Cell Biology & Molecular Genetics
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Item Simultaneous transcriptional profiling of Leishmania major and its murine macrophage host cell reveals insights into host-pathogen interactions(Springer Nature, 2015-12-29) Dillon, Laura A. L.; Suresh, Rahul; Okrah, Kwame; Corrada Bravo, Hector; Mosser, David M.; El-Sayed, Najib M.Parasites of the genus Leishmania are the causative agents of leishmaniasis, a group of diseases that range in manifestations from skin lesions to fatal visceral disease. The life cycle of Leishmania parasites is split between its insect vector and its mammalian host, where it resides primarily inside of macrophages. Once intracellular, Leishmania parasites must evade or deactivate the host's innate and adaptive immune responses in order to survive and replicate. We performed transcriptome profiling using RNA-seq to simultaneously identify global changes in murine macrophage and L. major gene expression as the parasite entered and persisted within murine macrophages during the first 72 h of an infection. Differential gene expression, pathway, and gene ontology analyses enabled us to identify modulations in host and parasite responses during an infection. The most substantial and dynamic gene expression responses by both macrophage and parasite were observed during early infection. Murine genes related to both pro- and anti-inflammatory immune responses and glycolysis were substantially upregulated and genes related to lipid metabolism, biogenesis, and Fc gamma receptor-mediated phagocytosis were downregulated. Upregulated parasite genes included those aimed at mitigating the effects of an oxidative response by the host immune system while downregulated genes were related to translation, cell signaling, fatty acid biosynthesis, and flagellum structure. The gene expression patterns identified in this work yield signatures that characterize multiple developmental stages of L. major parasites and the coordinated response of Leishmania-infected macrophages in the real-time setting of a dual biological system. This comprehensive dataset offers a clearer and more sensitive picture of the interplay between host and parasite during intracellular infection, providing additional insights into how pathogens are able to evade host defenses and modulate the biological functions of the cell in order to survive in the mammalian environment.Item ALTERED PORCINE ALVEOLAR MACROPHAGE PHENOTYPE BY ALL-TRANS RETINOIC ACID AND ASCARIS SUUM INFECTION(2011) Perry, Trinity Lynn; Mosser, David M; Urban, Joseph F; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ascaris suum is known to cause ascariasis of both pigs and humans. Ascariasis is associated with a reduced control of intracellular pathogens and decreased vaccine efficacy. All-trans retinoic acid (ATRA), an active metabolite of Vitamin A (VA), is known to mediate a diverse array of physiological and biological processes including immune and inflammatory responses. The phenotype of alveolar macrophages (AM), the most abundant cell in the lung, has not been critically examined during chronic infection with A. suum nor has the effect of administration of dietary ATRA alone or during infection. This combination of treatments is likely common in underdeveloped regions where infection with Ascaris is prevalent and public health initiatives to control nutrient deficiencies often include VA supplementation. In this study we showed that a low dose and repeated "trickle" infection with A. suum alone and with ATRA administration altered the AM cell surface antigen phenotype.Item The Role of Interleukin-19 in Interleukin-10 Production by Regulatory Macrophages(2010) Yahil, Ron Jonathan; Mosser, David M.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Interleukin-19 (IL-19) is a recently discovered member of the IL-10 family of Class II cytokines. Although it is known to be secreted by monocytes and has been associated with various models of disease, the biological function of IL-19 remains largely unknown. IL-19 does share many important characteristics with IL-10. Because of this, we hypothesized that IL-19 may be regulated in a manner similar to IL-10, and may provide insight into the molecular mechanism of IL-10 regulation. In addition, IL-19 has been reported to increase IL-10 production in monocytes, and we theorized that it may be able to do the same in macrophages. Like IL-10, IL-19 is expressed in regulatory macrophages. Also, IL-19 is itself able to increase IL-10 production in regulatory macrophages, and the mechanism is independent of ERK phosphorylation. This work suggests that IL-19 can play a central role in the anti-inflammatory processes of IL-10.Item Molecular Mechanisms of the Inhibition of Apoptosis by Mycobacterium tuberculosis(2009) Miller, Jessica Lynn; Briken, Volker; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The capacity of infected cells to undergo apoptosis upon insult with a pathogen is an ancient innate immune defense mechanism. Consequently, the ability of persistent intracellular pathogens, such as the human pathogen Mycobacterium tuberculosis (Mtb), to inhibit infection-induced apoptosis of macrophages is important for virulence and to achieve persistence in the host. The nuoG gene of Mtb, which encodes the NuoG subunit of the type I NADH dehydrogenase NDH-1, is important in Mtb-mediated inhibition of host macrophage apoptosis. Here I determine the molecular mechanisms of this host-pathogen interaction. Apoptosis induced by the nuoG deletion mutant (nuoG ) is caspase-8 and TNF-α dependent. This cell death was also reduced in the presence of neutralizers and inhibitors of reactive oxygen species (ROS) and in macrophages derived from NOX2 deficient mice, suggesting that DnuoG induced death is dependent upon NOX2 derived ROS. Correlatively, nuoG infected macrophages also produced more phagosomal ROS than those infected with Mtb, or cells derived from NOX2 deficient mice. NuoG also inhibited apoptosis in human alveolar macrophages in a NOX2 dependant manner. These data suggest that reduction of phagosomal ROS is important for inhibition of apoptosis. Consistent with this hypothesis, Mtb deficient in the ROS neutralizing catalase, KatG, also accumulated ROS in the phagosome and was pro-apoptotic in macrophages. The specific mechanism by which NuoG reduces phagosomal ROS is still unknown. We could not detect secretion of NuoG, so direct neutralization of ROS is unlikely. Interestingly, preliminary data suggests that nuoG may be defective in secretion of SodA and KatG, enzymes known to be important for neutralizing ROS. In conclusion, these studies revealed that Mtb inhibits macrophage apoptosis by neutralizing phagosomal ROS via the NuoG dependent secretion of SodA and KatG. Furthermore, this research suggests a novel function for NOX2 activity in innate immunity, which is the sensing of persisting intracellular pathogens and subsequent induction of host cell apoptosis as a second line of defense for pathogens resistant to the respiratory burst.Item The Influence of Parasite-derived Chemokines in Leishmaniasis(2006-12-13) Conrad, Sean Martin; Mosser, David M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Transgenic chemokine-secreting parasites were generated and used to actively recruit immune cells into Leishmania lesions. It was hypothesized that the chemokine induced cell migration would influence the magnitude and character of the immune response and thereby effect the outcome of disease. Two different transgenic chemokine-secreting parasites were engineered. One transgenic parasite secretes murine MCP-1, a CC chemokine primarily responsible for macrophage recruitment. The other transgenic parasite secretes murine IP-10, a CXC chemokine known to attract activated T-cells. Both transgenic parasites transcribed murine chemokine mRNA, translated murine chemokine protein, and infected and replicated inside resting peritoneal macrophages similar to wild-type parasites. However, the two transgenic parasites caused diverse phenotypes in infected mice. The MCP-1 secreting parasites caused little or no detectable lesions, while the IP-10 secreting parasites caused lesions that were significantly larger than the wild-type infected mice. The healing phenotype caused by MCP-1 secreting parasites was further analyzed. Infection of BALB/c, C57BL/6, or MCP-1 knockout (KO) mice with MCP-1 secreting parasites resulted in minimal lesion development compared to mice infected with wild-type parasites. MCP-1 secreting parasites caused substantial lesions with relatively high numbers of parasites in CCR2 KO mice indicating that the parasites are viable and healthy, and that the lack of lesion development is CCR2- dependent. The enumeration of transgenic MCP-1 parasites in lesions demonstrated a significant reduction in parasite numbers, which coincided with an increase in CCR2-positive macrophage migration on day 7. CCR2-positive macrophages isolated from ears of mice infected with transgenic MCP-1 parasites contained virtually no parasites, whereas infection with wild-type parasites resulted in heavily-infected macrophages in lesions. The lack of parasite survival in mice infected with MCP-1 secreting parasites suggests that parasite-derived MCP-1 is recruiting a population of CCR2-positive macrophages to the lesion that efficiently kill Leishmania parasites. In-vitro studies revealed that optimal parasite killing required the recruitment of CCR2-positive macrophages followed by stimulation with a combination of both MCP-1 and IFN-g. This work suggests that the parasite-derived MCP-1 can recruit a restrictive population of CCR2-positive macrophages into lesions that can be optimally stimulated by MCP-1 and IFN-g to efficiently kill Leishmania parasites.