Human macrophages differentiated in the presence of vitamin D3 restrict dengue virus infection and innate responses by downregulating mannose receptor expression

ABSTARCT: Severe dengue disease is associated with high viral loads and overproduction of pro-inflammatory cytokines, suggesting impairment in the control of dengue virus (DENV) and the mechanisms that regulate cytokine production. Vitamin D3 has been described as an important modulator of immune responses to several pathogens. Interestingly, increasing evidence has associated vitamin D with decreased DENV infection and early disease recovery, yet the molecular mechanisms whereby vitamin D reduces DENV infection are not well understood. METHODS AND PRINCIPAL FINDINGS: Macrophages represent important cell targets for DENV replication and consequently, they are key drivers of dengue disease. In this study we evaluated the effect of vitamin D3 on the differentiation of monocyte-derived macrophages (MDM) and their susceptibility and cytokine response to DENV. Our data demonstrate that MDM differentiated in the presence of vitamin D3 (D3-MDM) restrict DENV infection and moderate the classical inflammatory cytokine response. Mechanistically, vitamin D3-driven differentiation led to reduced surface expression of C-type lectins including the mannose receptor (MR, CD206) that is known to act as primary receptor for DENV attachment on macrophages and to trigger of immune signaling. Consequently, DENV bound less efficiently to vitamin D3-differentiated macrophages, leading to lower infection. Interestingly, IL-4 enhanced infection was reduced in D3-MDM by restriction of MR expression. Moreover, we detected moderate secretion of TNF-α, IL-1β, and IL-10 in D3-MDM, likely due to less MR engagement during DENV infection. CONCLUSIONS/SIGNIFICANCE: Our findings reveal a molecular mechanism by which vitamin D counteracts DENV infection and progression of severe disease, and indicates its potential relevance as a preventive or therapeutic candidate

Innate immune responses of monocytes/macrophages to DENV-2 infection

The pathogenesis of severe DENV infection is characterized by a dysregulated inflammatory response and a transient increase in endothelial permeability. Monocytes and macrophages play an essential role in the innate immune response towards DENV and also represent an important target cell for virus replication. In this thesis, we characterized the mechanisms by which monocytes and macrophages sense and respond to DENV infection. Furthermore, we evaluated how natural, host-intrinsic immunomodulatory compounds such as Vitamin D (VitD3) and LL-37 influence the innate immune response of macrophages to DENV infection. Altogether, the studies presented in this thesis can be summarized in two key messages. First, TLR2 on CM plays an important role in DENV pathogenesis. Second, VitD3 and LL37 show both antiviral and immunomodulatory effects against DENV infection in human primary cells and show potential as therapeutic candidates

Regulation of innate immune responses in macrophages differentiated in the presence of vitamin D and infected with dengue virus 2

A dysregulated or exacerbated inflammatory response is thought to be the key driver of the pathogenesis of severe disease caused by the mosquito-borne dengue virus (DENV). Compounds that restrict virus replication and modulate the inflammatory response could thus serve as promising therapeutics mitigating the disease pathogenesis. We and others have previously shown that macrophages, which are important cellular targets for DENV replication, differentiated in the presence of bioactive vitamin D (VitD3) are less permissive to viral replication, and produce lower levels of pro-inflammatory cytokines. Therefore, we here evaluated the extent and kinetics of innate immune responses of DENV-2 infected monocytes differentiated into macrophages in the presence (D(3)-MDMs) or absence of VitD3 (MDMs). We found that D(3)-MDMs expressed lower levels of RIG I, Toll-like receptor (TLR)3, and TLR7, as well as higher levels of SOCS-1 in response to DENV-2 infection. D(3)-MDMs produced lower levels of reactive oxygen species, related to a lower expression of TLR9. Moreover, although VitD3 treatment did not modulate either the expression of IFN-α or IFN-β, higher expression of protein kinase R (PKR) and 2′-5′-oligoadenylate synthetase 1 (OAS1) mRNA were found in D(3)-MDMs. Importantly, the observed effects were independent of reduced infection, highlighting the intrinsic differences between D(3)-MDMs and MDMs. Taken together, our results suggest that differentiation of MDMs in the presence of VitD3 modulates innate immunity in responses to DENV-2 infection

HIV Infection of Macrophages Is Restricted by Mannose Receptor

HIV infects several cell types in the body including CD4+ T cells and macrophages. The viral accessory protein Vpr is highly conserved in primate lentiviruses and promotes infection of both T cells and macrophages in vivo by unknown mechanisms. Previous studies demonstrated that Vpr enhances expression of HIV Env in macrophages and that this effect accelerates viral spread in macrophage cultures. Interesting, Vpr did not significantly enhance HIV infection in T cell cultures. The discrepancy between Vpr’s effect on T cell infection in vivo and in vitro was, until recently, unexplained. In a series of studies, we determined that Vpr and its cellular co-factor, DCAF1, are necessary to enhance infection of macrophages and co- cultured CD4+ T cells. Remarkably, we found evidence that Vpr counteracted a macrophage-specific, intrinsic antiviral pathway that targeted Env protein and Env-containing virions to lysosomes. When infected macrophages were co-cultured with uninfected, autologous CD4+ T cells we observed efficient spread via virological synapses, structures that form when Env on an infected macrophage binds CD4 on an uninfected T cell. By enhancing Env expression, Vpr enhanced formation of virological synapses and viral spread from macrophages to T cells. We found that the restriction of Env we observed in macrophages was mediated through detrimental interactions between mannose residues on Env and the macrophage mannose receptor (MR). Vpr counteracted this effect by reducing transcription of MR. Silencing MR or genetically deleting mannose residues on Env rescued Env expression in HIV- 1-infected macrophages lacking Vpr and increased spread from macrophages to T lymphocytes. Surprisingly, these experiments also demonstrated that disrupting interactions between Env and MR reduced initial infection of macrophages by cell-free virus, indicating that Env’s interaction with MR promotes viral entry. Together these results reveal a Vpr-Nef-Env axis that hijacks a host defense mechanism to boost viral entry then disables MR to alleviate detrimental interactions that inhibit viral egress.PHDMicrobiology & ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163064/1/jaylubow_1.pd

Studies of effector functions in mycobacterium tuberculosis-infected macrophages with implications for host-directed therapies

Tuberculosis infection remains a global health problem and the emergence of multidrugresistant TB (MDR-TB) adds further challenges in the battle to stop the spread of infection. Macrophages are the primary host cells to be infected with Mycobacterium tuberculosis (Mtb), and are therefore key cells in the initial control of TB infection. However, to survive and persist in activated macrophages, Mtb has developed strategies to counteract antimicrobial immunity. This thesis work aimed to explore macrophage responses in Mtbinfected cells, the nature of innate effector mechanisms and how these can be enhanced. For this purpose, we used an in vitro macrophage infection model as well as an organotypic lung tissue model system. The thesis work is based on four papers, Study I and III, involved studies on the effects of the immunomodulatory compounds, vitamin D3 (VitD3) and phenylbutyrate (PBA), on the induction of antimicrobial peptides, primarily human cathelicidin LL-37, and the ability to kill intracellular Mtb. In Study I, we observed that Mtb can down-regulate the expression of LL-37 in infected macrophages, but this effect was effectively counteracted by treatment with VitD3 and/or PBA. PBA and/or VitD3 prevented intracellular Mtb growth via induction of LL-37 as well as activation of autophagy in Mtb-infected macrophages. In Study III, we further demonstrated that PBA+VitD3 enhanced intracellular Mtb killing of both MDR-TB and drug-susceptible strains and this effect was dependent on LL-37. In Study IV, we explored the role of VitD3 in polarization of macrophages in comparison to activation with conventional stimuli such as GM-CSF (polarization of inflammatory M1 macrophages) or MCSF (polarization of anti-inflammatory M2 macrophages). The findings from Study IV, suggested that polarization with VitD3 enhanced the ability of Mtb-infected macrophages to control intracellular Mtb growth in comparison to M1 and M2 subsets. Enhanced Mtb growth control was associated with elevated levels of pro-inflammatory cytokines and LL-37, but reduced Mtb-induced expression of the immunosuppressive enzyme IDO (indoleamine 2,3-dioxygenase). Study II, aimed to investigate the expression and function of matrix metalloproteinases (MMPs) in early TB granuloma formation using the organotypic lung tissue model that enabled studies on macrophage-Mtb interactions in a more physiological environment in tissue. In this study, we found that pre-treatment of macrophages with the global MMP inhibitor, marimastat, resulted in effective inhibition of TB granuloma formation, which was associated with reduced Mtb growth detected in the lung tissue model. Thus, reduced degradation of extracellular matrix proteins, could prevent Mtb multiplication and spread of the infection. The main conclusion from this thesis work is that immunomodulatory compounds with the ability to boost or block innate effector mechanisms in Mtb-infected macrophages may be used as adjunct host-directed therapies that could support standard anti-TB drugs to enhance clinical recovery from TB

Innate Immune Responses in HIV-1 Infected Macrophages

In this study M-CSF differentiated human monocyte derived macrophages were used to investigate HIV-1 interactions with macrophage innate immune responses. Macrophages may be an important host cell for HIV-1. HIV-1 can infect and replicate within these cells without causing host cell cytopathicity unlike in T cells. Macrophages also aid in the spread of the virus and are likely to act as a viral reservoir protected from antivirals and immune responses due to the unique localisation of the virus within these cells. Surprisingly HIV-1 infection has little effect on the steady state transcriptome of MDM and despite the role of macrophages to detect incoming pathogens; no innate immune response to HIV-1 could be detected, in contrast with other viruses tested. The lack of immune response was not due to active viral suppression and addition of exogenous IFN or activation of the innate immune response at the time of HIV-1 infection can restrict viral infection in these cells, despite HIV-1 having a full complement of accessory proteins known to counteract IFN inducible restriction factors. This HIV-1 restriction induced by IFN was long lasting, likely for the lifetime of the MDM. IFN treatment of MDM with established HIV-1 infection however only transiently suppressed viral replication. Comparisons of MDM with other cell types which do show an innate immune IFN response to HIV-1 showed that MDM have relatively low levels of TLR7 gene expression, suggesting that MDM may lack one of the PRRs for detecting HIV-1. HIV-1 infection of MDM was found to attenuate NFκB activation in response to TLR stimulation and this attenuation could be reversed by priming the MDM with IFNγ. However this attenuation of the NFκB signal did not translate into decreased protein expression for a selection of proinflammatory cytokines examined

Herpesvirus and HIV-1 co-infection of human macrophages

HSV-1 and HIV-1 co-infection of human macrophages represents a clinically relevant model with which to investigate the host-pathogen interactions between macrophages and viruses. In this thesis, I demonstrate that HSV-1 productively infects human monocyte derived macrophages, with associated cell death and type I IFN responses, and additionally, that a proportion of macrophages support latent HSV-1 infection. I de ne latency as the absence of lytic gene transcription, virion production and cell death, in the presence of persistent expression of the HSV-1 latency associated transcript (LAT). I also demonstrate that HSV-1 super-infection increases HIV-1 transcription, and that latent HSV-1 can be reactivated by HIV-1. HSV-1 latent infection of neurons is well established, but this is the rst report, to my knowledge, of HSV-1 latent infection of myeloid lineage cells. The potential for macrophage reservoirs of latent HSV-1 may be an important factor for the clinical management of persistent reactive HSV-1 disease. Furthermore, HIV-1 infection in vivo is known to increase the frequency of HSV-1 reactivation in the host through the indirect mechanism of immune system suppression. However, a direct interaction between cells latently infected with HSV and HIV-1 has not previously been observed. Reactivation of latent HSV by HIV-1 therefore provides a novel mechanism for the well-established clinical synergy between these viruses

Lipid-specific IgM antibodies in multiple sclerosis and viral immunity

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) and the leading cause of disability in young adults. Over the last 20 years, a plethora of drugs have been approved for the treatment of MS, many of which target the immune response to reduce CNS inflammation. Although effective, many of these therapies have been associated with an increased risk of developing progressive multifocal leukoencephalopathy (PML), a fatal infection of the CNS by the John Cunningham virus. As a result, these effective therapeutics are used with caution pending the establishment of reliable risk stratification tools. A recent association between intrathecal lipid-reactive IgM synthesis and reduced incidence of PML in natalizumab-treated MS prompted our investigation into the antiviral properties of lipid-reactive IgM antibodies. Using murine heterogenous CNS cultures, we investigated the antiviral properties of both human and mouse IgMs. Initial investigations focused on the sulfatide-reactive IgM O4, a major target of the IgM response in MS patients. Using microarray analysis, RT-qPCR, and fluorescence in situ hybridisation, alongside genetic knock-out cultures and a range of pharmacological inhibitors, it was deduced that O4 could stimulate microglia to upregulate interferon beta (IFN-β) in a cGAS-STING-dependent manner, triggering global interferon stimulated gene (ISG) expression in major cell-types of the CNS. This interferon (IFN) response limited the replication of two genetically unrelated viruses, with final experiments focused on developing an in vivo model to investigate these antiviral IgMs. The data presented in this thesis provide a plausible explanation for the association between intrathecal IgM synthesis and reduced incidence of PML in natalizumab-treated MS patients. This research will aid risk stratification of MS patients, allowing safe access to effective therapeutics. Further understanding of the mechanism of this antibody-mediated response could lead to the development of an antiviral therapy for use as a co-treatment in MS or as a broad-spectrum therapeutic for the treatment of viral encephalitis

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

This work was supported by the National Institute of General Medical Sciences [GM131919].In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.PostprintPeer reviewe