WT Bone Marrow-derived Macrophages Research Articles

STING inhibition alleviates bone resorption in apical periodontitis.

The goal of this study was to investigate the potential effects of an immunotherapeutic drug targeting STING to suppress the overreactive innate immune response and relieve the bone defect in apical periodontitis. We established an apical periodontitis mouse model in Sting-/- and WT mice in vivo. The progression of apical periodontitis was analysed by micro-CT analysis and H&E staining. The expression level and localization of STING in F4/80+ cells were identified by IHC and immunofluorescence staining. RANKL in periapical tissues was tested by IHC staining. TRAP staining was used to detect osteoclasts. To clarify the effect of STING inhibitor C-176 as an immunotherapeutic drug, mice with apical periodontitis were treated with C-176 and the bone loss was identified by H&E, TRAP, RANKL staining and micro-CT. Bone marrow-derived macrophages (BMMs) were isolated from Sting-/- and WT mice and induced to osteoclasts in a lipopolysaccharide (LPS)-induced inflammatory environment in vitro. Moreover, WT BMMs were treated with C-176 to determine the effect on osteoclast differentiation by TRAP staining. The expression levels of osteoclast-related genes were tested using qRT-PCR. Compared to WT mice, the bone resorption and inflammatory cell infiltration were reduced in exposed Sting-/- mice. In the exposed WT group, STING was activated mainly in F4/80+ macrophages. Histological staining revealed the less osteoclasts and lower expression of osteoclast-related factor RANKL in Sting-/- mice. The treatment of the STING inhibitor C-176 in an apical periodontitis mice model alleviated inflammation progression and bone loss, similar to the effect observed in Sting-/- mice. Expression of RANKL and osteoclast number in periapical tissues were also decreased after C-176 administration. In vitro, TRAP staining showed fewer positive cells and qRT-PCR reflected decreased expression of osteoclastic marker, Src and Acp5 were detected during osteoclastic differentiation in Sting-/- and C-176 treated BMMs. STING was activated and was proven to be a positive factor in bone loss and osteoclastogenesis in apical periodontitis. The STING inhibitor C-176 administration could alleviate the bone loss via modulating local immune response, which provided immunotherapy to the treatment of apical periodontitis.

Two-pore potassium channel TREK-1 (K2P2.1) regulates NLRP3 inflammasome activity in macrophages.

Because of the importance of potassium efflux in inflammasome activation, we investigated the role of the two-pore potassium (K2P) channel TREK-1 in macrophage inflammasome activity. Using primary alveolar macrophages (AMs) and bone marrow-derived macrophages (BMDMs) from wild-type (wt) and TREK-1-/- mice, we measured responses to inflammasome priming [using lipopolysaccharide (LPS)] and activation (LPS + ATP). We measured IL-1β, caspase-1, and NLRP3 via ELISA and Western blot. A membrane-permeable potassium indicator was used to measure potassium efflux during ATP exposure, and a fluorescence-based assay was used to assess changes in membrane potential. Inflammasome activation induced by LPS + ATP increased IL-1β secretion in wt AMs, whereas activation was significantly reduced in TREK-1-/- AMs. Priming of BMDMs using LPS was not affected by either genetic deficiency or pharmacological inhibition of TREK-1 with Spadin. Cleavage of caspase-1 following LPS + ATP treatment was significantly reduced in TREK-1-/- BMDMs. The intracellular potassium concentration in LPS-primed wt BMDMs was significantly lower compared with TREK-1-/- BMDMs or wt BMDMs treated with Spadin. Conversely, activation of TREK-1 with BL1249 caused a decrease in intracellular potassium in wt BMDMs. Treatment of LPS-primed BMDMs with ATP caused a rapid reduction in intracellular potassium levels, with the largest change observed in TREK-1-/- BMDMs. Intracellular K+ changes were associated with changes in the plasma membrane potential (Em), as evidenced by a more depolarized Em in TREK-1-/- BMDMs compared with wt, and Em hyperpolarization upon TREK-1 channel opening with BL1249. These results suggest that TREK-1 is an important regulator of NLRP3 inflammasome activation in macrophages.NEW & NOTEWORTHY Because of the importance of potassium efflux in inflammasome activation, we investigated the role of the two-pore potassium (K2P) channel TREK-1 in macrophage inflammasome activity. Using primary alveolar macrophages and bone marrow-derived macrophages from wild-type and TREK-1-/- mice, we measured responses to inflammasome priming (using LPS) and activation (LPS + ATP). Our results suggest that TREK-1 is an important regulator of NLRP3 inflammasome activation in macrophages.

Brd4 Inhibition Abrogates Inflammation and Self-Renewal in a Murine Model of Tet2 Mutated Clonal Hematopoiesis

Clonal hematopoiesis (CH) is characterized by the somatic acquisition of malignancy associated mutations in hematopoietic stem cells (HSCs). Loss-of-function mutations in TET2 occur in one third of CH cases and are highly associated with the subsequent development of myeloid malignancies. Tet2 KO mice demonstrate increased HSC self-renewal, expansion of myeloid precursors, elevated inflammation, and progression to leukemia with long latencies, similar to CH to AML progression in humans. Additionally, leukemia development in Tet2 KO mice is dependent on increased production of pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α through hyperactive toll-like-receptor signaling (TLR). Despite extensive characterization of the adverse effects related to TET2 mutations, no effect intervention or pharmacologic therapy has been developed to slow its known progression. To investigate altered pathways in Tet2 mutant CH that might be amenable to pharmacologic targeting, we performed RNA-seq on sorted Lin -cKIT + (LK) myeloid progenitors and Lin -cKIT +Sca-1 + (LSK) stem cells from Tet2 KO vs WT mice. We found significant upregulation of hallmark JAK/STAT3 and TNF-α signaling pathways in Tet2 KO cells. The epigenetic reader protein bromo- and extra-terminal domain 4 (Brd4) interacts with both NF-κB and STAT3 to promote transcriptional activity and regulate inflammation. We hypothesized inhibition of Brd4 would reverse abnormal hematopoiesis in Tet2 KOs via reduction in inflammatory signaling. Therefore, we generated double KO mice by intercrossing Mx1-Cre mice with Tet2 conditional KO and Brd4 conditional KO mice. While deletion of Brd4 during fetal hematopoiesis is lethal, we have successfully induced deletion of Brd4 both alone and in combination with Tet2 in young adult animals without any observed lethality for 6 months. To study pharmacologic Brd4 inhibition in Tet2 KO mice, we utilized PLX51107, a structurally distinct BET inhibitor previously reported on by our laboratory. As expected, Tet2 KO marrow demonstrated increased colony formation and serial replating ability compared to WT controls. However, colony formation by Brd4 Tet2 DKO marrow was significantly decreased when compared to Tet2 KO ( p = 0.0003, n=5) or WT mice ( p = 0.0048, n=5) (Figure 1A). Similarly, PLX51107 treatment reduced Tet2 KO colony formation ( p = 0.0001, n=5) and abrogated serial replating ability ( p < 0.01, n=5). When Brd4 KO or Brd4 Tet2 DKO marrow was treated with PLX51107, which also inhibits Brd2 and Brd3, there was no significant change in colony formation compared to untreated marrow, suggesting Brd4 has a non-redundant role in colony formation. Collectively, these results suggest Brd4 plays a key role in self-renewal of Tet2 KO HSCs. To further characterize the role of Brd4 in Tet2 mutated CH, single cell RNA-seq analysis of Brd4 KO and Brd4 Tet2 DKO marrow populations is in progress. In murine models of Tet2 CH, disease progression is associated with emergence of an aberrant population of inflammatory macrophages. We therefore investigated functional differences in macrophages from WT, single, and double KO mice. LPS-stimulated bone marrow derived macrophages (BMDMs) from Brd4 Tet2 DKO mice produced significantly less TNF-α, IL-1β, IL12p40, and IL-18 when compared to single KO or WT controls ( p < 0.001, n=3) (Figure 1B). Similarly, we found mRNA expression of these cytokines to be decreased in LPS-stimulated Brd4 Tet2 DKO BMDMs when compared to single KO and WT controls ( p < 0.001, n=3). Similar results were observed in LPS-stimulated Tet2 KO and WT BMDMs treated with PLX51107 ( p < 0.005, n=3). Additionally, PLX51107 treatment reduced protein levels pSTAT3, NLRP3, and phosphorylated p65, key downstream targets of TLR signaling, in LPS-stimulated BMDMs. Together, these results suggest that Brd4 is required for the hyperinflammation characteristic of Tet2 mutated macrophages that drive progression to myeloid malignancies. In summary, we report that genetic or pharmacologic inhibition of Brd4 reduces the increased self-renewal and elevated inflammation in a murine model of Tet2 mutated CH. Our novel inducible double knockout mouse model of CH achieved simultaneous knockout of Brd4 and Tet2, which has never been investigated. Collectively, our data provides rationale for further pre-clinical investigation of BET inhibitors to prevent progression of Tet2 CH.

Role of GPER in the expression and secretion of pro-inflammatory cytokines and calcium signaling in bone marrow-derived macrophages

Macrophages play an important role in mediating inflammation in numerous disease states by secreting pro-inflammatory cytokines including IL-1β and IL-18. Macrophages are driven into the M1-proinflammatory phenotype upon detection of pathogen-associated molecular patterns (PAMPs), or damage-associated molecular patterns (DAMPs). As macrophages are polarized into the M1 phenotype, they upregulate the expression of pro-inflammatory cytokines and components of the NLRP3 inflammasome. An additional activating stimulus is required to induce the assembly of the NLRP3 inflammasome, which facilitates the activation of pro-caspase 1 and the subsequent conversion of the pro-forms of both IL-18 and IL-1β into their mature forms for their secretion through gasdermin D membrane pores. A canonical activator of the NLRP3 inflammasome is extracellular ATP. ATP activation of the P2X7 receptor induces potassium efflux and calcium influx, both of which are necessary for NLRP3 inflammasome activation. Previous studies suggest that the G protein-coupled receptor (GPER) agonist G-1 reduced inflammation in animal disease models. However, the GPER-dependent effect of G-1 on pro-inflammatory cytokine expression and NLRP3 inflammasome activation in macrophages is unknown. In the current study, we utilized bone marrow-derived macrophages (BMDMs) from either WT or GPER-/- mice to test the hypothesis that G-1 reduces inflammatory cytokine production and NLRP3 inflammasome expression in a GPER-dependent manner. We found that the treatment of WT BMDMs with 100 nM G-1 for 24 hrs inhibited LPS-induced NLRP3, pro-IL-1β, IL-6, CCL5 and expression, and IL-1β secretion. Importantly, G-1 had no effect on GPER-/- BMDMs. To begin to determine if GPER modulates NLRP3 inflammasome activation, we characterized ATP-induced Ca2+ signals in both WT and GPER BMDMs with and without LPS pretreatment. No differences were observed between unprimed WT and GPER-/- BMDMs with respect to basal Ca2+ level, ATP-induced intracellular Ca2+ release, and subsequent bulk Ca2+ signal upon reestablishment of extracellular Ca2+. However, priming with LPS significantly increased ATP-induced intracellular Ca2+ release in both WT and GPER-/- BMDMs. Compared to WT cells, LPS-primed GPER-/- BMDMs exhibit significantly higher basal Ca2+ yet significantly lower ATP-induced Ca2+ release. Consistently, 15-minute treatment with 100 nM G-1 agonist strongly reduces ATP-induced Ca2+ release in WT BMDMs but had no effect in GPER-/- BMDMs. These results suggest a role for GPER in mediating Ca2+ uptake into IP3-sensitive intracellular compartments in BMDMs during LPS priming. In keeping with the known role for mobilization of intracellular Ca2+ in ATP-induced secretion of pro-inflammatory cytokines, preliminary data suggests that short-term G-1 pretreatment reduced IL-1β secretion induced by ATP in LPS-primed BMDMs. Overall, these data indicate a role for GPER in the priming and activation of the NLRP3 inflammasome. This research was supported by Des Moines University IOER Research & Grant Award #03-22-10. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mkp-1 deficiency strengthens immune defense against fungal infection

Abstract Fungal infections are a serious threat to people with a compromised immune system. The recognition of fungal pathogens relies on pathways mediated by TLRs and C-type lectin receptors. Activation of these TLR and C-type lectin receptor pathways converge on both NF-κB and MAPKs to initiate cytokine and chemokine production, leading to phagocyte recruitment and pathogen elimination. Mkp-1 is a negative feedback regulator of the p38 and JNK MAPKs. To understand the role of Mkp-1 in immune defense against fungal pathogens, we assessed the effects of Mkp-1 deficiency on the immune response to C. albicans, the most common fungal pathogen. Contrary to our previous observation with E. coli or bacterial products such as LPS, Mkp1−/− mice were protected from a lethal dose of C. albicans. In response to C. albicans challenge, Mkp1−/− mice produced greater amounts of TNF-α, had lower kidney fungal burdens and smaller fungal lesions. In wildtype mice large number of neutrophils were found in the kidneys, forming clusters surrounding fungal lesions. In contrast, far less neutrophils were found in the kidneys of Mkp-1−/− mice, although these neutrophils appeared to express greater levels of neutrophil elastase and form tighter clusters surrounding the fungi. Upon stimulation with heat-killed C. albicans yeasts and hyphae Mkp-1−/− bone marrow-derived macrophages (BMDM) also produced greater amounts of TNF-α and had higher p38 and JNK activities than did WT BMDM. TNF-α production in both wildtype and Mkp-1−/− BMDM in response to heat-killed C. albicans was attenuated by the pharmacological inhibitors of MEK1, JNK, p38, and Syk. Taken together, our results suggest that during fungal infection Mkp-1 acts to hamper the immune response against fungal pathogens. *The studies were supported by NIH grants (AI113930 and AI124029 to Y.L., and AI121196 and AI123253 to J.Z.).

Targeting Arg-1 and PD-L1 in M2-Tumor Associated Macrophages Impairs Juvenile Myelomonocytic Leukemia (JMML) Cell Proliferation and Migration

Background and Hypothesis: Tumor-associated macrophages (TAMs) are a key component of tumor-infiltrating immune cells. They are largely characterized into M1 or M2 types. TAMs express an anti-inflammatory M2-like phenotype, promote tumor progression. However, the role of M2-TAMs in driving disease pathogenesis in patients with Juvenile myelomonocytic leukemia (JMML), a rare form of pediatric leukemia driven to a large extent by mutations in the PTPN11 gene, which encodes the phosphatase SHP2 is unclear. We hypothesized that in JMML, inflammatory myeloid cells including neutrophils and M2-TAMs express higher levels of arginase-1 (Arg-1) and PD-L1, which may contribute to the local suppression of immune responses and support the development of JMML.
 Methods: To study how alterations in M1/M2 macrophages contribute to JMML development, we utilized a mouse model bearing Shp2E76K mutation (Ptpn11E76K/+) which manifests the cardinal features of human JMML. We hypothesized that Shp2E76K/+ mutations enhance the function of bone marrow derived macrophages (BMDMs), including M2-TAMs and contribute to T-cell suppression.
 Results: Our analysis of the bulk RNA-sequence data from 90 JMML patients showed increase in the expression of Arg-1 and PD-1. Furthermore, single cell RNA-seq analysis of macrophages from 4 JMML patients revealed higher expression of M2-macrophage markers/genes. Our results show that in M2-TAMs, Arg-1 and PD-L1 levels are elevated in BM and spleens of Shp2E76K/+ mice compared to WT. Moreover, M2-TAMs, Arg-1 and PD-L1 levels were also higher in BMDMs derived from Shp2E76K/+ mice compared to WT. The BMDMs from Shp2E76K/+ mice have greater proliferation and migration potential compared to WT BMDMs, which was significantly reduced by inhibiting the function of Arg-1 and PD-L1.
 Conclusion: Our results show that M2-TAMs, arginase-1, and PD-L1 create a pro-tumor microenvironment, which likely contributes to the growth of JMML cells. Inhibition of Arg-1 and PD-L1 is a novel therapeutic approach to treat patients with JMML.

The role of plasminogen activator inhibitor-2 in pneumococcal meningitis

Pneumococcal meningitis is associated with dysregulation of the coagulation cascade. Previously, we detected upregulation of cerebral plasminogen activator inhibitor-2 (PAI-2) mRNA expression during pneumococcal meningitis. Diverse functions have been ascribed to PAI-2, but its role remains unclear. We analyzed the function of SERPINB2 (coding for PAI-2) in patients with bacterial meningitis, in a well-established pneumococcal meningitis mouse model, using Serpinb2 knockout mice, and in vitro in wt and PAI-2-deficient bone marrow-derived macrophages (BMDMs). We measured PAI-2 in cerebrospinal fluid of patients, and performed functional, histopathological, protein and mRNA expression analyses in vivo and in vitro. We found a substantial increase of PAI-2 concentration in CSF of patients with pneumococcal meningitis, and up-regulation and increased release of PAI-2 in mice. PAI-2 deficiency was associated with increased mortality in murine pneumococcal meningitis and cerebral hemorrhages. Serpinb2−/− mice exhibited increased C5a levels, but decreased IL-10 levels in the brain during pneumococcal infection. Our in vitro experiments confirmed increased expression and release of PAI-2 by wt BMDM and decreased IL-10 liberation by PAI-2-deficient BMDM upon pneumococcal challenge. Our data show that PAI-2 is elevated during in pneumococcal meningitis in humans and mice. PAI-2 deficiency causes an inflammatory imbalance, resulting in increased brain pathology and mortality.

Stimulator of Interferon Genes (STING) Promotes Staphylococcus aureus-Induced Extracellular Traps Formation via the ROS-ERK Signaling Pathway.

Stimulator of interferon genes (STING) is a cytosolic DNA sensor or directly recognizes bacterial cyclic dinucleotides, which is required for the detection of microbial infection. Extracellular traps (ETs) are known to be part of the antimicrobial defense system. However, the implication of STING in ETs formation during microbial infection remains unknown. Here, we showed that STING contributed to Staphylococcus aureus (S. aureus)-induced ETs formation through the ROS-ERK signaling. STING deficiency exhibited decreased cell-free DNA (cfDNA) level, reduced expression of citrullinated histone H3 (CitH3), and diminished DNA colocalization with CitH3 and myeloperoxidase (MPO). Interestingly, NADPH oxidase-derived reactive oxygen species (ROS) promoted ETs formation, accompanied by increased activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in S. aureus-stimulated bone marrow-derived macrophages (BMDMs). Corresponding to less ROS production, decreased ERK1/2 activation was shown in STING-/- BMDMs after S. aureus infection. Importantly, inhibiting the ROS-ERK signal reduced the ETs formation and the differences disappeared between WT and STING-/- BMDMs after S. aureus infection. Moreover, STING-/- BMDMs exhibited significantly increased levels of extracellular bacteria compared to WT BMDMs regardless of phagocytosis. In addition, such differences disappeared after DNase I treatment. DNase I treatment also facilitated pathogen colonization without affecting the inflammatory cells infiltration and pro-inflammatory factors secretion following pulmonary S. aureus infection. Furthermore, STING-/- mice presented decreased levels of cfDNA and CitH3, along with increased bacterial colonization compared to WT mice. Altogether, these findings highlighted that STING promoted ETs formation via the ROS-ERK signal for host defense against S. aureus infection.

LincRNA-EPS impairs host antiviral immunity by antagonizing viral RNA-PKR interaction.

LincRNA‐EPS is an important regulator in inflammation. However, the role of lincRNA‐EPS in the host response against viral infection is unexplored. Here, we show that lincRNA‐EPS is downregulated in macrophages infected with different viruses including VSV, SeV, and HSV‐1. Overexpression of lincRNA‐EPS facilitates viral infection, while deficiency of lincRNA‐EPS protects the host against viral infection in vitro and in vivo. LincRNA‐EPS −/− macrophages show elevated expression of antiviral interferon‐stimulated genes (ISGs) such as Mx1, Oas2, and Ifit2 at both basal and inducible levels. However, IFN‐β, the key upstream inducer of these ISGs, is downregulated in lincRNA‐EPS −/− macrophages compared with control cells. RNA pulldown and mass spectrometry results indicate that lincRNA‐EPS binds to PKR and antagonizes the viral RNA–PKR interaction. PKR activates STAT1 and induces antiviral ISGs independent of IFN‐I induction. LincRNA‐EPS inhibits PKR‐STAT1‐ISGs signaling and thus facilitates viral infection. Our study outlines an alternative antiviral pathway, with downregulation of lincRNA‐EPS promoting the induction of PKR‐STAT1‐dependent ISGs, and reveals a potential therapeutic target for viral infectious diseases.

DDX3X coordinates host defense against influenza virus by activating the NLRP3 inflammasome and type I interferon response

Viruses and hosts have coevolved for millions of years, leading to the development of complex host–pathogen interactions. Influenza A virus (IAV) causes severe pulmonary pathology and is a recurrent threat to human health. Innate immune sensing of IAV triggers a complex chain of host responses. IAV has adapted to evade host defense mechanisms, and the host has coevolved to counteract these evasion strategies. However, the molecular mechanisms governing the balance between host defense and viral immune evasion is poorly understood. Here, we show that the host protein DEAD-box helicase 3 X-linked (DDX3X) is critical to orchestrate a multifaceted antiviral innate response during IAV infection, coordinating the activation of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome, assembly of stress granules, and type I interferon (IFN) responses. DDX3X activated the NLRP3 inflammasome in response to WT IAV, which carries the immune evasive nonstructural protein 1 (NS1). However, in the absence of NS1, DDX3X promoted the formation of stress granules that facilitated efficient activation of type I IFN signaling. Moreover, induction of DDX3X-containing stress granules by external stimuli after IAV infection led to increased type I IFN signaling, suggesting that NS1 actively inhibits stress granule–mediated host responses and DDX3X-mediated NLRP3 activation counteracts this action. Furthermore, the loss of DDX3X expression in myeloid cells caused severe pulmonary pathogenesis and morbidity in IAV-infected mice. Together, our findings show that DDX3X orchestrates alternate modes of innate host defense which are critical to fight against NS1-mediated immune evasion strategies during IAV infection.

HUWE1 mediates inflammasome activation and promotes host defense against bacterial infection.

The mechanism by which inflammasome activation is modulated remains unclear. In this study, we identified an AIM2-interacting protein, the E3 ubiquitin ligase HUWE1, which was also found to interact with NLRP3 and NLRC4 through the HIN domain of AIM2 and the NACHT domains of NLRP3 and NLRC4. The BH3 domain of HUWE1 was important for its interaction with NLRP3, AIM2, and NLRC4. Caspase-1 maturation, IL-1β release, and pyroptosis were reduced in Huwe1-deficient bone marrow-derived macrophages (BMDMs) compared with WT BMDMs in response to stimuli to induce NLRP3, NLRC4, and AIM2 inflammasome activation. Furthermore, the activation of NLRP3, NLRC4, and AIM2 inflammasomes in both mouse and human cells was remarkably reduced by treatment with the HUWE1 inhibitor BI8622. HUWE1 mediated the K27-linked polyubiquitination of AIM2, NLRP3, and NLRC4, which led to inflammasome assembly, ASC speck formation, and sustained caspase-1 activation. Huwe1-deficient mice had an increased bacterial burden and decreased caspase-1 activation and IL-1β production upon Salmonella, Francisella, or Acinetobacter baumannii infection. Our study provides insights into the mechanisms of inflammasome activation as well as a potential therapeutic target against bacterial infection.

Fibrinogen-like protein 2 aggravates nonalcoholic steatohepatitis via interaction with TLR4, eliciting inflammation in macrophages and inducing hepatic lipid metabolism disorder.

Rationale: The functions of fibrinogen-like protein 2 (fgl2) have been studied in many inflammatory and neoplastic diseases, but the role of fgl2 in nonalcoholic fatty liver disease has not yet been elucidated. In this study, we sought to investigate the role of fgl2 in the pathogenesis of nonalcoholic steatohepatitis (NASH).Methods: Hepatic fgl2 expression was tested in patients with nonalcoholic fatty liver (NAFL) or NASH and controls. Wild-type and fgl2-/- C57BL/6 mice were subjected to a methionine/choline-deficient (MCD) diet or a high-fat diet (HFD) to establish NASH models. Bone marrow-derived macrophages (BMDMs) stimulated with LPS or free fatty acids were used for the in vitro study.Results: In both humans and mice with NASH, macrophage accumulation was concomitant with significantly increased fgl2 expression in the liver. Fgl2 deficiency attenuated liver steatosis and inflammation in diet-induced murine models of NASH. In both liver tissues and BMDMs from NASH mice, fgl2 deficiency resulted in reduced levels of proinflammatory cytokines and reactive oxygen species (ROS) compared with levels in wild-type controls. Activation of NF-κB, p38-MAPK and NLRP3 inflammasomes was also suppressed upon fgl2 disruption. Moreover, lipogenic genes (Fasn and SREBP-2) were downregulated while lipolytic genes (PPAR and CPT1A) were upregulated in the livers of fgl2-/- NASH mice. Primary hepatocytes incubated with the medium collected from fgl2-/- BMDMs showed less fat deposition than those incubated with WT BMDMs. Furthermore, we discovered that fgl2 combined with TLR4 mediates the activation of the Myd88-dependent signaling pathway, which may contribute to inflammation and lipid metabolism disorders.Conclusions: These data suggest that fgl2 aggravates the progression of NASH through activation of NF-κB, p38-MAPK and NLRP3 inflammasomes in macrophages, which consequently induces overproduction of proinflammatory cytokines and lipid metabolism disorders. An interaction of fgl2 and TLR4 may in part contribute to the activation of inflammatory signaling pathways in macrophages.

Macrophage HIF-2α regulates tumor-suppressive Spint1 in the tumor microenvironment.

In solid tumors, tumor-associated macrophages (TAMs) commonly accumulate within hypoxic areas. Adaptations to such environments evoke transcriptional changes by the hypoxia-inducible factors (HIFs). While HIF-1α is ubiquitously expressed, HIF-2α appears tissue-specific with consequences of HIF-2α expression in TAMs only being poorly characterized. An E0771 allograft breast tumor model revealed faster tumor growth in myeloid HIF-2α knockout (HIF-2αLysM-/- ) compared with wildtype (wt) mice. In an RNA-sequencing approach of FACS sorted wt and HIF-2α LysM-/- TAMs, serine protease inhibitor, Kunitz type-1 ( Spint1) emerged as a promising candidate for HIF-2α-dependent regulation. We validated reduced Spint1 messenger RNA expression and concomitant Spint1 protein secretion under hypoxia in HIF-2α-deficient bone marrow-derived macrophages (BMDMs) compared with wt BMDMs. In line with the physiological function of Spint1 as an inhibitor of hepatocyte growth factor (HGF) activation, supernatants of hypoxic HIF-2α knockout BMDMs, not containing Spint1, were able to release proliferative properties of inactive pro-HGF on breast tumor cells. In contrast, hypoxic wt BMDM supernatants containing abundant Spint1 amounts failed to do so. We propose that Spint1 contributes to the tumor-suppressive function of HIF-2α in TAMs in breast tumor development.

Induction of IL‐10 in Bone‐Marrow Derived Macrophages by Helicobacter Hepaticus Supernatant is Mediated by Gpr109a

Interleukin 10 (IL‐10) is an anti‐inflammatory cytokine, and its polymorphism is linked to inflammatory bowel diseases (IBDs) in humans. Interactions between gut microbiota and the host's the immune cells play a key role in shaping immune responses and intestinal health. Helicobacter hepaticus is a member of mouse gut microbiota that is tolerated by the host but can induce colonic inflammation in mice in absence of an intact IL‐10 signaling. H. hepaticus secretes a polysaccharide that signals through Toll‐like receptor 2 (TLR2) to induce IL‐10 production. Gpr109a is a G‐protein coupled receptor that is expressed at a very high level on innate immune cells, and it is thought to interact with gut microbe‐derived molecules to induce production of anti‐inflammatory cytokines and suppress colonic inflammation. It is hypothesized that H. hepaticus induction of IL‐10 through TLR2 is mediated by Gpr109a. To test whether Gpr109a is involved in IL‐10 induction by H. hepaticus secretions, bone‐marrow derived macrophages (BMDMs) of WT and Gpr109a KO mice were stimulated by H. hepaticus supernatant then IL‐10 mRNA and protein levels were determined. WT BMDMs were found to produce significantly more IL‐10 mRNA and protein compared to Gpr109a KO BMDMs. In addition, ERK1/2 is a protein complex that gets activated when the TLR2 is stimulated by H. hepaticus to induce IL‐10. We tested whether ERK1/2 activation by H. hepaticus supernatant is dependent on Gpr109a by using an immunoprecipitation assay for total and pERK1/2 upon stimulation with H. hepaticus supernatant for WT and Gpr109a KO BMDMs. Our data suggests that Gpr109a plays a critical role in activation of ERK1/2 downstream of TLR2 signaling leading to induction of IL‐10. These findings will be of a value in delineating the molecular mechanism of Gpr109a signaling and investigating Gpr109a role in preventing H. hepaticus induced colitis and intestinal cancers.Support or Funding InformationThis projected was funded by Augusta University (The STAR Program) and performed in Dr. Nagendra Singh's laboratory and under his supervision with technical training and support by Brinda Bhatt and Dr. Huabin Zhu.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

LPS PROMOTES ASC‐SPECK FORMATION IN TREK‐1‐DEFICIENT MACROPHAGES

BACKGROUNDTREK‐1 is a 2‐pore potassium channel that we previously showed played a protective role against hyperoxia‐induced lung injury. To determine whether this protective effect was related to reduced inflammasome activation, we investigated LPS‐induced responses. Our preliminary studies with macrophages from TREK‐1−/− mice indicated a reduction in NLRP3 inflammasome priming and activation compared with wt mice, but we found that prolonged LPS exposure promoted increased IL‐1β release. We therefore investigatElied non‐canonical inflammasome pathways.METHODSWe stimulated alveolar macrophages (AMs) and bone marrow derived macrophages (BMDMs) using both a short‐term LPS+/−ATP model of inflammasome priming and activation and a prolonged‐LPS exposure. wt, TREK‐1−/−, and NLRP3−/− AMs and BMDMs were pre‐treated with a TREK‐1‐inhibitor (SPADIN), a TREK‐1‐activator, a Pannexin inhibitor, a P2X channel inhibitor, an ASC‐oligomerization inhibitor, or a JNK‐inhibitor. We measured IL‐1β, NLRP3, caspase‐1, ASC, TNFα as well as caspase−8, −7, and −11, and phosphorylated‐JNK using western blot, immunostaining, and ELISA. We used a membrane permeable potassium indicator to measure potassium efflux during ATP exposure. Using a caspase‐luciferace assay, we then measured caspase‐activity +/− MG132 (a proteasome inhibitor).RESULTSATP treatment of LPS‐primed TREK‐1‐inhibited and ‐deficient BMDMs exaggerated caspase‐7 and caspase‐11 activity compared to wt BMDMs, but both were dampened with co‐treatment with Pannexin and P2X inhibitors. Both TREK1 inhibition and deficiency caused elevated potassium efflux after short term LPS + ATP. With prolonged LPS exposure, both wt and TREK‐1−/− BMDMs contained more intracellular potassium than unstimulated wt BMDMs, with the LPS‐treated TREK‐1−/− BMDMs containing the highest concentration. Prolonged LPS exposure increased casp1‐p20, IL‐1β, and ASC‐speck formation in TREK‐1−/− BMDMs and AMs, compared with wt. Similarly, TREK‐1 inhibition with SPADIN increased ASC‐speck formation in both wt and NLRP3−/− BMDMs after prolonged LPS exposure. Additionally, MG132 completely blocked caspase activity in wt BMDMs treated with SPADIN after LPS treatment. JNK inhibition decreased ASC‐oligomerization in both wt and TREK‐1−/− BMDMs. However, prolonged LPS exposure led to increased caspase‐7 and JNK activity in TREK‐1−/− BMDMs compared to wt. Additionally, TREK‐1 inhibition increased caspase‐7 and caspase‐8 activity after LPS exposure in both wt and NLRP3−/− BMDMs.CONCLUSIONThese results suggest that the protective effect of TREK‐1 may be associated with regulation of apoptotic association ASC‐speck formation.Support or Funding InformationSupported by NIH grants HL123540, HL131526, and HL118118, and by the Le Bonheur Children's Hospital Research Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hydrogen sulfide inhibits NLRP3 inflammasome activation and reduces cytokine production both in vitro and in a mouse model of inflammation

A variety of stimuli, including monosodium urate (MSU) crystals, activate the NLRP3 inflammasome, and this activation involves several molecular mechanisms including xanthine oxidase (XO) up-regulation and mitochondrial dysfunction. Upon oligomerization of apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1 becomes active and cleaves the proinflammatory cytokine IL-1β into its active secreted form. Hydrogen sulfide (H2S), a gasotransmitter mainly produced by cystathionine γ-lyase (CSE) in macrophages, could modulate inflammation. Here, we sought to investigate the effects of exogenous and endogenous H2S on NLRP3 inflammasome activation in vitro and in vivo Primed bone marrow-derived macrophages (BMDM) isolated from wildtype (wt) or CSE-deficient mice and human macrophages (THP1 cells and primary macrophages), were stimulated with MSU crystals in the presence or absence of a H2S donor, sodium thiosulfate (STS) or GYY4137 (GYY). In murine and human macrophages in vitro, both STS and GYY inhibited MSU crystal-induced IL-1β secretion in a dose-dependent manner. Moreover, the H2S donors inhibited MSU crystal-induced XO/caspase-1 activities, mitochondrial reactive oxygen species (ROS) generation, and ASC oligomerization. Accordingly, IL-1β secretion and XO/caspase-1 activities were higher in CSE-deficient BMDMs than in wt BMDMs. For in vivo studies, we experimentally induced peritonitis by intraperitoneal injection of MSU crystals into mice. GYY pretreatment ameliorated inflammation, evidenced by decreased IL-6/monocyte chemoattractant protein-1 (MCP-1) released into peritoneal lavages. Taken together, our results suggest that both exogenous (via H2S donors) and endogenous (via CSE) H2S production may represent approaches for managing, for example, acute gout or other inflammation conditions.

PI3K p85\u03b1 Subunit-deficient Macrophages Protect Mice from Acute Colitis due to the Enhancement of IL-10 Production

We investigated the role of the PI3K p85α subunit in the development of acute colitis with a focus on intestinal macrophages. Experimental acute colitis was induced using 3% dextran sulfate sodium (DSS) in drinking water for 7 days. The severity of DSS-induced acute colitis was significantly attenuated in p85α hetero-deficient (p85α+/−) mice compared with WT mice. The expression of proinflammatory mediators in intestinal macrophages isolated from the inflamed colonic mucosa was significantly suppressed in p85α+/− colitis mice compared with WT colitis mice. Interestingly, we found that bone marrow-derived macrophages (BMDMs) from p85α+/− mice produced a significantly higher amount of IL-10 than BMDMs from WT mice. The adoptive transfer of p85α+/− BMDMs, but not WT BMDMs, significantly improved the severity in WT colitis mice, and this effect was reversed by anti-IL-10 antibody. Furthermore, the expression of IL-10 in the intestinal macrophages of p85α+/− normal colonic mucosa was significantly higher than that in the intestinal macrophages of WT normal colonic mucosa. The present results demonstrate that p85α+/− mice exhibit a reduced susceptibility to DSS-induced acute colitis. Our study suggests that a deficiency of PI3K p85α enhances the production of IL-10 in intestinal macrophages, thereby suppressing the development of DSS-induced acute colitis.

Extracellular stimulation of VSIG4/complement receptor Ig suppresses intracellular bacterial infection by inducing autophagy

ABSTRACTVSIG4/CRIg (V-set and immunoglobulin domain containing 4) is a transmembrane receptor of the immunoglobulin superfamily that is expressed specifically on macrophages and mature dendritic cells. VSIG4 signaling accelerates phagocytosis of C3-opsonized bacteria, thereby efficiently clearing pathogens within macrophages. We found that VSIG4 signaling triggered by C3-opsonized Listeria (opLM) or by agonistic anti-VSIG4 monoclonal antibody (mAb) induced macrophages to form autophagosomes. VSIG4-induced autophagosomes were selectively colocalized with the intracellular LM while starvation-induced autophagosomes were not. Consistent with these results, the frequency of autophagosomes induced by infection with opLM was lower in VSIG4-deficient bone marrow-derived macrophages (BMDMs) than in WT BMDMs. Furthermore, when VSIG4 molecules were overexpressed in HeLa cells, which are non-macrophage cells, VSIG4 triggering led to efficient uptake of LM, autophagosome formation, and killing of the infected LM. These findings suggest that VSIG4 signaling not only promotes rapid phagocytosis and killing of C3-opsonized intracellular bacteria, as previously reported, but also induces autophagosome formation, eliminating the LM that have escaped from phagosomes. We conclude that VSIG4 signaling provides an anti-immune evasion mechanism that prevents the outgrowth of intracellular bacteria in macrophages.

Abstract 19419: Anti Atherogenic Effects of Micro RNA - 21

Micro RNA-21 (miR-21), an evolutionary conserved micro RNA has been implicated in the pathogenesis of restenosis, myocardial infarction and heart failure. However, little is known about the role of miR-21 in atherosclerosis. Our data show that in vitro, LDL, oxidized LDL, acetylated LDL and LPS induced miR-21 by 2-3-fold (P<0.05) and down regulated its target protein PDCD4 in bone marrow derived macrophages (BMDM). Feeding the LDL receptor-knockout (LDLR-KO) mice with western diet (WD, 8-20 weeks) increased the abundance of miR-21 in BMDM by 1.5-fold (P<0.05). Basally, BMDM isolated from miR-21-KO mice showed induction of TNF alpha, interferon gamma, M-CSF, RANTES, IP10 and LIF by (1.5-3.0-fold); increased early and late apoptosis (2-3-fold, P<0.05); and induced PDCD4 and PTEN. We also observed 40% decrease in the survival of F4/80+ cells during differentiation of bone marrow derived cells isolated from miR-21-KO mice. Stimulation of miR-21-KO BMDM with interferon gamma+LPS polarized the macrophages to pro-inflammatory M1 phenotype (increased expression of CD11c and CD86) and decreased IL-10 formation as compared with WT BMDM. Staurosporin and oxidized lipids derived aldehyde 4-hydroxynonenal significantly increased both early and late apoptosis of miR-21-KO BMDM (2-4-fold, P<0.05). This was accompanied by increased cleavage of caspase 3. Characterization of miR-21-KO mice showed 30% decrease in white blood cells and neutrophils in KO mice. However, levels of circulating immune cells and common progenitor cells in bone marrow of miR-21-KO mice were comparable with wild type mice. Transplantation of bone marrow cells from miR-21-KO into LDLR-KO mice, followed by 12 weeks of WD increased the lesion formation (1.7-fold, P<0.05), apoptosis (3-fold, P<0.05) and necrosis (1.6-fold, P<0.05) in the aortic valve of the chimeric mice. This was accompanied by increased accumulation of macrophages in the non-necrotic areas of the lesion and decrease in lesional smooth muscle cells. Plasma cholesterol levels, and lesional collagen and T-cell levels in the miR-21 chimeric mice were comparable with wild type chimeric mice. Collectively, these data suggest that miR-21 prevents atherosclerosis by inhibiting macrophage apoptosis, necrosis and inflammation.

Abstract 583: Receptor for Advanced Glycation End Products ( Ager ) and Diaphanous-1 ( Drf1) Suppress Macrophage Reverse Transendothelial Migration and Regression of Diabetic Atherosclerosis

Macrophages display complex trafficking properties in vivo , involving interaction with vascular endothelial cells. Macrophage migration is an important mechanism linked to the progression and regression of atherosclerosis. The underlying mechanisms involved in these processes are not fully defined. RAGE is a multiligand cell surface macromolecule, which binds distinct ligands. The RAGE cytoplasmic domain interacts with Diaphanous-1 ( Drf1 ), both in vitro and in vivo and this interaction is essential for RAGE ligand-mediated signaling in macrophages. We tested the hypothesis that RAGE and Diaphanous-1 suppress regression of diabetic atherosclerosis; at least in part by impaired reduction of plaque macrophage content in an AGE-enriched diabetic environment. In a aorta transplantation model, we examined the morphologic changes in LDLr-/- donor plaques found in Ager -/-, Drf1 -/- or WT recipient diabetic mice. After lipid normalization into Ager -/- recipient diabetic mice, we observed reduced plaque macrophage density (CD68 -46%;p<0.05), but increased plaque collagen (PSR+43%;p<0.06), compared to WT diabetic recipients. Oil Red O staining for fatty deposit suggests a decrease (-51%;p<0.05) in plaque area stained and lower AGE staining (-32%;p<0.05) in diabetic Drf1 -/- recipient mice compared to WT diabetic recipients. We employed a monocyte bead-tracking model in this in vivo aorta transplantation study. In WT recipient mice, when the donors were LDLr-/- mice fed a western diet, bead frequency per plaque section was reduced (22%;p< 0.05) on day 5 post-transplant compared with baseline. In contrast, bead frequency per plaque section was significantly reduced by (41%;p< 0.05) compared with baseline in the absence of Ager (gene encoding RAGE). In vitro, in a reverse transendothelial migration assay using primary aortic endothelial cells and Ager or Drf1 deficient bone marrow derived macrophages (BMDMs), revealed in the setting of treatment with RAGE ligand AGEs, deletion of Ager or Drf1 in BMDMs facilitated macrophage reverse migration vs. that observed in WT BMDMs. Therefore, we propose that deletion of Ager or Drf1 in recipient diabetic mice accelerates atherosclerotic plaque regression, via reduction in lesional macrophage content.