Deficient Bone Marrow-derived Macrophages Research Articles

Abstract P480: Myeloid YAP Inhibition Improves Cardiac Phenotype During Pressure Overload Stress

Inflammation is a component of cardiovascular disease and is thought to contribute to cardiac dysfunction in ischemic and non-ischemic models of heart failure. While ischemia-induced inflammation has been extensively studied in the heart, relatively less is known regarding cardiac inflammation during non-ischemic stress. Recent work has implicated a role for Yes-associated protein (YAP), a transcriptional co-factor, in modulating cardiac inflammation and remodeling after myocardial infarction. We hypothesized that YAP mediates a pro-inflammatory response during pressure overload (PO)-induced non-ischemic injury, and that targeted YAP inhibition is cardioprotective. PO in mice elicits an immune response characterized by infiltration of myeloid cells that precedes cardiac dysfunction. Myeloid cells isolated from the heart after 7d PO showed evidence of increased YAP activity. Myeloid-specific YAP knockout mice (YAP F/F ;LysM Cre ) were subjected to PO stress. After 4 weeks, cardiac hypertrophy was similar between YAP KO mice and controls. However, systolic dysfunction, cardiac fibrosis, and indicators of pathological remodeling were all attenuated in YAP KO mice compared to controls. Additionally, inflammatory gene expression and macrophage infiltration to the myocardium were significantly attenuated in YAP KO mice after PO, indicating reduced inflammation compared to controls. Experiments using RAW264.7 macrophages and primary bone marrow-derived macrophages (BMDMs) from YAP KO and control mice demonstrated that increased YAP expression enhanced, while YAP suppression attenuated, inflammatory gene expression. The inflammasome is a multiprotein complex and important facilitator of cytokine processing that mediates inflammation in the PO heart. We observed attenuated inflammasome priming and function in YAP deficient BMDMs, as well as in YAP KO hearts following PO, indicating disruption of inflammasome induction. Together these data implicate YAP as an important mediator of inflammasome function and cardiac inflammation during PO stress and suggest that selective inhibition of YAP in the myeloid compartment may prove a novel therapeutic target in non-ischemic heart disease.

Abstract MP42: Macrophage Chromatin Remodeling Complex Subunit Baf60a In Atherosclerosis

Objectives: Atherosclerosis is a primary medical concern due to the increasing prevalence and lack of effective treatment that reverses the disease progression. While multiple genetic mutations/variations have been associated with atherosclerotic risk, the development of atherosclerosis in response to risk factors is also contributed by factors at the epigenetic and chromatin level. The SWI/SNF chromatin remodeling complex alters DNA accessibility and chromatin structure to modulate gene expression in response to external and internal cellular environments, potentially serving as an underexplored compartment of CVD and atherosclerosis development. Approach and Results: In an effort to identify key chromatin remodeling compartments involved in atherosclerotic foam cell formation, primary bone marrow-derived macrophages (BMDMs) were exposed to a pro-atherogenic environment, specifically to oxidized Low-density lipoprotein (oxLDL). We observed drastically and time-dependent decrease in the protein abundance of BAF60a, a transcription factor interacting subunit of the SWI/SNF chromatin remodeling complex. Macrophage-specific Baf60a deletion in ApoE deficient mice showed over 30% increase in aortic plaque formation after 16-weeks of western diet feeding (N=12, p=0.0097). Integrated analysis of RNA-seq and ATAC-seq regulatory network in oxLDL treated BAF60a deficient BMDMs identified a shift of chromatin accessibility, transcription factor footprint, and a reshape in gene expression patterns that regulate extracellular matrix organization, focal adhesion, integrin signaling, inflammation, and lead to foam cell formation which ultimately contribute to the atherosclerotic plaque build-up. Conclusions: Our data demonstrated the role of macrophage BAF60a in atherogenesis and suggested a potential therapeutic target to treat atherosclerosis, presumably by compensating for the loss of Baf60a.

1989-P: Ablation of Ghrelin Receptor in Myeloid Cells Attenuates Diet-Induced NAFLD in Mice

Chronic low-grade inflammation is a hallmark of obesity, which is directly linked to nonalcoholic Fatty Liver Disease (NAFLD) and systemic insulin resistance. Ghrelin receptor, a growth hormone secretagogue receptor (GHS-R), mediates the stimulatory effect of ghrelin on food intake and fat deposition. We previously reported that global ablation of GHS-R in mice decreases adipose inflammation, and GHS-R knockdown in macrophage cell line reduces the expression of pro-inflammatory cytokine genes. To determine the physiological role of GHS-R in macrophages, we generated myeloid-specific GHS-R knockout mice (LysM-Cre;Ghsrf/f). Under diet-induced obesity (DIO), LysM-Cre;Ghsrf/f mice showed reduced circulating inflammatory cytokine levels, attenuated insulin resistance, but no change in body weight. Moreover, DIO-induced NAFLD was ameliorated in LysM-Cre;Ghsrf/f mice, showing reduced inflammation and lipid accumulation in the liver. To understand the regulatory mechanism of macrophage GHS-R in NAFLD pathogenesis and systemic inflammation and insulin sensitivity, we conducted ex vivo studies using bone marrow-derived macrophages (BMDMs). GHS-R deficient BMDMs showed reduced macrophage pro-inflammatory polarization and decreased expression of pro-inflammatory cytokine genes; consistently, exhibiting elevated mitochondrial respiration and improved mitochondrial dynamics. Moreover, the primary hepatocytes treated with conditioned media from BMDMs of LysM-Cre;Ghsrf/f mice showed gene expression of reduced inflammatory cytokines and increased insulin signaling. Taken together, our findings reveal that GHS-R is an important immunometabolic regulator and governs macrophage polarization through regulating mitochondrial function and cellular metabolism of macrophages, moderating hepatic and systemic inflammation and insulin sensitivity. GHS-R antagonist may present a novel therapeutic strategy for the metabolic disorders such as NAFLD. Disclosure D. Kim: None. J. Lee: None. Q. Pan: None. J. Noh: None. H. Wang: None. H. Han: None. C. Wu: None. S. Guo: None. Y. Sun: None. Funding American Diabetes Association (1-15- BS-177 to Y.S.); National Institutes of Health

MKP-1 negatively regulates LPS-mediated IL-1β production through p38 activation and HIF-1α expression

Interleukin 1 beta (IL-1β) is a pro-inflammatory cytokine that plays a major role in inflammatory diseases as well as cancer. The inflammatory response after Toll-like receptor (TLR) 4 activation is tightly regulated through phosphorylation of MAP kinases, including p38 and JNK pathways. The activation of MAP kinases is negatively regulated by MAPK phosphatases (MKPs). MKP-1 preferentially dephosphorylates p38 and JNK. IL-1β is regulated through the activation of MAPK, including p38 as well as several transcription factors. The oxygen-sensitive transcription factor HIF-1α is a known transcription factor for several inflammatory cytokines including IL-1β and IL-6. Here, we report that MKP-1 regulates HIF-1α expression in response to LPS. MKP-1 deficient bone marrow derived macrophages (BMDMs) exhibited increased reactive oxygen species (ROS) production and higher HIF-1α expression. In contrast, the expression of all three isoforms of prolyl hydroxylases (PHDs), which are important in destabilizing HIF-1α through hydroxylation, were significantly decreased in MKP-1 deficient BMDMs. LPS challenge of MKP-1 deficient BMDMs led to a substantial increase in IL-1β production. An inhibitor of HIF-1α significantly decreased LPS mediated IL-1β production both at the transcript and protein levels. Similarly, inhibition of p38 MAP kinase reduced LPS mediated pro-IL-1β and HIF-1α protein levels as well as ROS production in MKP-1 deficient BMDMs. These findings demonstrate a regulatory function for MKP-1 in modulating IL-1β expression through p38 activation, ROS production and HIF-1α expression.

Abstract 263: Role of Micro RNA-21 in Atherosclerosis

Micro RNAs (miR) are short non-coding RNAs that regulate several genes under pathophysiological conditions. Accumulating evidence suggest the involvement of miR in atherogenesis. However, limited information is available about atherogenic miR and the underling mechanisms by which miR affect atherogenesis. Our data shows that 12 weeks of western diet (WD) in LDL receptor-knockout (LDLR-KO) mice upregulated 99 and downregulated 50 miR in the aorta. Among the 41 differentially expressed miR associated with macrophage inflammation and apoptosis, expression of micro RNA-21 (miR-21) was increased by 1.4-fold (P<0.05). WD also increased the expression of miR-21 by 1.5-fold in bone marrow derived macrophages (BMDM). 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 BMDM. Basally, miR-21 deficient BMDM showed increased secretion of IL-6, IL-9 and CXCL-2,-3,-4, and -10 (P<0.05)); and increased early and late apoptosis (2-3-fold, P<0.05). 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 LPS significantly increased the activation of NF-κB and enhanced the secretion of several pro-inflammatory cytokines including TNFα, IL-6, IL-12 and CXCL-2 (2-10 fold; P<0.05); interferon gamma+LPS polarized the macrophages to pro-inflammatory M1 phenotype (increased expression of CD11c and CD86). 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, -7 and -9. 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. Collectively, these data suggest that miR-21 prevents atherosclerosis, at least in parts, by preventing macrophage apoptosis and inflammation.

108: The role of nucleotide-binding oligomerization domain 1 (Nod1) in cytokine production by macrophages in response to Mycobacterium tuberculosis

Mycobacterium tuberculosis infection is one of the leading causes of death worldwide. Recognition of pathogen by pattern recognition receptors is crucial for the activation of both innate and adaptive immune responses. Nucleotide-binding oligomerization domain (Nod) 1 and Nod2 are cytoplasmic receptors which detect unique muropeptide from bacterial peptidoglycan. Although Nod2 is critical for initiation of host immune responses against M. tuberculosis infection, the role of Nod1 remains largely unknown. In this study, we investigated the role of Nod1 on cytokines production by bone marrow-derived macrophages (BMDMs) in response to M. tuberculosis. We found that pro-inflammatory cytokines such as IL-6, TNF-α, and IL-1β were induced in BMDMs infected with M. tuberculosis, which was not affected by Nod1 deficiency. Activation of NF-κB and MAPKs by M. tuberculosis was also comparable between WT and Nod1-deficient BMDMs. Interestingly, IL-6 and IL-1β production was reduced in Nod1/Nod2-double deficient BMDMs, as compared with Nod2 single deficient cells. Furthermore, when TLRs signaling was inhibited by LPS pre-treatment, such cytokines production was diminished in Nod1-deficient BMDMs, as compared with WT cells. Our results indicate that Nod1 cooperates with Nod2 or TLRs to produce cytokines in macrophages in response to M. tuberculosis.

Redox Regulation of Lipopolysaccharide-mediated Sulfiredoxin Induction, Which Depends on Both AP-1 and Nrf2

Sulfiredoxin (Srx) is an enzyme that catalyzes the reduction of cysteine sulfinic acid of hyperoxidized peroxiredoxins and exerts a protective antioxidant role. Here we investigated the regulatory mechanism of Srx induction by lipopolysaccharide (LPS) in mouse macrophages. LPS up-regulated Srx expression on the transcriptional level. The promoter region of the Srx gene contained putative NF-κB and AP-1 (activator protein-1) sites, and the proximal site of three AP-1 sites was embedded within the antioxidant response element (ARE), a cis-acting element for Nrf2 (nuclear factor erythroid 2-related factor). Mutational analysis of the Srx promoter revealed that Srx induction is dependent on AP-1 sites and ARE but not on NF-κB sites. Consistently, both transcription factors, AP-1 and Nrf2, were required for LPS-mediated Srx induction, as revealed by chromatin immunoprecipitation using antibodies specific for c-Jun and c-Fos and little Srx induction in Nrf2-null bone marrow-derived macrophages. Among mitogen-activated protein kinases that mediate the signal transduction by LPS, JNK played a major role in Srx induction. Moreover, chemical antioxidants, such as N-acetylcysteine and butylated hydroxyanisole, and the NADPH oxidase inhibitor diphenyleneiodonium inhibited Srx induction as well as generation of reactive oxygen species, both of which were also suppressed in Nox2 (NADPH oxidase 2)-deficient bone marrow-derived macrophages. These results suggest that LPS-mediated Srx induction is dependent on both AP-1 and Nrf2, which is regulated by Nox2-derived reactive oxygen species.