The Many Hosts of Mycobacteria 9 (MHM9): A conference report.

The Many Hosts of Mycobacteria (MHM) meeting series brings together basic scientists, clinicians and veterinarians to promote robust discussion and dissemination of recent advances in our knowledge of numerous mycobacterial diseases, including human and bovine tuberculosis (TB), nontuberculous mycobacteria (NTM) infection, Hansen's disease (leprosy), Buruli ulcer and Johne's disease. The 9th MHM conference (MHM9) was held in July 2022 at The Ohio State University (OSU) and centered around the theme of "Confounders of Mycobacterial Disease." Confounders can and often do drive the transmission of mycobacterial diseases, as well as impact surveillance and treatment outcomes. Various confounders were presented and discussed at MHM9 including those that originate from the host (comorbidities and coinfections) as well as those arising from the environment (e.g., zoonotic exposures), economic inequality (e.g. healthcare disparities), stigma (a confounder of leprosy and TB for millennia), and historical neglect (a confounder in Native American Nations). This conference report summarizes select talks given at MHM9 highlighting recent research advances, as well as talks regarding the historic and ongoing impact of TB and other infectious diseases on Native American Nations, including those in Southwestern Alaska where the regional TB incidence rate is among the highest in the Western hemisphere.

Consequential drug combinations for tuberculosis treatments.

Improved therapies for tuberculosis will require the careful revision of complex, multi-drug regimens. In this issue of Cell Systems, Larkins-Ford et al. integrate extensive dose-response measurements of drug combinations, in vivo animal data, and computational analysis to provide a new predictive framework for the prioritization of specific antitubercular drug regimens.

Potentiation of P2RX7 as a host-directed strategy for control of mycobacterial infection.

Mycobacterium tuberculosis is the leading worldwide cause of death due to a single infectious agent. Existing anti-tuberculous therapies require long treatments and are complicated by multi-drug-resistant strains. Host-directed therapies have been proposed as an orthogonal approach, but few have moved into clinical trials. Here, we use the zebrafish-Mycobacterium marinum infection model as a whole-animal screening platform to identify FDA-approved, host-directed compounds. We identify multiple compounds that modulate host immunity to limit mycobacterial disease, including the inexpensive, safe, and widely used drug clemastine. We find that clemastine alters macrophage calcium transients through potentiation of the purinergic receptor P2RX7. Host-directed drug activity in zebrafish larvae depends on both P2RX7 and inflammasome signaling. Thus, targeted activation of a P2RX7 axis provides a novel strategy for enhanced control of mycobacterial infections. Using a novel explant model, we find that clemastine is also effective within the complex granulomas that are the hallmark of mycobacterial infection.

An explant technique for high-resolution imaging and manipulation of mycobacterial granulomas.

A central and critical structure in tuberculosis, the mycobacterial granuloma consists of highly organized immune cells, including macrophages that drive granuloma formation through a characteristic epithelioid transformation. Difficulties in imaging within intact animals and caveats associated with in vitro assembly models have severely limited the study and experimental manipulation of mature granulomas. Here we describe a new ex vivo culture technique, wherein mature, fully organized zebrafish granulomas are microdissected and maintained in three-dimensional (3D) culture. This approach enables high-resolution microscopy of granuloma macrophage dynamics, including epithelioid macrophage motility and granuloma consolidation, while retaining key bacterial and host characteristics. Using mass spectrometry, we find active production of key phosphotidylinositol species identified previously in human granulomas. We also describe a method to transfect isolated granulomas, enabling genetic manipulation, and provide proof-of-concept for host-directed small-molecule screens, identifying protein kinase C (PKC) signaling as an important regulator of granuloma macrophage organization.

Elemental Ingredients in the Macrophage Cocktail: Role of ZIP8 in Host Response to Mycobacterium tuberculosis.

Tuberculosis (TB) is a global epidemic caused by the infection of human macrophages with the world's most deadly single bacterial pathogen, Mycobacterium tuberculosis (M.tb). M.tb resides in a phagosomal niche within macrophages, where trace element concentrations impact the immune response, bacterial metal metabolism, and bacterial survival. The manipulation of micronutrients is a critical mechanism of host defense against infection. In particular, the human zinc transporter Zrt-/Irt-like protein 8 (ZIP8), one of 14 ZIP family members, is important in the flux of divalent cations, including zinc, into the cytoplasm of macrophages. It also has been observed to exist on the membrane of cellular organelles, where it can serve as an efflux pump that transports zinc into the cytosol. ZIP8 is highly inducible in response to M.tb infection of macrophages, and we have observed its localization to the M.tb phagosome. The expression, localization, and function of ZIP8 and other divalent cation transporters within macrophages have important implications for TB prevention and dissemination and warrant further study. In particular, given the importance of zinc as an essential nutrient required for humans and M.tb, it is not yet clear whether ZIP-guided zinc transport serves as a host protective factor or, rather, is targeted by M.tb to enable its phagosomal survival.

Zinc Modulates Endotoxin-Induced Human Macrophage Inflammation through ZIP8 Induction and C/EBPß Inhibition.

Two vital functions of the innate immune system are to initiate inflammation and redistribute micronutrients in favor of the host. Zinc is an essential micronutrient used in host defense. The zinc importer ZIP8 is uniquely induced through stimulation of the NF-κB pathway by LPS in monocytes and functions to regulate inflammation in a zinc-dependent manner. Herein we determined the impact of zinc metabolism following LPS-induced inflammation in human macrophages. We observed that ZIP8 is constitutively expressed in resting macrophages and strikingly elevated following LPS exposure, a response that is unique compared to the 13 other known zinc import proteins. During LPS exposure, extracellular zinc concentrations within the physiological range markedly reduced IL-10 mRNA expression and protein release but increased mRNA expression of TNFα, IL-8, and IL-6. ZIP8 knockdown inhibited LPS-driven cellular accumulation of zinc and prevented zinc-dependent reduction of IL-10 release. Further, zinc supplementation reduced nuclear localization and activity of C/EBPß, a transcription factor known to drive IL-10 expression. These studies demonstrate for the first time that zinc regulates LPS-mediated immune activation of human macrophages in a ZIP8-dependent manner, reducing IL-10. Based on these findings we predict that macrophage zinc metabolism is important in host defense against pathogens.

CD36-Mediated Uptake of Surfactant Lipids by Human Macrophages Promotes Intracellular Growth of Mycobacterium tuberculosis.

Mycobacterium tuberculosis imposes a large global health burden as the airborne agent of tuberculosis. Mycobacterium tuberculosis has been flourishing in human populations for millennia and is therefore highly adapted to the lung environment. Alveolar macrophages, a major host cell niche for M. tuberculosis, are not only phagocytose inhaled microbes and particulate matter but are also crucial in catabolizing lung surfactant, a lipid-protein complex that lines the alveolar spaces. Because macrophage host defense properties can be regulated by surfactant and M. tuberculosis can use host lipids as a carbon source during infection, we sought to determine the receptor(s) involved in surfactant lipid uptake by human macrophages and whether the presence of those lipids within macrophages prior to infection with M. tuberculosis enhances bacterial growth. We show that preformed scavenger receptor CD36 is redistributed to the cell membrane following exposure to surfactant lipids and surfactant protein A. Subsequently, surfactant lipids and/or surfactant protein A enhance CD36 transcript and protein levels. We show that CD36 participates in surfactant lipid uptake by human macrophages, as CD36 knockdown reduces uptake of dipalmitoylphosphatidylcholine, the most prevalent surfactant lipid species. Finally, exposing human macrophages to surfactant lipids prior to infection augments M. tuberculosis growth in a CD36-dependent manner. Thus, we provide evidence that CD36 mediates surfactant lipid uptake by human macrophages and that M. tuberculosis exploits this function for growth.

ZIP8 regulates host defense through zinc-mediated inhibition of NF-κB.

Activation of the transcription factor NF-κB is essential for innate immune function and requires strict regulation. The micronutrient zinc modulates proper host defense, and zinc deficiency is associated with elevated inflammation and worse outcomes in response to bacterial infection and sepsis. Previous studies suggest that zinc may regulate NF-κB activity during innate immune activation, but a mechanistic basis to support this has been lacking. Herein, we report that the zinc transporter SLC39A8 (ZIP8) is a transcriptional target of NF-κB and functions to negatively regulate proinflammatory responses through zinc-mediated down-modulation of IκB kinase (IKK) activity in vitro. Accordingly, fetal fibroblasts obtained from Slc39a8 hypomorphic mice exhibited dysregulated zinc uptake and increased NF-κB activation. Consistent with this, mice provided zinc-deficient dietary intakes developed excessive inflammation to polymicrobial sepsis in conjunction with insufficient control of IKK. Our findings identify a negative feedback loop that directly regulates innate immune function through coordination of zinc metabolism.