Proinflammatory Gene Expression In Macrophages Research Articles

Lipopolysaccharide regulation of antiinflammatory tristetraprolin family and proinflammatory gene expression in mouse macrophages

ObjectiveTristetraprolin (TTP/ZFP36) family proteins exhibit antiinflammatory effects by destabilizing proinflammatory mRNAs. Previous studies showed that bacterial endotoxin lipopolysaccharides (LPS) stimulated TTP and tumor necrosis factor (TNF) gene expression, but less was known about LPS effects on TTP homologues and other proinflammatory gene expression in macrophages. The objective was to investigate LPS regulation of TTP family gene and TTP-targeted gene expression in mouse RAW264.7 macrophages using much higher concentrations of LPS and much longer treatment time than previous studies.ResultsMTT assay showed that LPS was not toxic to the cells under LPS treatment up to 1000 ng/mL for 2–24 h. LPS mildly affected the soluble protein content in the cells. qPCR assay showed that LPS stimulated TTP mRNA rapidly but not sustainably with 40, 10, and 3 fold of the DMSO control after 2, 8 and 24 h treatment, respectively. Immunoblotting confirmed qPCR results on LPS stimulation of TTP gene expression in the mouse macrophages. LPS exhibited minimal effects on ZFP36L1, ZFP36L2 and ZFP36L3 mRNA levels. LPS increased mRNA levels of TNF, COX2, GM-CSF, INFγ and IL12b up to 311, 418, 11, 9 and 4 fold, respectively. This study demonstrated that LPS did not affect macrophage viability, dramatically increased antiinflammatory TTP gene expression as well as proinflammatory TNF and COX2 gene expression but had only mild effects on TTP homologues and other proinflammatory cytokine gene expression in the mouse macrophages.

BATF2 enhances proinflammatory cytokine responses in macrophages and improves early host defense against pulmonary Klebsiella pneumoniae infection.

Basic leucine zipper transcription factor ATF-like 2 (BATF2) is a transcription factor that is emerging as an important regulator of the innate immune system. BATF2 is among the top upregulated genes in human alveolar macrophages treated with LPS, but the signaling pathways that induce BATF2 expression in response to Gram-negative stimuli are incompletely understood. In addition, the role of BATF2 in the host response to pulmonary infection with a Gram-negative pathogen like Klebsiella pneumoniae (Kp) is not known. We show that induction of Batf2 gene expression in macrophages in response to Kp in vitro requires TRIF and type I interferon (IFN) signaling, but not MyD88 signaling. Analysis of the impact of BATF2 deficiency on macrophage effector functions in vitro showed that BATF2 does not directly impact macrophage phagocytic uptake and intracellular killing of Kp. However, BATF2 markedly enhanced macrophage proinflammatory gene expression and Kp-induced cytokine responses. In vivo, Batf2 gene expression was elevated in lung tissue of wild-type (WT) mice 24 h after pulmonary Kp infection, and Kp-infected BATF2-deficient (Batf2-/-) mice displayed an increase in bacterial burden in the lung, spleen, and liver compared with WT mice. WT and Batf2-/- mice showed similar recruitment of leukocytes following infection, but in line with in vitro observations, proinflammatory cytokine levels in the alveolar space were reduced in Batf2-/- mice. Altogether, these results suggest that BATF2 enhances proinflammatory cytokine responses in macrophages in response to Kp and contributes to the early host defense against pulmonary Kp infection.NEW & NOTEWORTHY This study investigates the signaling pathways that mediate induction of BATF2 expression downstream of TLR4 and also the impact of BATF2 on the host defense against pulmonary Kp infection. We demonstrate that Kp-induced upregulation of BATF2 in macrophages requires TRIF and type I IFN signaling. We also show that BATF2 enhances Kp-induced macrophage cytokine responses and that BATF2 contributes to the early host defense against pulmonary Kp infection.

Vascular endothelial cell-specific disruption of the profilin1 gene leads to severe multiorgan pathology and inflammation causing mortality.

Actin-binding protein Profilin1 is an important regulator of actin cytoskeletal dynamics in cells and critical for embryonic development in higher eukaryotes. The objective of the present study was to examine the consequence of loss-of-function of Pfn1 in vascular endothelial cells (ECs) in vivo. We utilized a mouse model engineered for tamoxifen-inducible biallelic inactivation of the Pfn1 gene selectively in EC (Pfn1EC-KO). Widespread deletion of EC Pfn1 in adult mice leads to severe health complications presenting overt pathologies (endothelial cell death, infarct, and fibrosis) in major organ systems and evidence for inflammatory infiltrates, ultimately compromising the survival of animals within 3 weeks of gene ablation. Mice deficient in endothelial Pfn1 exhibit selective bias toward the proinflammatory myeloid-derived population of immune cells, a finding further supported by systemic elevation of proinflammatory cytokines. We further show that triggering Pfn1 depletion not only directly upregulates proinflammatory cytokine/chemokine gene expression in EC but also potentiates the paracrine effect of EC on proinflammatory gene expression in macrophages. Consistent with these findings, we provide further evidence for increased activation of Interferon Regulatory Factor 7 (IRF7) and STAT1 in EC when depleted of Pfn1. Collectively, these findings for the first time demonstrate a prominent immunological consequence of loss of endothelial Pfn1 and an indispensable role of endothelial Pfn1 in mammalian survival unlike tolerable phenotypes of Pfn1 loss in other differentiated cell types.

Extracellular Vesicles Released after Doxorubicin Treatment in Rats Protect Cardiomyocytes from Oxidative Damage and Induce Pro-Inflammatory Gene Expression in Macrophages.

Doxorubicin (DOXO)-induced cardiomyopathy (DIC) is a lethal complication in cancer patients. Major mechanisms of DIC involve oxidative stress in cardiomyocytes and hyperactivated immune response. Extracellular vesicles (EVs) mediate cell-cell communication during oxidative stress. However, functions of circulating EVs released after chronic DOXO exposure on cardiomyocytes and immune cells are still obscured. Herein, we developed a DIC in vivo model using male Wistar rats injected with 3 mg/kg DOXO for 6 doses within 30 days (18 mg/kg cumulative dose). One month after the last injection, the rats developed cardiotoxicity evidenced by increased BCL2-associated X protein and cleaved caspase-3 in heart tissues, along with N-terminal pro B-type natriuretic peptide in sera. Serum EVs were isolated by size exclusion chromatography. EV functions on H9c2 cardiomyocytes and NR8383 macrophages were evaluated. EVs from DOXO-treated rats (DOXO_EVs) attenuated ROS production via increased glutathione peroxidase-1 and catalase gene expression, and reduced hydrogen peroxide-induced cell death in cardiomyocytes. In contrast, DOXO_EVs induced ROS production, interleukin-6, and tumor necrosis factor-alpha, while suppressing arginase-1 gene expression in macrophages. These results suggested the pleiotropic roles of EVs against DIC, which highlight the potential role of EV-based therapy for DIC with a concern of its adverse effect on immune response.

DOCK2 regulates antifungal immunity by regulating RAC GTPase activity.

Fungal infections cause ~1.5 million deaths each year worldwide, and the mortality rate of disseminated candidiasis currently exceeds that of breast cancer and malaria. The major reasons for the high mortality of candidiasis are the limited number of antifungal drugs and the emergence of drug-resistant species. Therefore, a better understanding of antifungal host defense mechanisms is crucial for the development of effective preventive and therapeutic strategies. Here, we report that DOCK2 (dedicator of cytokinesis 2) promotes indispensable antifungal innate immune signaling and proinflammatory gene expression in macrophages. DOCK2-deficient macrophages exhibit decreased RAC GTPase (Rac family small GTPase) activation and ROS (reactive oxygen species) production, which in turn attenuates the killing of intracellular fungi and the activation of downstream signaling pathways. Mechanistically, after fungal stimulation, activated SYK (spleen-associated tyrosine kinase) phosphorylates DOCK2 at tyrosine 985 and 1405, which promotes the recruitment and activation of RAC GTPases and then increases ROS production and downstream signaling activation. Importantly, nanoparticle-mediated delivery of in vitro transcribed (IVT) Rac1 mRNA promotes the activity of Rac1 and helps to eliminate fungal infection in vivo. Taken together, this study not only identifies a critical role of DOCK2 in antifungal immunity via regulation of RAC GTPase activity but also provides proof of concept for the treatment of invasive fungal infections by using IVT mRNA.

High-fat diet-induced metabolic syndrome increases ligature-induced alveolar bone loss in mice.

It has been well documented that metabolic syndrome (MetS) increases severity of periodontitis. In this study, we determined the effect of high-fat diet (HFD)-induced MetS on alveolar bone loss in a mouse model with ligature-induced periodontitis. To understand how MetS increases bone loss, we tested our hypothesis that palmitic acid (PA), a most abundant saturated fatty acid in the HFD, interacts with lipopolysaccharide (LPS) to promote osteoclastogenesis. We induced MetS by feeding mice HFD for 18weeks and induced periodontitis with ligature placement. After treatments, we assessed alveolar bone loss using micro-computed tomography and determined osteoclastogenesis using tartrate-resistant acid phosphatase (TRAP) staining. To explore the mechanisms, we treated macrophages with PA, LPS or both and analyzed the osteoclast formation and cytokine expression in macrophages. While ligature robustly induced periodontitis in mice with or without MetS, the mice with MetS had more bone loss than those without MetS. PA and LPS cooperatively induced osteoclast formation and stimulated the expression of inflammatory cytokines involved in osteoclastogenesis potentially via a FAT/CD36-dependent mechanism in macrophages. HFD-induced MetS increases alveolar bone loss in mice with ligature-induced periodontitis, and PA and LPS cooperatively stimulate osteoclast formation and proinflammatory gene expression in macrophages.

BHLHE40 promotes macrophage pro-inflammatory gene expression and functions.

Macrophages are the principal innate immune cells that populate all major organs and provide the first line of cellular defense against infections and/or injuries. The immediate and early-responding macrophages must mount a robust pro-inflammatory response to protect the host by eliminating deleterious agents. The effective pro-inflammatory macrophage response requires the activation of complex transcriptional programs that modulate the dynamic regulation of inflammatory and metabolic gene expression. Therefore, transcription factors that govern pro-inflammatory and metabolic gene expression play an essential role in shaping the macrophage inflammatory response. Herein, we identify the basic helix-loop-helix family member e40 (BHLHE40), as a critical transcription factor that promotes broad pro-inflammatory and glycolytic gene expression by elevating HIF1α levels in macrophages. Our in vivo studies revealed that myeloid-BHLHE40 deficiency significantly attenuates macrophage and neutrophil recruitment to the site of inflammation. Our integrated transcriptomics and gene set enrichment analysis (GSEA) studies show that BHLHE40 deficiency broadly curtails inflammatory signaling pathways, hypoxia response, and glycolytic gene expression in macrophages. Utilizing complementary gain- and loss-of-function studies, our analyses uncovered that BHLHE40 promotes LPS-induced HIF1α mRNA and protein expression in macrophages. More importantly, forced overexpression of oxygen stable form of HIF1α completely reversed attenuated pro-inflammatory and glycolytic gene expression in BHLHE40-deficient macrophages. Collectively, these results demonstrate that BHLHE40 promotes macrophage pro-inflammatory gene expression and functions by elevating HIF1α expression in macrophages.

Bifidobacterium breve CNCM I-4035, Lactobacillus paracasei CNCM I-4034 and Lactobacillus rhamnosus CNCM I-4036 Modulate Macrophage Gene Expression and Ameliorate Damage Markers in the Liver of Zucker-Lepr fa/fa Rats.

Non-alcoholic fatty liver disease (NAFLD) has reached pandemic proportions worldwide. We have previously reported that the probiotic strains Bifidobacterium breve CNCM I-4035, Lactobacillus paracasei CNCM I-4034 and Lactobacillus rhamnosus CNCM I-4036 exert anti-inflammatory effects in the intestine of Zucker-Leprfa/fa rats. In this work, we focused on their hepatic effects. M1 macrophages are related to inflammation and NAFLD pathogenesis, whereas M2 macrophages release anti-inflammatory mediators. We evaluated the effects of these 3 strains on macrophage polarization, inflammation and liver damage of Zucker-Leprfa/fa rats. The animals received either a placebo or 1010 CFU of probiotics orally for 30 days. Nos2 and Cd86 mRNA levels were determined as markers of M1 macrophages, and Cd163 and Arg1 as M2 markers, respectively, by qRT-PCR. Liver damage was determined by lipid peroxidation, leukocyte infiltration and myeloperoxidase activity. We evaluated a panoply of circulating chemokines, the hepatic ratio P-Akt/Akt, NF-kB and P-NF-kB protein levels. All 3 probiotic strains modulated macrophage polarization in liver and circulating levels of inflammation-related mediators. L. paracasei CNCM I-4034 increased the ratio P-Akt/Akt and NF-kB protein levels. B. breve CNCM I-4035, L. paracasei CNCM I-4034 and L. rhamnosus CNCM I-4036 decreased both pro-inflammatory macrophage gene expression and leukocyte infiltration in the liver.

A novel nitroalkene vitamin E analogue inhibits the NLRP3 inflammasome and protects against inflammation and glucose intolerance triggered by obesity

Chronic metabolic diseases, like obesity, type II diabetes and atherosclerosis often involve a low-grade and sterile systemic inflammatory state, in which activation of the pro-inflammatory transcription factor NF-kB and the NLRP3 inflammasome play a major role. It is well established that genetic inhibition of the NLRP3 inflammasome ameliorates acute and chronic inflammation. Indeed, accumulating experimental evidences in murine models and also in humans suggest that inhibition of the NLRP3 inflammasome might be a suitable approach to tackle the deleterious effects of chronic metabolic diseases. In this work, we explored our previously synthesized nitroalkene-Trolox™ derivative named NATx0, as a non-conventional anti-inflammatory strategy to treat chronic inflammatory diseases, such as obesity-induced glucose intolerance. We found that NATx0 inhibited NF-kB nuclear translocation and pro-inflammatory gene expression in macrophages in vitro. In addition, treatment with NATx0 prevented NLRP3 inflammasome activation after LPS/ATP stimulation in macrophages in vitro. When tested acutely in vivo, NATx0 inhibited neutrophil recruitment in zebrafish larvae, and also diminished IL-1β production after LPS challenge in mice. Finally, when NATx0 was administered chronically to diet-induced obese mice, it decreased muscle tissue inflammation and glucose intolerance, leading to improved glucose homeostasis. In conclusion, we propose that this novel nitroalkene-Trolox derivative is a suitable tool to tackle acute and chronic inflammation in vitro and in vivo mainly due to inhibition of NF-kB/NLRP3 activation.

CITED2 limits pathogenic inflammatory gene programs in myeloid cells.

Monocyte‐derived macrophages are the major innate immune cells that provide the first line of cellular defense against infections or injuries. These recruited macrophages at the site of inflammation are exposed to a broad range of cytokines that categorically incite a robust pro‐inflammatory response. However, macrophage pro‐inflammatory activation must be under exquisite control to avert unbridled inflammation. Thus, endogenous mechanisms must exist that rigorously preserve macrophage quiescence and yet, allow nimble pro‐inflammatory macrophage response with precise spatiotemporal control. Herein, we identify the CBP/p300‐interacting transactivator with glutamic acid/aspartic acid‐rich carboxyl‐terminal domain 2 (CITED2) as a critical intrinsic negative regulator of inflammation, which broadly attenuates pro‐inflammatory gene programs in macrophages. Our in vivo studies revealed that myeloid‐CITED2 deficiency significantly heightened macrophages and neutrophils recruitment to the site of inflammation. Our integrated transcriptomics and gene set enrichment analysis (GSEA) studies uncovered that CITED2 deficiency broadly enhances NFκB targets, IFNγ/IFNα responses, and inflammatory response gene expression in macrophages. Using complementary gain‐ and loss‐of‐function studies, we observed that CITED2 overexpression attenuate and CITED2 deficiency elevate LPS‐induced NFκB transcriptional activity and NFκB‐p65 recruitment to target gene promoter in macrophages. More importantly, blockade of NFκB signaling completely reversed elevated pro‐inflammatory gene expression in macrophages. Collectively, our findings show that CITED2 restrains NFκB activation and curtails broad pro‐inflammatory gene programs in myeloid cells.

Kruppel-like factor 6 and miR-223 signaling axis regulates macrophage-mediated inflammation.

Macrophage-mediated inflammation is an explicitly robust biologic response that plays a critical role in maintaining tissue homeostasis by eliminating deleterious agents. These tissue macrophages tailor appropriate responses to external cues by altering inflammatory gene expression. Therefore, transcription factors and regulators that modulate inflammatory gene expression play an essential role in shaping the macrophage inflammatory response. Here, we identify that Kruppel-like factor (KLF)6 promotes inflammation by restraining microRNA-223 (miR-223) expression in macrophages. We uncovered that pro- and anti-inflammatory agents oppositely regulate KLF6 and miR-223 expression in macrophages. Using complementary gain- and loss-of-function studies, we observed that overexpression of KLF6 attenuates and deficiency of KLF6 elevates miR-223 expression in macrophages. Furthermore, heightened miR-223 expression in KLF6-deficient macrophages significantly attenuates inducible proinflammatory gene expression. Concordantly, myeloid-Klf6 deficiency significantly curbs diet-induced adipose tissue inflammation, obesity, glucose intolerance, and insulin resistance. At the molecular level, KLF6 directly represses miR-223 expression by occupying its promoter region. More importantly, genetic inhibition of miR-223-3P in KLF6-deficient macrophages completely reversed attenuated proinflammatory gene expression in macrophages. Collectively, our studies reveal that KLF6 promotes proinflammatory gene expression and functions by repressing miR-223 expression in macrophages.-Kim, G.-D., Ng, H. P., Patel, N., Mahabeleshwar, G. H. Kruppel-like factor 6 and miR-223 signaling axis regulates macrophage-mediated inflammation.

Macrophages are Required for Recovery from Physiological Muscle Stress in the mdx Mouse Model of Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a lethal genetic disease associated with muscle degeneration and chronic inflammation. Current therapies are limited to corticosteroids which resolve inflammation at the expense of detrimental side effects, and exon skipping which is applicable only to a small subset of patients. Cardiosphere‐derived cells (CDCs) are cardiac progenitor/stromal cells with anti‐inflammatory, anti‐fibrotic, and regenerative properties. Recently, CDCs have been shown to reverse many of the abnormalities of heart and skeletal muscle in mdx mice, motivating the HOPE‐Duchenne clinical trial (Aminzadeh M, Rogers R et al., Stem Cell Reports 2018). Several lines of evidence now support the idea that CDCs exert their bioactivity by secreting nanoparticles called exosomes (CDC‐exos), which mediate cell‐cell transfer of genetic information. A single intravenous dose of CDC‐exos dramatically improves contractile function of the mdx mouse soleus, and restores muscle architecture. Given the anti‐inflammatory effects of CDC‐exos, RNA‐sequencing unexpectedly revealed a striking upregulation of inflammatory pathways in the solei of CDC‐exo treated mdx mice, as confirmed by confocal imaging of tissue macrophages using CD68 immunohistochemistry. Thus, we investigated the role of macrophages in mdx pathology. Clodronate liposomes were used to deplete monocytes and macrophages in vivo, as confirmed in blood by flow cytometry and in tissue by immunohistochemistry. Macrophage‐depleted and control mdx mice were subjected to a single bout of downhill running to induce muscle stress. Maximal exercise capacity was recorded prior to, and every other day for two weeks, following downhill running. Macrophage‐depleted mdx mice failed to recover during the two‐week period, whereas control mdx mice had recovered by the third day. Further, macrophage depletion shifted the force‐frequency relationship to higher frequencies and reduced maximal force developed by the soleus by 24%. Cultured macrophages from mdx bone marrow (BMDMs) express 4‐fold higher levels of the pro‐inflammatory cytokine TNF‐α than those from age‐matched wild‐type mice. In vitro conditioning of mdx BMDMs with CDC‐exos reduced the expression of TNF‐α, suggesting that CDC‐exos may skew mdx macrophage polarization toward a tissue healing phenotype. This interpretation is consistent with the finding that CDC‐exos polarize macrophages to a phenotype with enhanced efferocytosis capacity, minimizing scar formation after acute myocardial infarction. Thus, macrophages are required for recovery from physiological muscle stress in mdx mice, and CDC‐exos decrease pro‐inflammatory gene expression in macrophages. The unexpected requirement for macrophages in mdx skeletal muscle recovery from injury suggests that a macrophage population promotes tissue regeneration. We are currently testing the hypothesis that CDC‐exos may modulate the pro‐inflammatory phenotype of recruited macrophages to stimulate myogenesis and support mdx muscle regeneration.Support or Funding InformationCoalition DuchenneNIH T32 HL116273This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Docosahexaenoic acid antagonizes the boosting effect of palmitic acid on LPS inflammatory signaling by inhibiting gene transcription and ceramide synthesis.

It is well known that saturated fatty acids (SFAs) and unsaturated fatty acid, in particular omega-3 polyunsaturated fatty acids (n-3 PUFAs), have different effects on inflammatory signaling: SFAs are pro-inflammatory but n-3 PUFAs have strong anti-inflammatory properties. We have reported that palmitic acid (PA), a saturated fatty acid, robustly amplifies lipopolysaccharide (LPS) signaling to upregulate proinflammatory gene expression in macrophages. We also reported that the increased production of ceramide (CER) via sphingomyelin (SM) hydrolysis and CER de novo synthesis plays a key role in the synergistic effect of LPS and PA on proinflammatory gene expression. However, it remains unclear if n-3 PUFAs are capable of antagonizing the synergistic effect of LPS and PA on gene expression and CER production. In this study, we employed the above macrophage culture system and lipidomical analysis to assess the effect of n-3 PUFAs on proinflammatory gene expression and CER production stimulated by LPS and PA. Results showed that DHA strongly inhibited the synergistic effect of LPS and PA on proinflammatory gene expression by targeting nuclear factor kappa B (NFκB)-dependent gene transcription. Results also showed that DHA inhibited the cooperative effect of LPS and PA on CER production by targeting CER de novo synthesis, but not SM hydrolysis. Furthermore, results showed that myriocin, a specific inhibitor of serine palmitoyltransferase, strongly inhibited both LPS-PA-stimulated CER synthesis and proinflammatory gene expression, indicating that CER synthesis is associated with proinflammatory gene expression and that inhibition of CER synthesis contributes to DHA-inhibited proinflammatory gene expression. Taken together, this study demonstrates that DHA antagonizes the boosting effect of PA on LPS signaling on proinflammatory gene expression by targeting both NFκB-dependent transcription and CER de novo synthesis in macrophages.

Bioenergetic state regulates innate inflammatory responses through the transcriptional co-repressor CtBP

The innate inflammatory response contributes to secondary injury in brain trauma and other disorders. Metabolic factors such as caloric restriction, ketogenic diet, and hyperglycemia influence the inflammatory response, but how this occurs is unclear. Here, we show that glucose metabolism regulates pro-inflammatory NF-κB transcriptional activity through effects on the cytosolic NADH:NAD+ ratio and the NAD(H) sensitive transcriptional co-repressor CtBP. Reduced glucose availability reduces the NADH:NAD+ ratio, NF-κB transcriptional activity, and pro-inflammatory gene expression in macrophages and microglia. These effects are inhibited by forced elevation of NADH, reduced expression of CtBP, or transfection with an NAD(H) insensitive CtBP, and are replicated by a synthetic peptide that inhibits CtBP dimerization. Changes in the NADH:NAD+ ratio regulate CtBP binding to the acetyltransferase p300, and regulate binding of p300 and the transcription factor NF-κB to pro-inflammatory gene promoters. These findings identify a mechanism by which alterations in cellular glucose metabolism can influence cellular inflammatory responses.

MicroRNA-181 Variants Regulate T Cell Phenotype in the Context of Autoimmune Neuroinflammation.

Recent studies have revealed that multiple sclerosis (MS) lesions have distinct microRNA (miRNA) expression profiles. miR-181 family members show altered expression in MS tissues although their participation in MS pathogenesis remains uncertain. Herein, we investigated the involvement of miR-181a and miR-181b in the pathogenesis of MS and its animal model, experimental autoimmune encephalomyelitis (EAE). miR-181a and -b levels were measured in the central nervous system (CNS) of patients with MS and mice with EAE as well as relevant leukocyte cultures by real-time RT-PCR. To examine the role of the miRNAs in leukocyte differentiation and function, miR-181a and -b mimic sequences were transfected into cultured primary macrophages and purified CD4+ T cells which were then analyzed by RT-PCR and flow cytometry. Luciferase reporter assays were performed to investigate the interaction of miR-181a and -b with the 3'-UTR of potential target transcripts, and the expression of target genes was measured in the CNS of EAE mice, activated lymphocytes, and macrophages. Expression analyses revealed a significant decrease in miR-181a and -b levels in brain white matter from MS patients as well as in spinal cords of EAE mice during the acute and chronic phases of disease. Suppression of miR-181a was observed following antigen-specific or polyclonal activation of lymphocytes as well as in macrophages following LPS treatment. Overexpression of miR-181a and -b mimic sequences reduced proinflammatory gene expression in macrophages and polarization toward M1 phenotype. miR-181a and -b mimic sequences inhibited Th1 generation in CD4+ T cells and miR-181a mimic sequences also promoted Treg differentiation. Luciferase assays revealed Suppressor of mothers against decapentaplegic 7 (Smad7), as a direct target of miR-181a and -b. Our data highlight the anti-inflammatory actions of miR-181a and -b in the context of autoimmune neuroinflammation. miR-181a and -b influence differentiation of T helper cell and activation of macrophages, providing potential therapeutic options for controlling inflammation in MS.

The role of transforming growth factor (TGF)-β in the infarcted myocardium.

The adult mammalian heart has negligible regenerative capacity. Following myocardial infarction, sudden necrosis of cardiomyocytes triggers an intense inflammatory reaction that clears the wound from dead cells and matrix debris, while activating a reparative program. A growing body of evidence suggests that members of the transforming growth factor (TGF)-β family critically regulate the inflammatory and reparative response following infarction. Although all three TGF-β isoforms (TGF-β1, -β2 and -β3) are markedly upregulated in the infarcted myocardium, information on isoform-specific actions is limited. Experimental studies have suggested that TGF-β exerts a wide range of actions on cardiomyocytes, fibroblasts, immune cells, and vascular cells. The findings are often conflicting, reflecting the context-dependence of TGF-β-mediated effects; conclusions are often based exclusively on in vitro studies and on associative evidence. TGF-β has been reported to modulate cardiomyocyte survival responses, promote monocyte recruitment, inhibit macrophage pro-inflammatory gene expression, suppress adhesion molecule synthesis by endothelial cells, promote myofibroblast conversion and extracellular matrix synthesis, and mediate both angiogenic and angiostatic effects. This review manuscript discusses our understanding of the cell biological effects of TGF-β in myocardial infarction. We discuss the relative significance of downstream TGF-β-mediated Smad-dependent and -independent pathways, and the risks and challenges of therapeutic TGF-β targeting. Considering the high significance of TGF-β-mediated actions in vivo, study of cell-specific effects and dissection of downstream signaling pathways are needed in order to design safe and effective therapeutic approaches.

Kruppel-like Factor 6 Promotes Macrophage-mediated Inflammation by Suppressing B Cell Leukemia/Lymphoma 6 Expression

Macrophages are the predominant innate immune cells recruited to tissues following injury or infection. These early-responding, pro-inflammatory macrophages play an essential role in the amplification of inflammation. However, macrophage pro-inflammatory gene expression should be tightly regulated to avert host tissue damage. In this study, we identify the Kruppel-like transcription factor 6 (KLF6)-B cell leukemia/lymphoma 6 (BCL6) signaling axis as a novel regulator of macrophage inflammatory gene expression and function. Utilizing complementary gain- and loss-of-function studies, we observed that KLF6 is essential for macrophage motility under ex vivo and in vivo conditions. Concordant with these observations, myeloid-specific deficiency of KLF6 significantly attenuates macrophage pro-inflammatory gene expression, recruitment, and progression of inflammation. At the molecular level, KLF6 suppresses BCL6 mRNA and protein expression by elevating PR domain-containing 1 with ZNF domain (PRDM1) levels in macrophages. Interestingly, pharmacological or genetic inhibition of BCL6 in KLF6-deficient macrophages completely abrogated the attenuation of pro-inflammatory cytokine/chemokine expression and cellular motility. Collectively, our observations reveal that KLF6 repress BCL6 to enhance macrophage inflammatory gene expression and function.

Chemical epigenetics to assess the role of HDAC1–3 inhibition in macrophage pro-inflammatory gene expression

Azobenzene ortho-aminoanilides inhibit HDACs 1–3 and possess anti-inflammatory properties in murine macrophages.

Metabolic Syndrome Exacerbates Inflammation and Bone Loss in Periodontitis

Clinical studies have shown that metabolic syndrome (MetS) is associated with increased risk of developing periodontitis. However, the underlying mechanisms remain largely unknown. Since it is known that lipopolysaccharide (LPS)–activated toll-like receptor 4 signaling pathways play a crucial role in periodontitis, we hypothesized that MetS enhances LPS-induced periodontal inflammation and alveolar bone loss. In this study, we induced MetS in C57BL/6 mice by feeding them high-fat diet (HFD), and we induced periodontitis by periodontal injection of Aggregatibacter actinomycetemcomitans LPS. We found that mice fed a HFD had significantly increased body weight, plasma lipids, insulin, and insulin resistance when compared with mice fed regular chow, indicating that the mice developed MetS. We also found that a HFD markedly increased LPS-induced alveolar bone loss, osteoclastogenesis, and inflammatory infiltration. Analysis of gene expression in periodontal tissue revealed that HFD and LPS injection cooperatively stimulated expression of cytokines that are known to be involved in periodontal tissue inflammation and osteoclastogenesis—such as interleukin 6, monocyte-chemotactic protein 1, receptor activator of nuclear factor kappa-B ligand, and macrophage colony-stimulating factor. To further understand the potential mechanisms involved in MetS-boosted tissue inflammation, our in vitro studies showed that palmitic acid—the most abundant saturated fatty acid (SFA) and the major SFA in the HFD used in our animal study—potently enhanced LPS-induced proinflammatory gene expression in macrophages. In sum, this study demonstrated that MetS was associated with increased periodontal inflammation and alveolar bone loss in an LPS-induced periodontitis animal model. This study also suggests that SFA palmitic acid may play an important role in MetS-associated periodontitis by enhancing LPS-induced expression of inflammatory cytokines in macrophages.

Histone Deacetylase 7 Promotes Toll-like Receptor 4-dependent Proinflammatory Gene Expression in Macrophages

Broad-spectrum inhibitors of histone deacetylases (HDACs) constrain Toll-like receptor (TLR)-inducible production of key proinflammatory mediators. Here we investigated HDAC-dependent inflammatory responses in mouse macrophages. Of the classical Hdacs, Hdac7 was expressed at elevated levels in inflammatory macrophages (thioglycollate-elicited peritoneal macrophages) as compared with bone marrow-derived macrophages and the RAW264 cell line. Overexpression of a specific, alternatively spliced isoform of Hdac7 lacking the N-terminal 22 amino acids (Hdac7-u), but not the Refseq Hdac7 (Hdac7-s), promoted LPS-inducible expression of Hdac-dependent genes (Edn1, Il-12p40, and Il-6) in RAW264 cells. A novel class IIa-selective HDAC inhibitor reduced recombinant human HDAC7 enzyme activity as well as TLR-induced production of inflammatory mediators in thioglycollate-elicited peritoneal macrophages. Both LPS and Hdac7-u up-regulated the activity of the Edn1 promoter in an HDAC-dependent fashion in RAW264 cells. A hypoxia-inducible factor (HIF) 1 binding site in this promoter was required for HDAC-dependent TLR-inducible promoter activity and for Hdac7- and HIF-1α-mediated trans-activation. Coimmunoprecipitation assays showed that both Hdac7-u and Hdac7-s interacted with HIF-1α, whereas only Hdac7-s interacted with the transcriptional repressor CtBP1. Thus, Hdac7-u positively regulates HIF-1α-dependent TLR signaling in macrophages, whereas an interaction with CtBP1 likely prevents Hdac7-s from exerting this effect. Hdac7 may represent a potential inflammatory disease target.