Search

Mycobacterium tuberculosis (Mtb) and Human Immunodeficiency virus (HIV) are important causes of death in infected patients worldwide, and act synergistically to worsen disease in those with co-infection. C-type lectin receptors (CLRs) are an important component of innate immunity that can recognize and respond to pathogen-associated signals. The macrophage galactose-type lectin (MGL, CLEC10) has been described as an immunomodulatory CLR associated with M2 macrophages and regulation of inflammation following infection. Our lab previously identified an important antibacterial role of murine MGL-1 in host immunity to Mtb. More recently we observed that human peripheral blood mononuclear cell-derived macrophages, and THP-1 cells, upregulated MGL following in vitro exposure to Mtb, Mycobacterium bovis BCG, and TLR2 agonists. Silencing of MGL in primary human macrophages permitted greater intracellular Mtb replication. Interestingly, we observed that MGL is suppressed by HIV in differential transcriptome analysis of lung from HIV-infected humanized mice, in lung and spleen of autopsy specimens from HIV-infected decedents, and upon in vitro infection of human macrophages. Using a recombinant human MGL/Fc chimeric molecule we observed that Mtb, and not HIV, directly bound MGL. MGL surface expression was reduced in lung of mice with chronic HIV infection compared to those with Mtb, and variable in co-infected animals. These results indicate that human MGL is an important CLR for pathogen recognition and initiation of protective immunity by host macrophages to Mtb. The suppression of MGL due to HIV may increase susceptibility to Mtb in people with HIV or exacerbate disease progression in those with co-infection. Supported by grants from NIH (R33 AI138328, R61 AI138328, U24 MH100930)

Cytokines, such as type-I Interferons (IFN-I), are essential to mount an effective antiviral response. The production of IFN-I is regulated by multiple mechanisms, including ubiquitination of host signaling factors. However, viruses can hijack the ubiquitin (Ub) system to promote viral replication. For example, we recently reported that the E3-Ub ligase TRIM7 ubiquitinates the envelope protein of Zika virus, which increases virus binding to its receptor favoring virus entry. Previous studies suggest that TRIM7 may play dual roles during infection by promoting or inhibiting IFN-I induction depending on the conditions. Here, we used our recently generated Trim7−/− mice to study its physiological role during SARS-CoV-2 (SCoV2) infection. We found that Trim7−/− mice have increased weight loss and lung virus titers compared to WT controls upon infection with SCoV2. We also found a dysregulated innate immune response, which included increased IFN-I induction and reduced neutrophil recruitment and production of inflammatory cytokines such as IL-6 in Trim7−/− mice. We observed a higher frequency of apoptotic cells in Trim7−/− compared to WT mice, specifically in CD45− lung epithelial cells. Mechanistically, using co-immunoprecipitation studies, we confirmed previous reports that TRIM7 interacts with the viral membrane protein (M), which can cause apoptosis. Using recombinant mutants that lack ubiquitination sites on M, we found that TRIM7-mediated ubiquitination of M protects cells against death. This suggests that TRIM7 restricts SCoV2 infection via a novel non-degradative mechanism involving ubiquitination of M. Understanding the role of TRIM7 in inhibiting SCoV2 replication could help develop treatments for COVID-19. Supported by grants from NIH R01 AI166668

Tuberculosis (TB) kills an estimated 1–2 million people per year and is the leading cause of death in people with HIV (PWH). The CD4+ T helper (Th) populations play significant roles in protective immunity to Mycobacterium tuberculosis (Mtb), and are also important host cells for HIV proliferation and persistence. Emerging evidence in PWH suggests that Th17 and Th22 cells may be preferentially depleted during HIV infection, an outcome that could pose a risk for Mtb containment during co-infection. We employed our humanized mouse model of co-infection to assess pulmonary and peripheral changes in Th17 and Th22 frequency and function due to infection with HIV, Mtb, or both. As determined with multi-plex ELISA, co-infection with HIV suppressed the cytokine response to Mtb including IL-17A, IL-22, IL-6, and IL-1β. Multi-parameter flow cytometric analysis revealed that Mtb and HIV/Mtb infection led to increased Th17+ cells in lung and decreased Th17+ cells in spleen. Following HIV or HIV/Mtb infection, Th22+ cells were significantly decreased in both lung and spleen. Importantly, HIV preferentially infected Th17 cells compared to Th22 and other Th subpopulations. Greater HIV viral load was also observed in the lung, but not spleen, of animals with Mtb/HIV co-infection compared to those with HIV mono-infection. Overall, these results suggest that HIV may compromise Th22 immunity, and exploit Th17+ cells to promote viral pathogenesis, in the setting of Mtb and HIV co-infection. Y.B. Martinez-Martinez is supported by Conacyt-I2T2 in Mexico, Contex, and the James W. McLaughlin Fellowship Fund. Grant support was provided by NIH R01AI147948 award to J. Endsley

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is responsible for approximately 10 million new infections and 1.5 million deaths each year. Pattern recognition receptors (PRRs) expressed on the surface of macrophages play an important role in orchestrating the immune response to pathogens. Amongst these, the C-type lectin receptors (CLRs) are an important PRR system that is increasingly understood to mediate innate immunity to mycobacteria. We recently described a new anti-bacterial and anti-inflammatory role for the macrophage galactose-type lectin-1 (MGL-1) in a murine model of experimental TB. MGL is a type II CLR that is known to recognize galactose and N-acetylgalactosamine monosaccharides. We observed that exposure to Mtb activated expression of both murine MGL orthologues, MGL-1 and MGL-2. The specific pathogen-associated molecular patterns that activate MGL orthologue transcription, however, have yet to be determined. Here we demonstrate that expression of MGL-1 and MGL-2 are differentially regulated by cytokines, TLR agonists, and mycobacterial ligands. MGL-1 expression is activated by dexamethasone, IL-4, IL-10, and the mycobacterial cell wall glycolipid Trehalose 6,6′-dimycolate. In contrast, MGL-2 expression is activated by TLR2/6 and TLR2/1 agonists and total lipid extracts of mycobacteria. Use of a MyD88 inhibitor further showed that molecular regulation of MGL-1 and MGL-2 transcription, following exposure of macrophages to mycobacteria, is TLR pathway-dependent. These results identify overlapping and unique host immune pathways that differentially activate MGL orthologues following exposure to Mtb and could be targeted through immune modulation.

The study of HIV infection and pathogenicity in physical reservoirs requires a biologically relevant model which resembles the human immune system and physiology. The human immune system (HIS) mouse is well established as a model of HIV, but defects in immune tissue reconstitution remain a challenge for examining pathology in tissues. Herein we show that exogenous injection of the human cytokine FLT-3L into the HSC cord blood HIS mouse model significantly expanded the total area of axial lymph nodes and the circulating percentage of human T cells, thus enabling us to visualize and quantify HIV infectivity in the secondary lymphoid tissues of the spleen and axial node. Further, we detected cell death and T cell depletion in tissues, consistent with HIV pathogenesis. Treatment with the caspase-1 inhibitor VX-765 significantly decreased viral load as measured by gag qPCR, and apoptosis as measured by TUNEL assay, in the spleen. A significant restoration of CD4+ T cells in the spleen due to VX-765 treatment was observed via flow cytometry. In situ hybridization further demonstrated a significant decrease in viral RNA in both the spleen and axial lymph nodes. Transcriptomic analysis further revealed an upregulation in host HIV restriction factors such as APOBR, SAMHD1 and APOBEC3A as a result of VX-765 administration. These findings demonstrate FLT-3L as a mechanism whereby the human immune environment in HIS mice can be enhanced to support investigations of HIV pathogenesis and immune outcomes in tissue compartments. Preliminary results indicate that targeting inflammasome pathways with VX-765 in HIS mice treated with FLT-3L, and infected with HIV, preserved T cell populations and decreased viral load. Supported by R01 A1HL129881, NIH/NHLBI

Pattern recognition receptors, such as the C-type lectin receptor (CLR) family, play an important role in the generation of the innate immune response against a variety of pathogens. The CLR Macrophage galactose-type lectin (MGL), also known as CLEC10, is one member of this family which we and others found can bind SARS-CoV-2. MGL is known to increase in expression during SARS-CoV-2 infection and, in concert with other CLRs, is associated with elevated proinflammatory immune responses. Dexamethasone, a clinical treatment commonly used to mitigate the hyperinflammatory response in individuals with COVID-19, is also a positive stimulus for MGL expression. We therefore sought to investigate the role of MGL in disease pathogenesis using MGL-1 receptor deficient mice. Mice were intranasally infected with mouse adapted SARS-CoV-2 strain CMA3p20 and euthanized 3- or 7-days post infection. Following acute infection at 3 days, MGL-1 deficiency did not affect the viral burden in the lungs of mice. This was supported by multiplex cytokine and chemokine data which revealed similar acute inflammatory responses in the lung of infected WT and MGL-1 knockout (KO) mice. Interestingly, MGL-1 KO mice did display a significant increase in the lung viral burden when compared to WT mice 7 days post-infection. These results suggest a role for MGL-1 in resolution of SARS-CoV-2 at later stages of infection. Further investigations to determine differences in cellular recruitment and lung pathology in the absence of MGL-1 are warranted. In addition, assessment of a role for the MGL-2 orthologue expressed by murine myeloid cells may identify roles for MGL pathways in SARS-CoV-2 disease severity and pathogenesis. Supported by UTMB Institute for Human Infection and Immunity COVID-19 Pilot Grant

In the era of highly active antiretroviral therapy, HIV infection still produces 800,000 deaths and 1.2 million new infections per year. People living with HIV (PLWH) are vulnerable to several co-morbidities and co-infections, due to depletion of CD4+T cells and other incompletely characterized immune disturbances. To address the mechanisms involved, lung mRNAs from humanized mice co-infected with HIV and Mycobacterium tuberculosis (Mtb) were screened. Macrophage Galactose-type Lectin (MGL) was differentially regulated in co-infected animals. When MGL protein was quantified in biobanked frozen human lung specimens from 47 HIV-infected decedents, it was lower in the lung specimens with actively replicating HIV-1, as compared to those lacking detectable viral load. Lung MGL levels were negatively correlated with HIV RNA copies in the lung, and in the spleen, which is a key lymphoid organ that influences systemic immunity. Peripheral blood leukocytes from HIV infected patients showed that MGL activation was abnormal in those cells; comparable results were obtained in cultured HIV-infected macrophages. Molecular silencing of MGL in human monocyte-derived macrophages in vitro produced increased growth of Mtb, suggesting that lower MGL expression in PLWH could compromise their innate immunity to opportunistic pathogens such as Mtb. Macrophage MGL is a novel target to elucidate how HIV infection disrupts innate immunity and increases vulnerability to tuberculosis.

Pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is characterized by inflammatory pathology that is regulated by poorly understood mechanisms of innate immunity. Pattern recognition receptors (PRR) expressed on the surface of macrophages regulate the balance of inflammatory and antimicrobial functions that determine disease outcome. Our work identifies a novel role for the C-type lectin receptor Macrophage Galactose-type Lectin (MGL) PRR in a murine model of experimental tuberculosis. Bone marrow derived macrophages (BMDM) upregulate MGL-1, but not MGL-2, following in vitro exposure to both heat killed and live Mtb. Macrophages from MGL-1-deficient mice display greater lipid accumulation, and increased production of pro-inflammatory cytokines (IL-1b, IL-6 and IFN-g). Mtb growth is also increased in MGL−/− BMDM, independent of the inflammatory outcomes and production of reactive nitrogen species. TB in mice lacking MGL-1 results in increased pro-inflammatory cytokines, more diffuse granulomatous inflammation, and increased accumulation of foamy macrophages. Assessment of disease kinetics suggests that MGL has an important role for controlling mycobacterial burden during the establishment stages of TB disease, and has less influence once the chronic disease stage is established. Macrophages in murine and human TB granulomas contain abundant MGL associated with inflammatory foci. These results suggest that MGL is a potential new therapeutic target against TB.

Human immunodeficiency virus (HIV) is the world’s sixth leading cause of death. Antiretroviral therapy (ART) is the current standard of treatment, but issues of accessibility and compliance pose additional hurdles. In 2018, there were 37.9 million people living with HIV, but only 62% were on ART. The development of a successful vaccine is imperative to combat the transmission of HIV, of which one of the major goals is to generate broadly neutralizing antibodies (bnAb). The membrane-proximal external region (MPER) of gp41 in the envelope glycoprotein (Env) is a largely conserved motif, a key player in fusion, and the target of bnAb in a subset of patients with long-term HIV infections. We previously demonstrated the efficacy of a nanofiber vaccine platform as evidenced by the generation of both strong humoral and cell-mediated responses against viral targets, and enhanced protection against infection. In recent studies, we observed that serial oral dosing of nanofibers (NF) bearing the MPER epitope (NF-MPER) in mice correlated with a significant increase in serum and mucosal neutralizing antibodies against HIV. The NF-MPER vaccine also augmented CD8+ memory T cell populations in the colon, which were further characterized as effector memory T cells (TEM), using surface marker phenotypes (CD69+/− CD103+ CD44+ CD62Llo). Additionally, we observed significant expansion of CD8+ TEM pools in the spleen, a reservoir for HIV, a subset of which were similar to tissue resident memory T cells (TRM) seen in human elite controllers. These results suggest that oral immunization with a NF-based vaccine elicits the production of bnAb and enhanced CD8+ TEM populations, in non-lymphoid and lymphoid tissues, that are necessary for a successful HIV vaccine.

Natural Killer cells (NK) are important in both antiviral, bacterial and tumor defense and constitute a major effector population of the innate immune defense. Activation of NK cells by specific cell surface expressed proteins in infected or tumor cells can induce the production of IFNg and cytotoxicity through release of cytotoxic granules such as perforin and granulysin. Bovine NK cells were recently phenotypically characterized but selective activation via cell surface receptors is not fully understood. Treatments using either inhibitory or stimulatory receptors that activate NKs can be used as a target for vaccine formulation against important bovine and human pathogens. In the present study we used lymph node-derived bovine NK cells that were activated with different conditions including activation via CD244 AND CD16 resulting in increasing IFNg and Perforin. Our studies demonstrate that specific in vitro activation conditions of purified bovine NK cells results in a rapid response via secretion of IFNg and cytotoxic granules. Insight into selective NK functional activities can be used in the development of better vaccines against rapidly replicating pathogens such as foot and mouth disease virus. keywords: NK cells, Bovine, CD16 Receptor, CD244 Receptor