A hallmark event in the development of atherosclerotic plaque is the accumulation of lipid-laden macrophages in the subendothelial layers of affected blood vessels. Macrophages are key players in all stages of atherogenesis, including plaque initiation, growth, and rupture, as well as healing of ruptured plaques. In this context, macrophages are the principal innate immune cells that modulate atherogenesis by engaging in various processes, such as inflammation, extracellular matrix degradation, phagocytosis, and efferocytosis. Here, this report shows that Kruppel-like factor 6 (KLF6) deficiency attenuates proinflammatory gene expression in macrophages and experimentally induced atherosclerotic plaque development. In vivo studies show that myeloid-KLF6 deficiency on Apoe-null background significantly curtails high-fat/high-cholesterol diet-induced atherosclerotic lesion formation and macrophage abundance in atherosclerotic plaques. Integrated transcriptomics and Gene Set Enrichment Analysis show that KLF6 deficiency significantly curtails a large number of tumor necrosis factor (TNF)-induced gene targets, TNF-induced interferon-γ response, interferon-α response, and inflammatory response signaling in macrophages. At the molecular level, KLF6 promotes IRF1 signaling to enhance TNF-induced proinflammatory gene expression in macrophages. Collectively, study results show that KLF6 promotes proinflammatory gene expression in macrophages and boosts experimentally induced atherosclerotic plaque formation in vivo.

Recent studies have documented the complexity of the intestinal fungal community (‘mycobiome’) in mice, and clinical and experimental observations have shown that the mycobiome influences both gut health and disease, e.g., inflammatory bowel disease (IBD). In fact, prior studies have shown that Crohn’s disease (CD) patients, compared to healthy controls, harbor higher levels of intestinal Candida tropicalis (Ct), which is the major fungal species detected in the colons of colitic mice after DSS challenge. More recently, Debaryomyces hansenii (also known as Candida famata) has been found in intestinal wounds of mice and inflamed mucosal tissues of CD patients. Moreover, proteins encoded by genes within IBD susceptibility loci, such as the pattern recognition receptor (PRR), NOD2, are known not only to recognize bacterial components, but also a fungal cell wall element, chitin. In fact, the role of PRRs in regulating immunity against intestinal fungi, and how fungi influence IBD remains poorly defined. We challenged DSS colitic WT and Nod2-/- mice with Ct 2 days before DSS administration and subsequently on day (d)0, 3 and 6. Our data confirms previous studies that Ct challenge does not exacerbate colitis in DSS-treated C57BL/6 wildtype (WT) mice, however, Ct-infected Nod2-/- mice possess a higher fungal burden and exhibit worse colitis symptoms, such as weight loss, decreased stool consistency, and presence of blood in stools, vs. Ct-infected WT mice, indicating an essential and protective role for NOD2 during colitis recovery after Ct challenge. Our results also show that Ct-infected Nod2-/- mice display a marked reduction in colonic Il22 and Il17, which are cytokines previously reported to be important in maintaining epithelial barrier integrity during DSS colitis, These data were confirmed in colons of Ct infected DSS challenged, ileitis-prone SAMP1/YitFc (SAMP) mice that were deficient in NOD2. Moreover, Our in vitro data show, a decrease in Il1b and Il23 in bone marrow-derived macrophages from SAMP Nod2-/- mice compared to WT after 2h of exposure to chitin. IL-17+ innate lymphoid cells (ILCs) are also known to control fungal burden during opportunistic fungal infections, and interestingly, our findings indicate that Ct infection of Nod2-/- vs. WT mice results in a decreased frequency of mesenteric lymph node–derived type 3 ILCs (ILC3s), suggesting that delay in fungal clearance and recovery in Nod2-/- mice may be due to the inability to mount protective type 3 immune responses. Taken together, the data so far collected suggests that NOD2 is essential to maintain gut mycobiome homeostasis and drives protective innate immune responses, via a macrophage-mediated ILC3 recruitment and IL-17 mechanism, by preventing the overgrowth of opportunistic fungi that may contribute to chronic intestinal inflammation, such as that observed in CD.

Locally injected mesenchymal stem cells (MSCs) are now an approved therapy in European Union for perianal Crohn’s disease (CD) fistulas, however, clinical studies have shown limited efficacy of systemic MSC therapy for luminal CD. Thus, we studied the SAMP-1/YitFc (SAMP), a chronic and spontaneous murine model of small intestine (SI) inflammation for treatment with human bone marrow derived MSC (hMSC). We previously reported that hMSCs injected intraperitoneally (i.p) into SAMP resulted in histologic, mucosal and radiologic healing (Dave M et al. Gastroenterology 154 (6), S-438). The aim of this study was to determine the mechanism(s) by which hMSCs mediate immunosuppression in SAMP. hMSCs were transduced with lentivirus vector containing shRNA for COX-2 (hMSCshCOX-2) to knockdown secretion of Prostaglandin E2 (PGE2) and lentivirus with scrambled shRNA was used as control (hMSC-S control). Using NanoString immune cell profiling assay, SAMP mice treated with hMSC i.p had increased abundance of macrophages and decreased abundance of lymphocytes in the SI. In MLRs set up with T lymphocytes from SAMP mice, hMSCs profoundly suppressed T cell proliferation in a dose dependent manner as measured by [3H] thymidine incorporation (95% CI of difference: 8,573 – 14,335 cpm; ANOVA P<0.0001 for 2X105 hMSCs). By enzyme immunoassay we determined that hMSCs basally secrete PGE2 (4850 ± 86 pg/mL per 100,000 cells). Indomethacin, a COX-1/2 inhibitor (10 μM) abrogated the suppression of T cell proliferation in MLR and MLR performed with PGE2 inhibited hMSC (hMSCshCOX-2) in comparison to hMSC-S control again demonstrated abrogation of suppression of T cell proliferation (95% CI of difference: 3,885 – 10,456 cpm; ANOVA P<0.001 for hMSC-S control and 95% CI of difference: -1,859 – 7580 cpm; ANOVA P not sig. for hMSCshCOX-2). In co-culture assay hMSCs reprogrammed SAMP macrophages (MØ) to anti-inflammatory phenotype with higher gene expression of arginase I (28.9 fold increase, P<0.0001) and lower gene expression of proinflammatory IL-6 (0.2-fold decrease, P<0.0001), TNF-α (0.2-fold decrease, P<0.0001), IL-1α (0.3-fold decrease, P<0.0001), and IL-1β (0.4-fold decrease, P<0.0001) compared to untreated SAMP MØ. The supernatants from MØ co-cultured with hMSCs contained higher amounts of PGE2 than MØ alone (4100 ± 394 pg/mL vs.81.6 ± 70.7 pg/mL; P<0.001). Flow sorting for C11b+ and F4/80+ MØ four weeks after treatment, demonstrated that MØ from SAMP mice treated with i.p hMSC had an anti-inflammatory phenotype characterized by higher gene expression of arginase-I (13.6-fold increase, P=.01, n=8) and lower gene expression of TNF-α (0.76-fold decrease, P=0.1, n=8) compared to untreated mice. hMSCs mediate immunosuppression by secreting PGE2 and reprogramming macrophages to anti-inflammatory phenotype in SAMP model of experimental CD.

The determinants of HIV-1-associated lymphadenopathy are poorly understood. We hypothesized that lymphocytes could be sequestered in the HIV-1+ lymph node (LN) through impairments in sphingosine-1-phosphate (S1P) responsiveness. To test this hypothesis, we developed novel assays for S1P-induced Akt phosphorylation and actin polymerization. In the HIV-1+ LN, naïve CD4 T cells and central memory CD4 and CD8 T cells had impaired Akt phosphorylation in response to S1P, whereas actin polymerization responses to S1P were impaired dramatically in all LN maturation subsets. These defects were improved with antiretroviral therapy. LN T cells expressing CD69 were unable to respond to S1P in either assay, yet impaired S1P responses were also seen in HIV-1+ LN T cells lacking CD69 expression. Microbial elements, HIV-1, and interferon α - putative drivers of HIV-1 associated immune activation all tended to increase CD69 expression and reduce T-cell responses to S1P in vitro. Impairment in T-cell egress from lymph nodes through decreased S1P responsiveness may contribute to HIV-1-associated LN enlargement and to immune dysregulation in a key organ of immune homeostasis.

The vascular endothelium plays a fundamental role in the health and disease of the cardiovascular system. The molecular mechanisms regulating endothelial homeostasis, however, remain incompletely understood. CCN3, a member of the CCN (Cyr61, Ctgf, Nov) family of cell growth and differentiation regulators, has been shown to play an important role in numerous cell types. The function of CCN3 in endothelial cells has yet to be elucidated. Immunohistochemical analysis of CCN3 expression in mouse tissues revealed robust immunoreactivity in the endothelium of large arteries, small resistance vessels, and veins. We found that CCN3 expression in human umbilical vein endothelial cells (HUVECs) is transcriptionally induced by laminar shear stress (LSS) and HMG CoA-reductase inhibitors (statins). Promoter analyses identified the transcription factor Kruppel-like factor 2 (KLF2) as a direct regulator of CCN3 expression. In contrast to LSS, proinflammatory cytokines reduced CCN3 expression. Adenoviral overexpression of CCN3 in HUVEC markedly inhibited the cytokine-mediated induction of vascular adhesion molecule-1 (VCAM-1). Consistent with this observation, CCN3 significantly reduced monocyte adhesion. Conversely, CCN3 knockdown in HUVECs resulted in enhancement of cytokine-induced VCAM-1 expression. Concordant effects were observed on monocyte adhesion. Gain and loss-of-function mechanistic studies demonstrated that CCN3 negatively regulates nuclear factor kappaB (NF-κB) activity by reducing its translocation into the nucleus and subsequent binding to the VCAM-1 promoter, suggesting that CCN3’s anti-inflammatory effects occur secondary to inhibition of NF-κB nuclear accumulation. This study identifies CCN3 as a novel regulator of endothelial proinflammatory activation.

Background : Selective permeability is a life sustaining function of endothelial cells. Defects in permeability are implicated in several diseases such as sepsis, ischemia reperfusion injury and diabetes. Previous studies by our group and others have identified KLF2 as a molecular switch regulating endothelial gene expression and function. However the role of KLF2 in vascular permeability remains unknown. Methods and Results : Adenoviral overexpression of KLF2 in HUVECs strongly attenuated the increase of endothelial permeability by thrombin (1U/ml), histamine (200 μ M) and hydrogen peroxide (500 μ M) as measured by two independent assays – transendothelial electrical resistance (TER) and FITC-Dextran passage using a transwell system. Conversely, KLF2 deficiency in HUVECs (by siRNA knockdown) and primary mouse endothelial cells (derived from KLF2 +/+ and KLF2+/− mice) exhibited a marked increase in thrombin-induced permeability. To assess the effect of KLF2 in vascular permeability in vivo, we measured the leakage of Evans Blue dye into interstitial tissues of the mouse ear (expressed as μ g of dye/mg of ear tissue) after treatment with mustard oil. Consistent with our in vitro observation, by comparison to KLF2+/+ mice, KLF2 +/− mice exhibited a significantly higher degree of vascular leak (53.4±2.68 μ g/mg in KLF2+/+ versus 72.36±4.71 μ g/mg in KLF2+/−, p<0.05). Mechanistically, our gain and loss-of-function studies indicate that KLF2 differentially affects key endothelial targets that regulate barrier function. Specifically, KLF2 markedly attenuated agonist- induced increase in Myosin Light Chain (MLC) phosphorylation (a key signaling molecule that promotes endothelial barrier permeability), increased Rac1 activation (a small GTPase that has been shown to confer barrier protection properties), and increased the expression of occludin and zona occludens-1 (tight junction proteins that promote barrier function). Conclusions : These observations identify KLF2 as an essential regulator of endothelial barrier function. Targeting of KLF2 function may be beneficial in disorders characterized by excessive vascular permeability such as sepsis. This research has received full or partial funding support from the American Heart Association, Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).

Introduction: Hypoxia-inducible factor 1 (HIF-1) is a central regulator of the hypoxic response in many cell types. In endothelial cells, HIF-1 induces the expression of key pro-angiogenic factors such as vascular endothelial growth factor (VEGF) to induce new blood vessel formation. Recent studies have identified Kruppel-like Factor 2 (KLF2) as a potent inhibitor of angiogenesis. However, the role of KLF2 in regulating the expression and function of HIF-1 has not been evaluated. Hypothesis: KLF2 inhibits HIF-1α expression and function. Methods and Results: Adenoviral overexpression of KLF2 in human umbilical vein endothelial cells (HUVECs) inhibited hypoxia-induced expression of HIF-1α, VEGF secretion, and matrigel tube formation. Conversely, siRNA-mediated knockdown of KLF2 in HUVECs increased HIF-1α expression and VEGF secretion. Consistent with this observation, KLF2−/− mouse embryonic fibroblasts (MEFs) showed accelerated HIF-1α accumulation in response to hypoxia and a marked induction of VEGF secretion (90.4±7.4 pg/ml in KLF2+/+ versus 234.6±6.1 pg/ml in KLF2−/−, p<0.01). From a mechanistic standpoint, KLF2-mediated reduction of HIF-1α level was attenuated by three distinct proteasome inhibitors (MG132, ALLN, or lactacystin), demonstrating that KLF2 mediated reduction of HIF-1α protein was proteasome-dependent. In addition, KLF2 maintained its ability to inhibit HIF-1α protein levels in VHL−/− cells, thermo-sensitive E1 deficient cells, and p53−/− cells. Finally, co-immunoprecipitation experiments revealed that KLF2 overexpression disrupted the interaction between HIF-1α and its chaperone Hsp90, suggesting that KLF2 promotes degradation of HIF-1α by affecting its folding and maturation. Conclusions: These observations identify KLF2 as a novel inhibitor of HIF-1α expression and function through a VHL/p53 independent but proteasome dependent pathway. As such, KLF2 may be a target for modulating the angiogenic response in disease states.

Angiogenesis, the formation of new blood vessels from preexisting vasculature, occurs in a variety of physiological and pathological conditions. The process of angiogenesis is known to be regulated by vascular endothelial growth factor (VEGF) and its receptors (VEGFR‐2) in coordination with extracellular matrix interacting molecules such as integrins. Integrins have been reported to exhibit extracellular matrix dependent regulation of several tyrosine kinases on endothelial cell surfaces. In this study we have revealed the role of β3 integrin cytoplasmic tyrosine motifs in the regulation of protein tyrosine phosphatase (PTPs), associated with VEGFR‐2, in a tyrosine phosphorylation‐dependent manner. SHP‐2 association with VEGFR‐2 regulates the extent of basal and growth factor‐induced VEGFR‐2 phosphorylation in endothelial cells. Additionally, phosphorylated β3 integrin cytoplasmic tyrosine motifs provide binding sites for SHP‐2 and protect VEGFR‐2 from dephosphorylation. Moreover, DiYF endothelial cells, in which β3 integrin cytoplasmic tyrosine motifs are mutated to phenylalanine, exhibit deficient SHP‐2 binding to β3 integrin and VEGF stimulation, also display a modest decrease in SHP‐2 dissociation from VEGFR‐2 and decreased pathological angiogenesis in vivo. Further, we have also demonstrated that SHP‐2 binds to phosphorylated tyrosine residue 773 of the β3 cytoplasmic domain, and that β3 integrin cytoplasmic‐derived cell permeable prephosphorylated peptide 773Yp reduced VEGF‐induced VEGFR‐2 tyrosine phosphorylation and down stream signaling, endothelial cell migration, tube formation, ex vivo and in vivo angiogenesis. We have for the first time demonstrated this novel mechanism which regulates processes of VEGFR‐2 activation and pathological angiogenesis.