Publications

Coinfection of Mycobacterium tuberculosis (Mtb) and human immunodeficiency virus-1 (HIV) is a significant public health concern. Treatment is challenging due to prolonged duration of therapy and drug interactions between antiretroviral therapy (ART) and anti-TB drugs. Noniron gallium meso-tetraphenyl porphyrin (GaTP), a heme mimetic, has shown broad antimicrobial activity. Here, we investigated the efficacy of nanoparticle encapsulating GaTP (GaNP) for the treatment of HIV and Mtb coinfection or single infection in in vitro granuloma structures. GaNP significantly reduced viable Mtb within primary human in vitro granuloma structures infected with Mtb H37Rv-lux and significantly reduced levels of HIV in CD4+ T cells infected with the virus axenically. Similarly, GaNP exhibited significant antimicrobial activity against HIV/Mtb-coinfected granuloma structures created in vitro, which contain the primary immune cells seen in human TB granulomas, including CD4+ T cells and macrophages, as assessed by a luciferase assay for Mtb and p24 ELISA for HIV detection. Furthermore, mechanistic studies revealed that GaTP increases the level of reactive oxygen species and inhibits catalase in Mtb. A significant increase in Mtb nitrate reductase activity was also observed when Mtb was incubated with GaTP and sodium nitrate. Overall, increased oxidative stress and nitrite levels induced by GaTP are consistent with the possibility that GaTP inhibits Mtb aerobic respiration, which leads to incomplete O2 reduction and a shift to respiration using exogenous NO3. These cumulative data continue to support the potential for developing the noniron heme analog GaTP and its nanoparticle GaNP as new therapeutic approaches for the treatment of HIV/Mtb coinfection.

With devastating health and socioeconomic impact worldwide, much work is left to understand the Coronavirus Disease 2019 (COVID-19), with emphasis in the severely affected elderly population. Here, we present a proteomics study of lung tissue obtained from aged vs. young rhesus macaques (Macaca mulatta) and olive baboons (Papio Anubis) infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using age as a variable, we identified common proteomic profiles in the lungs of aged infected non-human primates (NHPs), including key regulators of immune function, as well as cell and tissue remodeling, and discuss the potential clinical relevance of such parameters. Further, we identified key differences in proteomic profiles between both NHP species, and compared those to what is known about SARS-CoV-2 in humans. Finally, we explored the translatability of these animal models in the context of aging and the human presentation of the COVID-19.

Neutrophils are increasingly implicated in chronic inflammation and metabolic disorders. Here, we show that visceral adipose tissue (VAT) from individuals with obesity contains more neutrophils than in those without obesity and is associated with a distinct bacterial community. Exploring the mechanism, we gavaged microbiome-depleted mice with stool from patients with and without obesity during high-fat or normal diet administration. Only mice receiving high-fat diet and stool from subjects with obesity show enrichment of VAT neutrophils, suggesting donor microbiome and recipient diet determine VAT neutrophilia. A rise in pro-inflammatory CD4+ Th1 cells and a drop in immunoregulatory T cells in VAT only follows if there is a transient spike in neutrophils. Human VAT neutrophils exhibit a distinct gene expression pattern that is found in different human tissues, including tumors. VAT neutrophils and bacteria may be a novel therapeutic target for treating inflammatory-driven complications of obesity, including insulin resistance and colon cancer.

Human alveolar macrophages are a unique myeloid subset critical for understanding pulmonary diseases and are difficult to access. Here, we present a protocol to generate human alveolar macrophage-like (AML) cells from fresh peripheral blood mononuclear cells or purified monocytes. We describe steps for cell isolation, incubation in a defined cocktail of pulmonary surfactant and lung-associated cytokines, phenotype analysis, and validation with human alveolar macrophages. We then detail procedures for quality control and technical readouts for monitoring microbial response. For complete details on the use and execution of this protocol, please refer to Pahari et al.1 and Neehus et al.2.

The elderly population is highly susceptible to developing respiratory diseases, including tuberculosis, a devastating disease caused by the airborne pathogen Mycobacterium tuberculosis (M.tb) that kills one person every 18 seconds. Once M.tb reaches the alveolar space, it contacts alveolar lining fluid (ALF), which dictates host-cell interactions. We previously determined that age-associated dysfunction of soluble innate components in human ALF leads to accelerated M.tb growth within human alveolar macrophages. Here we determined the impact of human ALF on M.tb infection of alveolar epithelial type cells (ATs), another critical lung cellular determinant of infection. We observed that elderly ALF (E-ALF)-exposed M.tb had significantly increased intracellular growth with rapid replication in ATs compared to adult ALF (A-ALF)-exposed bacteria, as well as a dampened inflammatory response. A potential mechanism underlying this accelerated growth in ATs was our observation of increased bacterial translocation into the cytosol, a compartment that favors bacterial replication. These findings in the context of our previous studies highlight how the oxidative and dysfunctional status of the elderly lung mucosa determines susceptibility to M.tb infection, including dampening immune responses and favoring bacterial replication within alveolar resident cell populations, including ATs, the most abundant resident cell type within the alveoli.

AIM2 (absent in melanoma 2), an inflammasome component, mediates IL-1β release in murine macrophages and cell lines. AIM2 and IL-1β contribute to murine control of Mycobacterium tuberculosis (M.tb) infection, but AIM2's impact in human macrophages, the primary niche for M.tb, remains unclear. We show that M.tb, Mycobacterium bovis bacillus Calmette-Guérin (BCG), and M. smegmatis induce AIM2 expression in primary human macrophages. M.tb-induced AIM2 expression is peroxisome proliferator-activated receptor γ (PPARγ)-dependent and M.tb ESX-1-independent, whereas BCG- and M. smegmatis-induced AIM2 expression is PPARγ-independent. PPARγ and NLRP3, but not AIM2, are important for IL-1β release in response to M.tb, and NLRP3 colocalizes with M.tb. This is in contrast to the role for AIM2 in inflammasome activation in mice and peritoneal macrophages. Altogether, we show that mycobacteria induce AIM2 expression in primary human macrophages, but AIM2 does not contribute to IL-1β release during M.tb infection, providing further evidence that AIM2 expression and function are regulated in a cell- and/or species-specific manner.

We describe a human lung disease caused by autosomal recessive, complete deficiency of the monocyte chemokine receptor C-C motif chemokine receptor 2 (CCR2). Nine children from five independent kindreds have pulmonary alveolar proteinosis (PAP), progressive polycystic lung disease, and recurrent infections, including bacillus Calmette Guérin (BCG) disease. The CCR2 variants are homozygous in six patients and compound heterozygous in three, and all are loss-of-expression and loss-of-function. They abolish CCR2-agonist chemokine C-C motif ligand 2 (CCL-2)-stimulated Ca2+ signaling in and migration of monocytic cells. All patients have high blood CCL-2 levels, providing a diagnostic test for screening children with unexplained lung or mycobacterial disease. Blood myeloid and lymphoid subsets and interferon (IFN)-γ- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-mediated immunity are unaffected. CCR2-deficient monocytes and alveolar macrophage-like cells have normal gene expression profiles and functions. By contrast, alveolar macrophage counts are about half. Human complete CCR2 deficiency is a genetic etiology of PAP, polycystic lung disease, and recurrent infections caused by impaired CCL2-dependent monocyte migration to the lungs and infected tissues.

Tuberculosis is the leading cause of death for people living with HIV (PLWH). We hypothesized that altered functions of innate immune components in the human alveolar lining fluid of PLWH (HIV-ALF) drive susceptibility to Mycobacterium tuberculosis (M.tb) infection. Our results indicate a significant increase in oxidation of innate proteins and chemokine levels and significantly lower levels and function of complement components and Th1/Th2/Th17 cytokines in HIV-ALF versus control-ALF (non-HIV-infected people). We further found a deficiency of surfactant protein D (SP-D) and reduced binding of SP-D to M.tb that had been exposed to HIV-ALF. Primary human macrophages infected with M.tb exposed to HIV-ALF were significantly less capable of controlling the infection, which was reversed by SP-D replenishment in HIV-ALF. Thus, based on the limited number of participants in this study, our data suggest that PLWH without antiretroviral therapy (ART) have declining host innate defense function in their lung mucosa, thereby favoring M.tb and potentially other pulmonary infections.

Tuberculosis (TB) accounts for 1.6 million deaths annually and over 25% of deaths due to antimicrobial resistance. Mycobacterium tuberculosis (M.tb) drives MCL-1 expression (family member of anti-apoptotic BCL-2 proteins) to limit apoptosis and grow intracellularly in human macrophages. The feasibility of re-purposing specific MCL-1 and BCL-2 inhibitors to limit M.tb growth, using inhibitors that are in clinical trials and FDA-approved for cancer treatment has not be tested previously. We show that specifically inhibiting MCL-1 and BCL-2 induces apoptosis of M.tb-infected macrophages, and markedly reduces M.tb growth in human and murine macrophages, and in a pre-clinical model of human granulomas. MCL-1 and BCL-2 inhibitors limit growth of drug resistant and susceptible M.tb in macrophages and act in additive fashion with the antibiotics isoniazid and rifampicin. This exciting work uncovers targeting the intrinsic apoptosis pathway as a promising approach for TB host-directed therapy. Since safety and activity studies are underway in cancer clinics for MCL-1 and BCL-2 inhibitors, we expect that re-purposing them for TB treatment should translate more readily and rapidly to the clinic. Thus, the work supports further development of this host-directed therapy approach to augment current TB treatment.