result in a reduction in bacterial numbers. In vivo, Arg1 mRNA was upregulated in the spleen, but not in the liver of C57BL/6N mice following intraperitoneal S.tm infection. The genetic deletion of ARG1 (Tie2Cre+/−ARG1fl/fl) or its pharmacological inhibition with CB-1158 neither affected the numbers of S.tm in spleen, liver and blood nor the expression of host response genes such as iNOS, IL-6 or tumour necrosis factor (TNF). Furthermore, ARG1 was dispensable for pathogen control irrespective of the presence or absence of the phagolysosomal natural resistance-associated macrophage protein 1 (NRAMP1). Thus, unlike the detrimental function of ARG1 seen during infections with other intraphagosomal microorganisms, ARG1 did not support bacterial survival in systemic salmonellosis, indicating differential roles of arginine metabolism for host immune response and microbe persistence depending on the type of pathogen.

Knockout of MAPK Phosphatase-1 Exaggerates Type I IFN Response during Systemic Escherichia coli Infection.

We have previously shown that Mkp-1-deficient mice produce elevated TNF-α, IL-6, and IL-10 following systemic Escherichia coli infection, and they exhibited increased mortality, elevated bacterial burden, and profound metabolic alterations. To understand the function of Mkp-1 during bacterial infection, we performed RNA-sequencing analysis to compare the global gene expression between E. coli-infected wild-type and Mkp-1 -/- mice. A large number of IFN-stimulated genes were more robustly expressed in E. coli-infected Mkp-1 -/- mice than in wild-type mice. Multiplex analysis of the serum cytokine levels revealed profound increases in IFN-β, IFN-γ, TNF-α, IL-1α and β, IL-6, IL-10, IL-17A, IL-27, and GMSF levels in E. coli-infected Mkp-1 -/- mice relative to wild-type mice. Administration of a neutralizing Ab against the receptor for type I IFN to Mkp-1 -/- mice prior to E. coli infection augmented mortality and disease severity. Mkp-1 -/- bone marrow-derived macrophages (BMDM) produced higher levels of IFN-β mRNA and protein than did wild-type BMDM upon treatment with LPS, E. coli, polyinosinic:polycytidylic acid, and herring sperm DNA. Augmented IFN-β induction in Mkp-1 -/- BMDM was blocked by a p38 inhibitor but not by an JNK inhibitor. Enhanced Mkp-1 expression abolished IFN-β induction by both LPS and E. coli but had little effect on the IFN-β promoter activity in LPS-stimulated RAW264.7 cells. Mkp-1 deficiency did not have an overt effect on IRF3/7 phosphorylation or IKK activation but modestly enhanced IFN-β mRNA stability in LPS-stimulated BMDM. Our results suggest that Mkp-1 regulates IFN-β production primarily through a p38-mediated mechanism and that IFN-β plays a beneficial role in E. coli-induced sepsis.

Nogo-A Is Critical for Pro-Inflammatory Gene Regulation in Myocytes and Macrophages

Nogo-A (Rtn 4A), a member of the reticulon 4 (Rtn4) protein family, is a neurite outgrowth inhibitor protein that is primarily expressed in the central nervous system (CNS). However, previous studies revealed that Nogo-A was upregulated in skeletal muscles of Amyotrophic lateral sclerosis (ALS) patients. Additionally, experiments showed that endoplasmic reticulum (ER) stress marker, C/EBP homologous protein (CHOP), was upregulated in gastrocnemius muscle of a murine model of ALS. We therefore hypothesized that Nogo-A might relate to skeletal muscle diseases. According to our knocking down and overexpression results in muscle cell line (C2C12), we have found that upregulation of Nogo-A resulted in upregulation of CHOP, pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α, while downregulation of Nogo-A led to downregulation of CHOP, IL-6 and TNF-α. Immunofluorescence results showed that Nogo-A and CHOP were expressed by myofibers as well as tissue macrophages. Since resident macrophages share similar functions as bone marrow-derived macrophages (BMDM), we therefore, isolated macrophages from bone marrow to study the role of Nogo-A in activation of these cells. Lipopolysaccharide (LPS)-stimulated BMDM in Nogo-KO mice showed low mRNA expression of CHOP, IL-6 and TNF-α compared to BMDM in wild type (WT) mice. Interestingly, Nogo knockout (KO) BMDM exhibited lower migratory activity and phagocytic ability compared with WT BMDM after LPS treatment. In addition, mice experiments data revealed that upregulation of Nogo-A in notexin- and tunicamycin-treated muscles was associated with upregulation of CHOP, IL-6 and TNF-α in WT group, while in Nogo-KO group resulted in low expression level of CHOP, IL-6 and TNF-α. Furthermore, upregulation of Nogo-A in dystrophin-deficient (mdx) murine model, myopathy and Duchenne muscle dystrophy (DMD) clinical biopsies was associated with upregulation of CHOP, IL-6 and TNF-α. To the best of our knowledge, this is the first study to demonstrate Nogo-A as a regulator of inflammation in diseased muscle and bone marrow macrophages and that deletion of Nogo-A alleviates muscle inflammation and it can be utilized as a therapeutic target for improving muscle diseases.

KLF3 Represses the Inflammatory Response in Macrophages

Krüppel-like factors (KLFs) are a family of transcription factors that play essential roles in the development and differentiation of the hematopoietic system. These transcription factors possess highly conserved C-terminal zinc-finger motifs, which enable their binding to GC-rich, or CACC-box, motifs in promoter and enhancer regions of target genes. The N-terminal domains of these proteins are more varied and mediate the recruitment of various co-factors, which can form a complex with either activator or repressor function. Acting primarily as a gene repressor through its recruitment of CtBPs and histone deacetylases (HDACs) [1], we have recently shown that KLF3 competes with KLF1 bound sites in the genome to repress gene expression during erythropoiesis [2]. However, the function of Klf3 in other lineages has been less well studied. This widely expressed transcription factor has reported roles in the differentiation of marginal zone B cells, eosinophil function and inflammation [3]. We utilised the Klf3-null mouse model [4] to more closely examine the role of Klf3 in innate inflammatory cells. These mice exhibit elevated white cell counts, including monocytes (Figure 1A), and inflammation of the skin. Conditional knockout of Klf4 in myeloid cells leads to a deficiency of inflammatory macrophages [5]. To test our hypothesis KLF3 normally represses inflammation, perhaps by antagonising the action of KLF4, bone-marrow derived macrophages (BMDM) were generated from wild-type or Klf3-null mice and stimulated with the bacterial toxin lipopolysaccharide (LPS). In wild type BMDM, LPS induces Klf3 gene expression and activation then delayed repression of target genes such as Lgals3 (galectin-3) over a 21 hour time course (Figure 1B). Quantitative real-time PCR and mRNA-seq of WT v Klf3-null macrophages identified ~100 differentially expressed genes involved in proliferation, macrophage activation and inflammation. We transduced the monocyte cell line, RAW264.7 (that expresses Klf4, Klf3 and Klf2), with a retroviral vector expressing a tamoxifen-inducible KLF3-ER fusion construct. KLF3 induced cell cycle arrest and macrophage differentiation. We will report on KLF3-induced gene expression changes (repression and activation), and ChIP-seq for KLF3, in RAW cells. The results shed light on the mechanism by which KLF3 normally represses monocyte/macrophage responses to infection. This study highlights the importance of key transcriptional regulators that tightly control gene expression during inflammation. Loss of Klf3 leads to alterations in this process, resulting in hyper-activation of inflammatory macrophages, increased white cell counts and inflammation of the skin. A greater knowledge of the inflammatory process and how it is regulated is important for our understanding of acute infection and inflammatory disease. Further studies are planned to investigate the role of the KLF3 transcription factor in response to inflammation in vivo.

Enhanced Expression of Catalase in Mitochondria Modulates NF-κB-Dependent Lung Inflammation through Alteration of Metabolic Activity in Macrophages.

NF-κB is a reduction-oxidation-sensitive transcription factor that plays a key role in regulating the immune response. In these studies, we intended to investigate the role of mitochondrial-derived reactive oxygen species in regulating NF-κB activation by studying transgenic mice that overexpress mitochondrial-targeted human catalase (mCAT). We treated wild-type (WT) and mCAT mice with intratracheal instillation of Escherichia coli LPS and found that mCAT mice had exaggerated NF-κB activation in the lungs, increased neutrophilic alveolitis, and greater lung inflammation/injury compared with WT mice. Additional studies using bone marrow chimeras revealed that this hyperinflammatory phenotype was mediated by immune/inflammatory cells. Mechanistic studies using bone marrow-derived macrophages (BMDMs) showed that LPS treatment induced a sustained increase in NF-κB activation and expression of NF-κB-dependent inflammatory mediators in mCAT BMDMs compared with WT BMDMs. Further investigations showed that cytoplasmic, but not mitochondrial, hydrogen peroxide levels were reduced in LPS-treated mCAT BMDMs. However, mCAT macrophages exhibited increased glycolytic and oxidative metabolism, coupled with increased ATP production and an increased intracellular NADH/NAD+ ratio compared with BMDMs from WT mice. Treatment of BMDMs with lactate increased the intracellular NADH/NAD+ ratio and upregulated NF-κB activation after LPS treatment, whereas treatment with a potent inhibitor of the mitochondrial pyruvate carrier (UK5099) decreased the NADH/NAD+ ratio and reduced NF-κB activation. Taken together, these findings point to an increased availability of reducing equivalents in the form of NADH as an important mechanism by which metabolic activity modulates inflammatory signaling through the NF-κB pathway.