Interleukin 33 Triggers Early Eosinophil-Dependent Events Leading to Metaplasia in a Chronic Model of Gastritis-Prone Mice.

BACKGROUND & AIMS: Interleukin (IL)33/IL1F11 is an important mediator for the development of type 2 T-helper cell (Th2)-driven inflammatory disorders and has also been implicated in the pathogenesis of gastrointestinal (GI)-related cancers, including gastric carcinoma. We therefore sought to mechanistically determine IL33's potential role as a critical factor linking chronic inflammation and gastric carcinogenesis using gastritis-prone SAMP1/YitFc (SAMP) mice. METHODS: SAMP and (parental control) AKR mice were assessed for baseline gastritis and progression to metaplasia. Expression/localization of IL33 and its receptor, ST2/IL1R4, were characterized in corpus tissues, and activation and neutralization studies were both performed targeting the IL33/ST2 axis. Dissection of immune pathways leading to metaplasia was evaluated, including eosinophil depletion studies using anti-IL5/anti-CCR3 treatment. RESULTS: Progressive gastritis and, ultimately, intestinalized spasmolytic polypeptide-expressing metaplasia (SPEM) was detected in SAMP stomachs, which was absent in AKR but could be moderately induced with exogenous, recombinant IL33. Robust peripheral (bone marrow) expansion of eosinophils and local recruitment of both eosinophils and IL33-expressing M2 macrophages into corpus tissues were evident in SAMP. Interestingly, IL33 blockade did not affect bone marrow-derived expansion and local infiltration of eosinophils, but markedly decreased M2 macrophages and SPEM features, while eosinophil depletion caused a significant reduction in both local IL33-producing M2 macrophages and SPEM in SAMP. CONCLUSIONS: IL33 promotes metaplasia and the sequelae of eosinophil-dependent downstream infiltration of IL33-producing M2 macrophages leading to intestinalized SPEM in SAMP, suggesting that IL33 represents a critical link between chronic gastritis and intestinalizing metaplasia that may serve as a potential therapeutic target for preneoplastic conditions of the GI tract.

Macrophage-Hypoxia-Inducible Factor-1α Signaling in Carotid Artery Stenosis.

Macrophages play crucial and diverse roles in the pathogenesis of inflammatory vascular diseases. Macrophages are the principal innate immune cells recruited to arterial walls to govern vascular homeostasis by modulating the proliferation of vascular smooth muscle cells, the reorganization of extracellular matrix components, the elimination of dead cells, and the restoration of normal blood flow. However, chronic sterile inflammation within the arterial walls draws inflammatory macrophages into intimal/neointimal regions that may contribute to disease pathogenesis. In this context, the accumulation and aberrant activation of macrophages in the neointimal regions govern the progression of inflammatory arterial wall diseases. Herein, we report that myeloid-hypoxia-inducible factor-1α (HIF1α) deficiency attenuates vascular smooth muscle cells and macrophage abundance in stenotic arteries and abrogates carotid neointima formation in vivo. The integrated transcriptomics, Gene Set Enrichment Analysis, metabolomics, and target gene evaluation showed that HIF1α represses oxidative phosphorylation, tricarboxylic acid cycle, fatty acid metabolism, and c-MYC signaling pathways while promoting inflammatory, glycolytic, hypoxia response gene expression in stenotic artery macrophages. At the molecular level, proinflammatory agents utilized STAT3 signaling pathways to elevate HIF1α expression in macrophages. Collectively, this study uncovers that macrophage-HIF1α deficiency restrains the pathogenesis of carotid artery stenosis by rewiring inflammatory and metabolic signaling pathways 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.

CITED2 inhibits STAT1-IRF1 signaling and atherogenesis.

Macrophages are the principal component of the innate immune system. They play very crucial and multifaceted roles in the pathogenesis of inflammatory vascular diseases. There is an increasing recognition that transcriptionally dynamic macrophages are the key players in the pathogenesis of inflammatory vascular diseases. In this context, the accumulation and aberrant activation of macrophages in the subendothelial layers govern atherosclerotic plaque development. Macrophage-mediated inflammation is an explicitly robust biological response that involves broad alterations in inflammatory gene expression. Thus, cell-intrinsic negative regulatory mechanisms must exist which can restrain inflammatory response in a spatiotemporal manner. In this study, we identified CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 2 (CITED2) as one such cell-intrinsic negative regulator of inflammation. Our in vivo studies show that myeloid-CITED2-deficient mice on the Apoe-/- background have larger atherosclerotic lesions on both control and high-fat/high-cholesterol diets. Our integrated transcriptomics and gene set enrichment analyses studies show that CITED2 deficiency elevates STAT1 and interferon regulatory factor 1 (IRF1) regulated pro-inflammatory gene expression in macrophages. At the molecular level, our studies identify that CITED2 deficiency elevates IFNγ-induced STAT1 transcriptional activity and STAT1 enrichment on IRF1 promoter in macrophages. More importantly, siRNA-mediated knockdown of IRF1 completely reversed elevated pro-inflammatory target gene expression in CITED2-deficient macrophages. Collectively, our study findings demonstrate that CITED2 restrains the STAT1-IRF1 signaling axis in macrophages and limits the development of atherosclerotic plaques.

Kruppel-like factor 6 promotes macrophage inflammatory and hypoxia response.

Macrophages are the professional phagocytes that protect the host from infection or injury. Tissue microenvironment at the site of injury and inflammation is characterized by low oxygen concentration and poor supply of nutrients. The responding macrophages have to advance against oxygen and nutrient gradients to reach the site of inflammation to perform host protection, and tissue repair functions. Thus, evolution has fashioned macrophages to orchestrate a coordinated inflammatory and hypoxic gene program to mount an effective immune response. Here, we discovered that Kruppel-like factor 6 (KLF6) governs macrophage functions by promoting inflammatory and hypoxic response gene programming. Our in vivo studies revealed that myeloid-KLF6-deficient mice were highly resistant to endotoxin-induced systemic inflammatory response syndrome symptomatology and mortality. Using complementary gain- and loss-of-function studies, we observed that KLF6 overexpression elevate and KLF6 deficiency attenuate inducible HIF1α expression in macrophages. Our integrated transcriptomics and gene set enrichment analysis studies uncovered that KLF6 deficiency attenuates broad inflammatory and glycolytic gene expression in macrophages. More importantly, overexpression of oxygen stable HIF1α reversed attenuated proinflammatory and glycolytic gene expression in KLF6-deficient macrophages. Collectively, our studies uncovered that KLF6 govern inflammatory and hypoxic response by regulating HIF1α expression in macrophage.

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.

The myeloid transcription factor KLF2 regulates the host response to polymicrobial infection and endotoxic shock.

Precise control of myeloid cell activation is required for optimal host defense. However, this activation process must be under exquisite control to prevent uncontrolled inflammation. Herein, we identify the Kruppel-like transcription factor 2 (KLF2) as a potent regulator of myeloid cell activation in vivo. Exposure of myeloid cells to hypoxia and/or bacterial products reduced KLF2 expression while inducing hypoxia inducible factor-1α (HIF-1α), findings that were recapitulated in human septic patients. Myeloid KLF2 was found to be a potent inhibitor of nuclear factor-kappaB (NF-κB)-dependent HIF-1α transcription and, consequently, a critical determinant of outcome in models of polymicrobial infection and endotoxemia. Collectively, these observations identify KLF2 as a tonic repressor of myeloid cell activation in vivo and an essential regulator of the innate immune system.

Dual regulation of Stat1 and Stat3 by the tumor suppressor protein PML contributes to interferon α-mediated inhibition of angiogenesis.

IFNs are effective in inhibiting angiogenesis in preclinical models and in treating several angioproliferative disorders. However, the detailed mechanisms of IFNα-mediated anti-angiogenesis are not completely understood. Stat1/2/3 and PML are IFNα downstream effectors and are pivotal regulators of angiogenesis. Here, we investigated PML's role in the regulation of Stat1/2/3 activity. In Pml knock-out (KO) mice, ablation of Pml largely reduces IFNα angiostatic ability in Matrigel plug assays. This suggested an essential role for PML in IFNα's anti-angiogenic function. We also demonstrated that PML shared a large cohort of regulatory genes with Stat1 and Stat3, indicating an important role of PML in regulating Stat1 and Stat3 activity. Using molecular tools and primary endothelial cells, we demonstrated that PML positively regulates Stat1 and Stat2 isgylation, a ubiquitination-like protein modification. Accordingly, manipulation of the isgylation system by knocking down USP18 altered IFNα-PML axis-mediated inhibition of endothelial cell migration and network formation. Furthermore, PML promotes turnover of nuclear Stat3, and knockdown of PML mitigates the effect of LLL12, a selective Stat3 inhibitor, on IFNα-mediated anti-angiogenic activity. Taken together, we elucidated an unappreciated mechanism in which PML, an IFNα-inducible effector, possess potent angiostatic activity, doing so in part by forming a positive feedforward loop with Stat1/2 and a negative feedback loop with Stat3. The interplay between PML, Stat1/Stat2, and Stat3 contributes to IFNα-mediated inhibition of angiogenesis, and disruption of this network results in aberrant IFNα signaling and altered angiostatic activity.

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.

ß3 phosphorylation of platelet αIIbß3 is crucial for stability of arterial thrombus and microparticle formation in vivo.

BACKGROUND: It is well accepted that functional activity of platelet integrin αIIbß3 is crucial for hemostasis and thrombosis. The ß3 subunit of the complex undergoes tyrosine phosphorylation shown to be critical for outside-in integrin signaling and platelet clot retraction ex vivo. However, the role of this important signaling event in other aspects of prothrombotic platelet function is unknown. METHOD: Here, we assess the role of ß3 tyrosine phosphorylation in platelet function regulation with a knock-in mouse strain, where two ß3 cytoplasmic tyrosines are mutated to phenylalanine (DiYF). We employed platelet transfusion technique and intravital microscopy for observing the cellular events involved in specific steps of thrombus growth to investigate in detail the role of ß3 tyrosine phosphorylation in arterial thrombosis in vivo. RESULTS: Upon injury, DiYF mice exhibited delayed arterial occlusion and unstable thrombus formation. The mean thrombus volume in DiYF mice formed on collagen was only 50% of that in WT. This effect was attributed to DiYF platelets but not to other blood cells and endothelium, which also carry these mutations. Transfusion of isolated DiYF but not WT platelets into irradiated WT mice resulted in reversal of the thrombotic phenotype and significantly prolonged blood vessel occlusion times. DiYF platelets exhibited reduced adhesion to collagen under in vitro shear conditions compared to WT platelets. Decreased platelet microparticle release after activation, both in vitro and in vivo, were observed in DiYF mice compared to WT mice. CONCLUSION: ß3 tyrosine phosphorylation of platelet αIIbß3 regulates both platelet pro-thrombotic activity and the formation of a stable platelet thrombus, as well as arterial microparticle release.

Kruppel-like transcription factor 6 regulates inflammatory macrophage polarization.

Accumulating evidence supports the importance of macrophage plasticity in a broad spectrum of biological processes operative in health and disease. A major locus of control regulating macrophage polarization is at the transcriptional level, and several major pathways have been elucidated in recent years. In this study, we identify the Kruppel-like transcription factor 6 (KLF6) as a molecular toggle controlling macrophage speciation. KLF6 expression was robustly induced by pro-inflammatory M1 stimuli (e.g. LPS and IFN-γ) and strongly suppressed by M2 stimuli (e.g. IL4 and IL-13) in human and murine macrophages. Gain- and loss-of-function studies suggest that KLF6 is required for optimal LPS-induced pro-inflammatory gene expression, acting cooperatively with NF-κB. Furthermore, KLF6 inhibits anti-inflammatory gene expression by negatively regulating peroxisome proliferator-activated receptor γ expression in macrophages. Collectively, these observations identify KLF6 as a novel transcriptional regulator of macrophage polarization.

Macrophage gene expression and foam cell formation are regulated by plasminogen.

BACKGROUND: Deciphering the molecular and cellular processes that govern macrophage foam cell formation is critical to understanding the basic mechanisms underlying atherosclerosis and other vascular pathologies. METHODS AND RESULTS: Here, we identify a pivotal role of plasminogen (Plg) in regulating foam cell formation. Deficiency of Plg inhibited macrophage cholesterol accumulation on exposure to hyperlipidemic conditions in vitro, ex vivo, and in vivo. Gene expression analysis identified CD36 as a regulated target of Plg, and macrophages from Plg(-/-) mice had decreased CD36 expression and diminished foam cell formation. The Plg-dependent CD36 expression and foam cell formation depended on conversion of Plg to plasmin, binding to the macrophage surface, and the consequent intracellular signaling that leads to production of leukotriene B4. Leukotriene B4 rescued the suppression of CD36 expression and foam cell formation arising from Plg deficiency. CONCLUSIONS: Our findings demonstrate an unanticipated role of Plg in the regulation of gene expression and cholesterol metabolism by macrophages and identify Plg-mediated regulation of leukotriene B4 as an underlying mechanism.

Kruppel-like factor 2 is a transcriptional regulator of chronic and acute inflammation.

Although myeloid cell activation is requisite for an optimal innate immune response, this process must be tightly controlled to prevent collateral host tissue damage. Kruppel-like factor 2 (KLF2) is a potent regulator of myeloid cell proinflammatory activation. As an approximately 30% to 50% reduction in KLF2 levels has been observed in human subjects with acute or chronic inflammatory disorders, we studied the biological response to inflammation in KLF2(+/-) mice. Herein, we show that partial deficiency of KLF2 modulates the in vivo response to acute (sepsis) and subacute (skin) inflammatory challenge. Mechanistically, we link the anti-inflammatory effects of KLF2 to the inhibition of NF-κB transcriptional activity. Collectively, the observations provide biologically relevant insights into KLF2-mediated modulation of these inflammatory processes that could potentially be manipulated for therapeutic gain.

MSCs mediate long-term efficacy in a Crohn's disease model by sustained anti-inflammatory macrophage programming via efferocytosis.

Mesenchymal stem cells (MSCs) are novel therapeutics for the treatment of Crohn's disease. However, their mechanism of action is unclear, especially in disease-relevant chronic models of inflammation. Thus, we used SAMP-1/YitFc (SAMP), a chronic and spontaneous murine model of small intestinal inflammation, to study the therapeutic effects and mechanism of action of human bone marrow-derived MSCs (hMSC). hMSC dose-dependently inhibited naïve T lymphocyte proliferation via prostaglandin E2 (PGE2) secretion and reprogrammed macrophages to an anti-inflammatory phenotype. We found that the hMSCs promoted mucosal healing and immunologic response early after administration in SAMP when live hMSCs are present (until day 9) and resulted in a complete response characterized by mucosal, histological, immunologic, and radiological healing by day 28 when no live hMSCs are present. hMSCs mediate their effect via modulation of T cells and macrophages in the mesentery and mesenteric lymph nodes (mLN). Sc-RNAseq confirmed the anti-inflammatory phenotype of macrophages and identified macrophage efferocytosis of apoptotic hMSCs as a mechanism that explains their long-term efficacy. Taken together, our findings show that hMSCs result in healing and tissue regeneration in a chronic model of small intestinal inflammation and despite being short-lived, exert long-term effects via sustained anti-inflammatory programming of macrophages via efferocytosis.

A myeloid hypoxia-inducible factor 1α-Krüppel-like factor 2 pathway regulates gram-positive endotoxin-mediated sepsis.

Although gram-positive infections account for the majority of cases of sepsis, the molecular mechanisms underlying their effects remains poorly understood. We investigated how cell wall components of gram-positive bacteria contribute to the development of sepsis. Experimental observations derived from cultured primary macrophages and the cell line indicate that gram-positive bacterial endotoxins induce hypoxia-inducible factor 1α (HIF-1α) mRNA and protein expression. Inoculation of live or heat-inactivated gram-positive bacteria with macrophages induced HIF-1 transcriptional activity in macrophages. Concordant with these results, myeloid deficiency of HIF-1α attenuated gram-positive bacterial endotoxin-induced cellular motility and proinflammatory gene expression in macrophages. Conversely, gram-positive bacteria and their endotoxins reduced expression of the myeloid anti-inflammatory transcription factor Krüppel-like transcription factor 2 (KLF2). Sustained expression of KLF2 reduced and deficiency of KLF2 enhanced gram-positive endotoxins induced HIF-1α mRNA and protein expression in macrophages. More importantly, KLF2 attenuated gram-positive endotoxins induced cellular motility and proinflammatory gene expression in myeloid cells. Consistent with these results, mice deficient in myeloid HIF-1α were protected from gram-positive endotoxin-induced sepsis mortality and clinical symptomatology. By contrast, myeloid KLF2-deficient mice were susceptible to gram-positive sepsis induced mortality and clinical symptoms. Collectively, these observations identify HIF-1α and KLF2 as critical regulators of gram-positive endotoxin-mediated sepsis.

A novel role for platelet secretion in angiogenesis: mediating bone marrow-derived cell mobilization and homing.

Angiogenesis alleviates hypoxic stress in ischemic tissues or during tumor progression. In addition to endothelial cell proliferation and migration, the angiogenic process requires bone marrow-derived cell (BMDC) recruitment to sites of neovascularization. However, the mechanism of communication between hypoxic tissues and the BM remains unknown. Using 2 models of hypoxia-induced angiogenesis (ischemic hindlimb surgery and subcutaneous tumor growth), we show that platelet infusion promotes BMDC mobilization into the circulation, BMDC recruitment into growing neovasculature, tumor vascularization, and blood flow restoration in ischemic limbs, whereas platelet depletion inhibits these effects. Thus, platelets are required for BMDC recruitment into ischemia-induced vasculature. Secretion of platelet α-granules, but neither dense granules nor platelet aggregation is crucial for BMDC homing and subsequent angiogenesis, as determined using VAMP-8(-/-), Pearl, and integrin Beta 3(-/-) platelets. Finally, platelets sequester tumor-derived promoters of angiogenesis and BMDC mobilization, which are counterbalanced by the antiangiogenic factor thrombospondin-1. A lack of thrombospondin-1 in platelets leads to an imbalance in proangiogenic and antiangiogenic factors and accelerates tumor growth and vascularization. Our data demonstrate that platelets stimulate BMDC homing in a VAMP-8-dependent manner, revealing a previously unknown role for platelets as key mediators between hypoxic tissues and the bone marrow during angiogenesis.

Myeloid Krüppel-like factor 4 deficiency augments atherogenesis in ApoE-/- mice--brief report.

OBJECTIVE: To investigate the role of Krüppel-like factor 4 (KLF4), an essential transcriptional regulator of macrophage polarization (M1/M2), in the pathogenesis of atherosclerosis. METHODS AND RESULTS: Despite the acknowledged importance of macrophages in atherosclerosis, the role of M1 (classically activated or proinflammatory) versus M2 (alternatively activated or anti-inflammatory) macrophages in this process remains incompletely understood. We recently identified KLF4 as a regulator of macrophage subset specification; that is, KLF4 promotes M2 and inhibits M1 phenotype. Here, we provide evidence that KLF4-deficient macrophages exhibit enhanced proinflammatory activation and foam cell formation in response to oxidized lipids. In vivo, myeloid KLF4-deficient mice (ApoE(-/-) background) develop significantly more vascular inflammation and atherosclerotic lesion formation. CONCLUSIONS: Our findings identify myeloid KLF4 as an essential regulator of vascular inflammation and experimental atherogenesis.

Myeloid-CITED2 Deficiency Exacerbates Diet-Induced Obesity and Pro-Inflammatory Macrophage Response.

Macrophages are the principal component of the innate immune system that are found in all tissues and play an essential role in development, homeostasis, tissue repair, and immunity. Clinical and experimental studies have shown that transcriptionally dynamic pro-inflammatory macrophages are involved in the pathogenesis of diet-induced obesity and insulin resistance. However, cell-intrinsic mechanisms must exist that bridle uncontrolled pro-inflammatory macrophage activation in metabolic organs and disease pathogenesis. In this study, we show that CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 2 (CITED2) is an essential negative regulator of pro-inflammatory macrophage activation and inflammatory disease pathogenesis. Our in vivo studies show that myeloid-CITED2 deficiency significantly elevates high-fat diet (HFD)-induced expansion of adipose tissue volume, obesity, glucose intolerance, and insulin resistance. Moreover, myeloid-CITED2 deficiency also substantially augments HFD-induced adipose tissue inflammation and adverse remodeling of adipocytes. Our integrated transcriptomics and gene set enrichment analyses show that CITED2 deficiency curtails BCL6 signaling and broadly elevates BCL6-repressive gene target expression in macrophages. Using complementary gain- and loss-of-function studies, we found that CITED2 deficiency attenuates, and CITED2 overexpression elevates, inducible BCL6 expression in macrophages. At the molecular level, our analyses show that CITED2 promotes BCL6 expression by restraining STAT5 activation in macrophages. Interestingly, siRNA-mediated knockdown of STAT5 fully reversed elevated pro-inflammatory gene target expression in CITED2-deficient macrophages. Overall, our findings highlight that CITED2 restrains inflammation by promoting BCL6 expression in macrophages, and limits diet-induced obesity and insulin resistance.

Mesenchymal stem cells ameliorate inflammation in an experimental model of Crohn's disease via the mesentery.

Objective: Mesenchymal stem cells (MSCs) are novel therapeutics for treatment of Crohn's disease. However, their mechanism of action is unclear, especially in disease-relevant chronic models of inflammation. Thus, we used SAMP-1/YitFc, a chronic and spontaneous murine model of small intestinal inflammation, to study the therapeutic effect and mechanism of human bone marrow-derived MSCs (hMSC). Design: hMSC immunosuppressive potential was evaluated through in vitro mixed lymphocyte reaction, ELISA, macrophage co-culture, and RT-qPCR. Therapeutic efficacy and mechanism in SAMP were studied by stereomicroscopy, histopathology, MRI radiomics, flow cytometry, RT-qPCR, small animal imaging, and single-cell RNA sequencing (Sc-RNAseq). Results: hMSC dose-dependently inhibited naïve T lymphocyte proliferation in MLR via PGE 2 secretion and reprogrammed macrophages to an anti-inflammatory phenotype. hMSC promoted mucosal healing and immunologic response early after administration in SAMP model of chronic small intestinal inflammation when live hMSCs are present (until day 9) and resulted in complete response characterized by mucosal, histological, immunologic, and radiological healing by day 28 when no live hMSCs are present. hMSC mediate their effect via modulation of T cells and macrophages in the mesentery and mesenteric lymph nodes (mLN). Sc-RNAseq confirmed the anti-inflammatory phenotype of macrophages and identified macrophage efferocytosis of apoptotic hMSCs as a mechanism of action that explains their long-term efficacy. Conclusion: hMSCs result in healing and tissue regeneration in a chronic model of small intestinal inflammation. Despite being short-lived, exert long-term effects via macrophage reprogramming to an anti-inflammatory phenotype. Data Transparency Statement: Single-cell RNA transcriptome datasets are deposited in an online open access repository 'Figshare' (DOI: https://doi.org/10.6084/m9.figshare.21453936.v1 ).