• Myeloid specific regulation of NF-kB and M-CSF signaling in HIV-1 and AML

      Rappaport, Jay; Wigdahl, Brian; Fischer-Smith, Tracy; Langford, Dianne; Hu, Wenhui (Temple University. Libraries, 2013)
      The HIV protein, Vpr, is a multifunctional accessory protein critical for efficient viral infection of target CD4+ T cells and macrophages. Vpr is incorporated into virus particles and functions to transport the preintegration complex into the nucleus where the process of viral integration into the host genome is completed. This action is particularly important in macrophages, which as a result of their terminal differentiation and non-proliferative status, would be otherwise more refractory to HIV infection. Vpr has several other critical functions including activation of HIV-1 LTR transcription, cell-cycle arrest due to DCAF-1 binding, and both direct and indirect contributions to T-cell dysfunction. The interactions of Vpr with molecular pathways in the context of macrophages, on the other hand, support accumulation of a persistent reservoir of HIV infection in cells of the myeloid lineage. The role of Vpr in the virus life cycle, as well as its effects on immune cells, appears to play an important role in the immune pathogenesis of AIDS and the development of HIV induced end-organ disease. In view of the pivotal functions of Vpr in virus infection, replication, and persistence of infection, this protein represents an attractive target for therapeutic intervention. Numerous studies have reported that Vpr alters NF-kappa B signaling in various cells, however, the findings have so far been largely conflicting with reports both stimulatory and inhibitory effects of Vpr. Our aim was to investigate the role of Vpr signaling in myeloid cells and address discrepancies that have been reported in the field. Our results show that Vpr expressed intracellularly is inhibitory to NF-kappa B, while extracelluar Vpr may have some stimulatory effects. Consistent with this notion, we report that Vpr has inhibitory effects that are specific to the TNF-alpha pathway, but not the LPS pathway, suggesting that multiple targets of Vpr may exist for NF-kappa B regulation. Further, we identify VprBP as one possible cellular component of Vpr's regulation of I-kappa B-alpha in response to TNF-alpha stimulation. We did not identify such a role for HSP27, which instead seems to inhibit Vpr functions. Finally, our findings suggest that NF-kappa B regulation by Vpr is further changed by the presence of other HIV-1 components within the cells, as U1 cells lacking Vpr were unexpectedly less responsive to TNF-alpha than those cells that had normal Vpr expression levels. This data suggests that Vpr may serve an important role in vivo by selectively inhibiting immune activation while stimulating NF-kappa B mediated viral production in HIV-1 infected T-cells and myeloid cells. M-CSF is a cytokine that promotes monocyte differentiation and survival. When over-expressed, M-CSF contributes to pathology in a wide variety of diseases including osteoporosis, obesity, certain human cancers, and in HIV-1 infection, particularly with respect to monocyte/macrophage infection and the development of HIV-1. In this study, our aim is to expand on the current knowledge of M-CSF regulation by NF-kappa B, a prominent transcription factor during inflammation and HIV-1 infection. Our results suggest that TNF-alpha promotes M-CSF secretion in macrophages and activates the -1310/+48 bp M-CSF promoter in Mono-Mac 1 cells. Inhibitors of the NF-kappa B pathway, diminish this response. We identified four putative NF-kappa B and four C/EBP-beta binding sites within the M-CSF promoter. Our findings using M-CSF promoter constructs mutated at individual NF-kappa B locations suggest these sites are redundant with respect to M-CSF promoter regulation. TNF-alpha treatment promoted NF-kappa B p65 binding to the M-CSF promoter in PMA treated U937 cells chronically infected with HIV-1 (U1 cells), but not in PMA treated uninfected U937 cells, suggesting that the presence of HIV-1 increases the NF-kappa B response. In conclusion, our findings demonstrate that NF-kappa B induces M-CSF expression on a promoter level via multiple functional NF-kappa B binding sites and that this pathway is likely relevant in HIV-1 infection of macrophages. The oncogenic potential of M-CSF receptor has been has been suggested over thirty years ago, however, few current studies have focused on the role of the receptor in AML. In a clinical trial for AML, Sunitinib was found to hold some efficacy for treating the disease. The authors hypothesized that the primary therapeutic target of Sunitinib in AML is FLT3 kinase. However, FLT3 inhibition alone has not been shown to recapitulate all the effects of Sunitinib in vitro and, furthermore, the drug is also known to have cross reactivity to other potential oncogenic receptors. In this study, we treated three myeloid cell lines, Mono-Mac 1, THP-1 and U937 with Sunitinib and a proprietary cFMS inhibitor from Johnson and Johnson to test the anti-cancer effect in of such treatment. We observed that only Mono-Mac 1 cells had diminished proliferation in vitro. Mono-Mac 1 cells had inhibited ERK as a result of cFMS inhibition and showed a dose dependent increase in cFMS expression with both Sunitinib and J&J cFMS-1 treatment. Our results suggest potential for cFMS as an important target of Sunitinib or other similar drugs AML, either independently or in combination with other targets. Alternatively, cFMS may be a marker for differentiation of AML and may be linked with responsiveness to certain therapeutics. In both cases, the future study of cFMS may produce more targeted therapeutic approaches and may be a suitable tool for the development of personalized medicine for AML.

      Houser, Steven R.; Sabri, Abdelkarim; Gallucci, Stefania; Kishore, Raj; Lindsey, Merry (Temple University. Libraries, 2020)
      Acute damage to the heart, as in the case of myocardial infarction (MI), triggers a robust inflammatory response to the sterile injury and requires a complex and highly organized wound healing processes for survival. Cortical bone stem cell (CBSC) therapy has been shown to attenuate the decline in cardiac function associated with MI in both mouse and swine models. However, the cellular changes brought about by CBSC treatment and their relationship to inflammation and the wound healing process are unknown. We observed that CBSCs secrete paracrine factors known to have immunomodulatory properties, most notably Macrophage Colony Stimulating Factor (M-CSF) and Transforming Growth Factor-b, but not IL-4. Macrophages treated with CBSC medium containing these factors polarized to a hybrid M2a/M2c phenotype characterized by increased CD206 expression but not CD206 and CD163 co-expression, increased efferocytic ability, increased IL-10, TGF-b and IL-1RA secretion, and increased mitochondrial respiration in the absence of IL-4. Media from these macrophages increased proliferation and decreased a-Smooth Muscle Actin expression in fibroblasts in vitro. In addition, CBSC therapy increased macrophages, CD4+ T-cells, and fibroblasts while decreasing myocyte, macrophage, and total apoptosis in an in vivo swine model of MI. From these data, we conclude that CBSCs are modulating the immune response to MI in favor of an anti-inflammatory reparative response, ultimately reducing cell death and altering fibroblast populations resulting in smaller scar and preserved cardiac geometry and function.

      Autieri, Michael V.; Scalia, Rosario; Houser, Steven R.; Eguchi, Satoru; Soprano, Dianne R. (Temple University. Libraries, 2015)
      BACKGROUND: Despite aggressive dietary modification, lipid lowering medications, and other medical therapy, vascular proliferative diseases continue to account for 50% of all mortality in the United States. It is a significant medical and socioeconomic problem contributing to the mortality of multiple diseases including myocardial infarction (MI), stroke, renal failure, and peripheral vascular disease. With a growing number of children becoming obese and an increase in the number of patients with co-morbidities such as metabolic syndrome and Type 2 diabetes mellitus, epidemiological studies project the morbidity and mortality of these diseases to increase. Among these vascular proliferative diseases are primary atherosclerosis, vascular restenosis, and allograft vasculopathy, all of which are the result of chronic inflammation believed to stem from initial endothelial injury. Once activated by any number of potential injurious agents, endothelial cells (ECs) secrete cytokines that act on multiple cell types. Stimulation of resident vascular smooth muscle cells (VSMCs) results in a phenotypic switch from a normally contractile state to a proliferative state. Following this phenotypic shift, VSMCs migrate from the media to the intima of the artery where they begin secretion of both pro- and anti-inflammatory cytokines. Vascular proliferative disease ensues as a result of the autocrine and paracrine signaling of these cytokines between many different cell types including ECs, VSMCs, macrophages, and T-cells. As a result of the integral role pro- and anti-inflammatory cytokines play in the development of vascular proliferative diseases, they have become the subject of intense study in the field of cardiovascular research. Interleukin-19 (IL-19) is a newly described member of the IL-10 sub-family of anti-inflammatory cytokines. Discovered in 2000, it was originally only thought to be basally expressed in monocytes and lymphocytes, however in 2005 our lab discovered that while uninjured arteries have no detectable IL-19, arteries of patients with vascular proliferative diseases have notable IL-19 expression. Since its discovery in multiple cell types of injured arteries, our lab has subsequently shown that IL-19 inhibits proliferation, migration, spreading, production of reactive oxygen species (ROSs), and expression of pro-inflammatory genes in VSMCs, while in ECs IL-19 has been shown to promote angiogenesis, proliferation, migration, and spreading. AIMS and HYPOTHESIS: The first aim of the current study is to show that IL-19 is expressed in atherosclerotic plaque, and to test that IL-19 can reduce experimental atherosclerosis in susceptible mice. The second aim of the study is to show that IL-19 can regulate development of intimal hyperplasia in a murine model of restenosis. For both aims, we sought to identify potential intracellular signaling mechanisms of IL-19 which produce the observed effect. These aims directed our overall hypothesis that the anti-inflammatory properties of IL-19 can attenuate the vascular response to injury in various animal models of vascular proliferative disease. METHODS and RESULTS: The first aim of this dissertation showed that LDLR-/- mice fed an atherogenic diet and injected with either 1.0ng/g/day or 10.0ng/g/day rmIL-19 had significantly less plaque area in the aortic arch compared with controls (p<0.0001). Weight gain and serum lipid levels were not significantly different. IL-19 could halt, but not reverse expansion of existing plaque. Gene expression in splenocytes from IL-19 treated mice demonstrated immune cell Th2 polarization, with decreased expression of T-bet, IFNgamma, IL-1β and IL-12β, and increased expression of GATA3 messenger ribonucleic acid (mRNA). A greater percentage of lymphocytes were Th2 polarized in IL-19 treated mice. Cellular characterization of plaque by immunohistochemistry demonstrated IL-19 treated mice have significantly less macrophage infiltrate compared with controls (p<0.001). Intravital microscopy revealed significantly less leukocyte adhesion in wild-type mice injected with IL-19 and fed an atherogenic diet compared with controls. Treatment of cultured EC, VSMC, and bone marrow-derived macrophages (BMDM) with IL-19 resulted in a significant decrease in chemokine mRNA, and in the mRNA-stability protein HuR. In the second aim of this dissertation we showed that IL-19 attenuates vascular restenosis in response to carotid artery ligation. Carotid artery ligation of hyper-responsive friend leukemia virus B (FVB) wild-type mice injected with 10ng/g/day rIL-19 had significantly lower neointima/media ratio (I/M) compared with phosphate buffered saline (PBS) controls (p=0.006). Conversely, carotid artery of IL-19-/- mice demonstrated significantly higher I/M ratio compared with wild-type mice (p=0.04). Importantly, the increased I/M ratio in the knockout mice could be rescued by injection of 10ng/g/day IL-19 (p=0.04). VSMC explanted from IL-19-/- mice proliferated significantly more rapidly compared with wild-type (p=0.04). Surprisingly, in this model, IL-19 does not modulate adoptive immunity. Rather, addition of IL-19 to cultured wild-type VSMC did not significantly decrease VSMC proliferation, but could rescue proliferation in IL-19-/- VSMC to wild-type levels (p=0.02). IL-19-/- VSMC expressed significantly greater levels of inflammatory mRNA including IL-1β, TNFα, and MCP-1 in response to TNFα stimulation (p<0.01 for all). No polarization of adaptive immunity was noted in these mice. CONCLUSIONS: These data are the first to report that IL-19 is a potent inhibitor of experimental atherosclerosis via diverse mechanisms including immune cell polarization, decrease in macrophage adhesion, and decrease in gene expression. In addition, these data are also the first to show that IL-19 plays a previously unrecognized protective role in vascular restenosis. Together, these data suggest IL-19 is both anti-atherogenic and anti-restenotic and may identify IL-19 as a novel therapeutic to limit vascular inflammation.