Browsing by Subject "macrophages"
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Item Characterization of the effects of Interferon gamma protein-10 (CXCL10) against the protozoan parasite Leishmania amazonesis(2007-06-26) Rene Ernesto Vasquez; Lynn Soong; Thomas K. Hughes; Randall Goldblum; Johnny W. Peterson; David CorryLeishmania amazonensis causes progressive disease in most inbred strains of mice. We have previously shown that L. amazonensis-infected C57BL/6 mice have profound impairments in expression of pro-inflammatory cytokines, chemokines and in activation of antigen-specific CD4+ T cells. These impairments are independent of IL-4. The precise mechanism of pathogenesis associated with L. amazonensis infection remains largely unresolved. Since chemokines are essential mediators of leukocyte recruitment and effector cell function, we hypothesized that these molecules are important for the initiation of early responses locally and the eventual control of the infection. In this study, we found that CXCL10-treated bone marrow-derived macrophages from both BALB/c and C57BL/6 mice showed decreased numbers of L. amazonensis parasites, which was partially due to increased nitric oxide production, as well as elevated production of pro-inflammatory chemokines. When susceptible C57BL/6 mice were locally injected with CXCL10 following L. amazonensis infection, there was a significant delay in lesion development and reduction in parasite burdens, accompanied by a 7- and 3.5-fold increase in IFN-γ and IL-12 secretion, respectively, in re-stimulated lymph node cells. This study confirms that CXCL10 assists in the reduction of intracellular parasites. To address the mechanism underlying this enhanced immunity we utilized stationary promastigotes to infect bone marrow-derived DCs of C57BL/6 mice and assessed the activation of DC subsets and the capacity of these DC subsets in priming CD4+ T cells in vitro. We found that CXCL10 induced IL-12p40, but reduced IL-10 production in DCs. Yet, L. amazonensis-infected DCs produced elevated levels of IL-10, despite CXCL10 treatment. Elimination of endogenous IL-10 led to increased responsiveness to CXCL10 treatment, as judged by increased IL-12 production in DCs, as well as increased proliferation and IFN-γ production by CD4+ T cells. In addition, CXCL10-treated CD4+ T cells became more responsive to IL-12 via increased expression of the IL-12Rβ2 chain and produced elevated IFN-γ. This study indicates the interplay between CXCL10 and IL-10 in the generation of Th1-favored, pro-inflammatory responses and further highlights the utility of CXCL10 as a potential therapeutic for the control of non-healing cutaneous leishmaniasis.Item Mechanism of local IL-6 production and its role in accelerating vascular inflammation leading to aortic diseases(2008-12-03) Brian Cuong Tieu; Ronald Tilton; Steven Weinman; Michael Boulton; Dianna Milewicz; Darrell CarneyVascular inflammation plays a significant role in aortic diseases and involves enhanced recruitment and local activation of circulating monocytes along with cytokine production, but the mechanisms responsible for these processes are unclear. The cytokine interleukin-6 (IL-6) is highly induced in aortic aneurysm and dissection and significantly increases the risk of aneurysm rupture and mortality due to cardiovascular diseases; however, it remains unknown where and how IL-6 is produced in the vascular wall and how it contributes to disease exacerbation. Using an Ang II-infusion mouse model, we found that 6 days of subcutaneous Ang II infusion into aged C57BL/6J mice induced aortic IL-6 and MCP-1 predominantly in the tunica adventitia. Likewise, IL-6 was detected mostly in the adventitia of sporadic aortic dissections in humans. There was concomitant macrophage recruitment, adventitial expansion, and development of thoracic and suprarenal aortic aneurysms and dissections in treated wild-type mice. In contrast, no dissections were produced with infusion into IL-6-/- or CCR2-/- mice over the same time period along with significantly reduced inductions of aortic IL-6 and MCP-1. Using flow cytometric quantification of aortic cellular constituents, we found that Ang II induced CCR2+ macrophage accumulation of a specific CD14hiCD11bhiF4/80- phenotype selectively in aortic dissections and not in aortas from IL-6-/- mice, which were CD14loCD11bloF4/80+. Adoptive transfer of CCR2+/+ monocytes into CCR2-/- mice resulted in selective monocyte uptake into the thoracic and suprarenal aorta with restoration of IL-6 and MCP-1 secretion and increased incidence of dissection. To elucidate a source of IL-6, we demonstrated that aortic adventitial fibroblasts (AoAFs) highly produce IL-6 and MCP-1 and Ang II treatment increased their expression. Ang II and monocytes stimulated AoAF proliferation also. In addition, coculture of monocytes and AoAFs strongly potentiated MCP-1 and IL-6, which differentiated monocytes to macrophages and up-regulated CD14 and CD11b as well as induced MCP-1 and MMP-9 expression. These results suggest that AoAFs are a source of IL-6 and that a leukocyte-fibroblast interaction in the aortic adventitia potentiates its production, leading to promotion of local monocyte recruitment and activation, thereby accelerating vascular inflammation, ECM remodeling and aortic destabilization.Item microRNA-223 Regulates Macrophage Polarization and Diet-induced Insulin Resistance(2013-05-01) Meng, CongMacrophage activation plays a crucial role in regulating adipose tissue inflammation and is a major contributor to the pathogenesis of obesity-associated cardiovascular diseases. On various types of stimuli, macrophages respond with either classic (M1) or alternative (M2) activation. M1- and M2-mediated signaling pathways and corresponding cytokine production profiles are not completely understood. The discovery of microRNAs provides a new opportunity to understand this complicated but crucial network for macrophage activation and adipose tissue function. We have examined the activity of microRNA-223 (miR-223) and its role in controlling macrophage functions in adipose tissue inflammation and systemic insulinresistance. miR-223-/- mice on a high-fat diet exhibited an increased severity of systemic insulin resistance compared with wild-type mice that was accompanied by a marked increase in adipose tissue inflammation. The specific regulatory effects of miR-223 in myeloid cell-mediated regulation of adipose tissue inflammation and insulin resistance were then confirmed by transplantation analysis. Moreover, using bone marrow-derived macrophages, we demonstrated that miR-223 is a novel regulator of macrophage polarization, which suppresses classic pro-inflammatory pathways and enhances the alternative anti-inflammatory responses. In addition, we identified Pknox1 as a genuine miR-223 target gene and an essential regulator for macrophage polarization. For the first time, this study demonstrates that miR-223 acts to inhibit Pknox 1,suppressing pro-inflammatory activation of macrophages; thus, it is a crucial regulator of macrophage polarization and protects against diet-induced adipose tissue inflammatory response and systemic insulin resistance.Item Parasite interactions with dendritic cells and macrophages: Implications for cutaneous Leishmaniasis caused by Leishmania amazonensis(2003-03-26) Hai Qi; Lynn Soong; Vivian L. Braciale; Rolf Konig; Joseph M. Vinetz; David M. Mosser; Barbara L. DoughtyProtozoan Leishmania is an important human pathogen that affects millions people worldwide. Investigation of experimental Leishmania infection in mice has been instrumental to our understanding of interactions between the parasite and the host immune system. Previous studies have established the model of Th1-Th2 paradigm: gamma interferon (IFN-g)-secreting Th1 cells protect the host from developing progressive diseases, while interleukin (IL)-4-producing Th2 cells drive the disease pathogenesis. Focused on L. amazonensis infection in mice, this dissertation study is mainly intended to understand the cellular mechanism underlying the generation of parasite-specific Th2 cells and to ascertain the role for IFN-g in parasite-macrophage interactions. We showed that L. amazonensis parasites infected and activated dendritic cells (DCs), a population of phagocytic antigen-presenting cells specialized in activating naïve T cells. We found that DCs from susceptible or resistant mice differentially responded to amastigotes in CD40-dependent cytokine production and that amastigote-infected DCs favor Th2 priming in susceptible but not resistant mice. IFN-g is believed to be crucial for activating macrophages to kill intracellular parasites such as L. major. However, we found that L. amazonensis amastigotes but not promastigotes could not only survive but also replicate better in IFN-g-activated macrophages. The promastigote was evidently killed in IFN-g-activated macrophages. On the other hand, macrophages activated with IFN-g and LPS were able to limit intracellular amastigote replication. When tested in vivo, endogenous IFN-g apparently exerted minimal effects on the course of amastigote infection. It is likely that IFN-g plays a bidirectional role during L. amazonensis infection: when optimally coupled with other factors, it can activate macrophages to control parasite infection; while in the absence of such synergy, it would promote amastigote propagation by itself. Collectively, results presented in this dissertation have pointed to the unique ability of L. amazonensis amastigotes to modulate host immune system to the advantage of their own survival.Item The role of tetraspanin pretein CD63 in humna immunodeficiency virus type 1 infection and replication(2008-05-05) Hui Chen; Monique R. Ferguson; William A O'Brien; Richard SuttonHuman Immunodeficiency Virus (HIV) infection typically involves interaction of Env with CD4 and a chemokine coreceptor, either chemokine (C-C motif) receptor 5 (CCR5) or chemokine (C-X-C motif) receptor (CXCR4). Other cellular factors supporting HIV replication have recently been characterized. Previous works demonstrated a role for CD63 in early HIV infection events in macrophages via inhibition by pretreatment of an anti-CD63 antibody, which did not inhibit HIV replication in peripheral blood lymphocytes or in two CD4+ cell lines tested. To confirm the requirement for CD63 in HIV replication, CD63 expression in cells was decreased by RNA interference using short interfering RNAs (siRNA). Inhibition of HIV replication was demonstrated in macrophages following CD63-specific siRNA treatment. The inhibition effect was also shown when the anti-CD63 antibody treatment was delayed 12 hours after HIV infection, or by CD63 siRNA treatment 72 hours post infection, suggesting that late HIV replication events may also be affected. In U373-MAGI cells engineered to stably express either CCR5 or CXCR4, CD63-specific siRNA treatment resulted in over 90% reduction in CD63, which was associated with decreased HIV replication, even though these cells were refractory to HIV inhibition by the anti-CD63 antibody treatment. Using an R5/X4 HIV-89.6, CD63 downregulation was shown to decrease HIV replication in the U373-MAGI cells expressing either CCR5 or CXCR4. Although anti-CD63 antibody was previously shown to inhibit early HIV infection events only in macrophages, current progress shows a role for CD63 in HIV replication in CD4+ cell lines. Further delineation of the role of CD63 in HIV replication may lead to development of novel therapeutic compounds.Item The role of CCL5 (RANTES) in the immune response against Mycobacterium tuberculosis in the guinea pig(Texas A&M University, 2005-02-17) Skwor, Troy ArthurTuberculosis is the second leading cause of morbidity and mortality worldwide due to an infectious disease. Development of a new tuberculosis (TB) vaccine would be facilitated by a better understanding of the mechanisms of protection induced by the current TB vaccine, Mycobacterium bovis BCG. Recombinant guinea pig (rgp)CCL5 and anti-rgpCCL5 were developed and characterized. The biological activity of rgpCCL5 was determined in a chemotaxis assay using T lymphocytes and pleural exudate cells. The specificity of rabbit anti-rgpCCL5 polyclonal antibody was confirmed by Western blot. RgpCCL5 was used to stimulate alveolar and peritoneal macrophages in vitro. and cytokine/chemokine gene expression was evaluated using real-time PCR. RgpCCL5 stimulated TNFα, IL-1β, CCL2, and CXCL8 mRNA expression and TNFα protein production (as assessed in the L929 cell bioassay) in macrophages. The effect of BCG-vaccination on CCL5 expression and production in leukocytes infected with M. tuberculosis was examined in vitro and in vivo. Polyclonal anti-rgpCCL5 was used to develop an ELISA assay to quantify gpCCL5 protein levels, and real-time PCR was used to detect CCL5 mRNA. Leukocytes isolated from BCG-vaccinated guinea pigs and infected in vitro with virulent M. tuberculosis demonstrated significantly elevated gpCCL5 mRNA and protein compared to cells from naive animals. The response of gpCCL5 to M. tuberculosis in vivo was studied in tuberculous pleural effusions, where peak levels of CCL5 mRNA and protein were reached at day 4 post-induction. Disease severity, cellular differentiation, histology, and cytokine/chemokine mRNA levels in pleural cells and granulomas were analyzed on day 4 in guinea pigs induced with tuberculous pleurisy and treated with either rgpCCL5 or anti-rgpCCL5 by direct intra-pleural injection. In these studies, neutralizing CCL5 resulted in reduced macrophage accumulation, diminished levels of IFNγ, TNFα, and CCL5 mRNA in pleural effusion cells, and reduced spontaneous lymphocyte proliferation. Together these studies suggest an important role for gpCCL5 in activating leukocytes during M. tuberculosis infection.