Publications

Vaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease would benefit from validated small animal models. Here, we show that transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18 hACE2) represent a susceptible rodent model. K18 hACE2 transgenic mice succumbed to SARS-CoV-2 infection by day 6, with virus detected in lung airway epithelium and brain. K18 ACE2 transgenic mice produced a modest TH1/2/17 cytokine storm in the lung and spleen that peaked by day 2, and an extended chemokine storm that was detected in both lungs and brain. This chemokine storm was also detected in the brain at day 6. K18 hACE2 transgenic mice are, therefore, highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of vaccines (prophylactic) and antivirals (therapeutics) for SARS-CoV-2 infection and associated severe COVID-19 disease.

The elderly population is more susceptible to pulmonary infections, including tuberculosis. In this article, we characterize the impact of aging on the phenotype of mouse alveolar macrophages (AMs) and their response to Mycobacterium tuberculosis. Uninfected AMs were isolated from bronchoalveolar lavage of young (3 mo) and old (18 mo) C57BL/6 mice. AMs from old mice expressed higher mRNA levels of CCL2, IFN-β, IL-10, IL-12p40, TNF-α, and MIF than young mice, and old mice contained higher levels of CCL2, IL-1β, IFN-β, and MIF in their alveolar lining fluid. We identified two distinct AM subpopulations, a major CD11c+ CD11b- population and a minor CD11c+ CD11b+ population; the latter was significantly increased in old mice (4-fold). Expression of CD206, TLR2, CD16/CD32, MHC class II, and CD86 was higher in CD11c+ CD11b+ AMs, and these cells expressed monocytic markers Ly6C, CX3CR1, and CD115, suggesting monocytic origin. Sorted CD11c+ CD11b+ AMs from old mice expressed higher mRNA levels of CCL2, IL-1β, and IL-6, whereas CD11c+ CD11b- AMs expressed higher mRNA levels of immune-regulatory cytokines IFN-β and IL-10. CD11c+ CD11b+ AMs phagocytosed significantly more M. tuberculosis, which expressed higher RNA levels of genes required for M. tuberculosis survival. Our studies identify two distinct AM populations in old mice: a resident population and an increased CD11c+ CD11b+ AM subpopulation expressing monocytic markers, a unique inflammatory signature, and enhanced M. tuberculosis phagocytosis and survival when compared with resident CD11c+ CD11b- AMs, which are more immune regulatory in nature.

Nuclear receptors (NRs) are ligand-activated transcription factors that are expressed in a wide variety of cells and play a major role in lipid signaling. NRs are key regulators of immune and metabolic functions in macrophages and are linked to macrophage responses to microbial pathogens. Pathogens are also known to induce the expression of specific NRs to promote their own survival. In this review, we focus on the NRs recently shown to influence macrophage responses to Mycobacterium tuberculosis (M.tb), a significant cause of morbidity and mortality worldwide. We provide an overview of NR-controlled transcriptional activity and regulation of macrophage activation. We also discuss in detail the contribution of specific NRs to macrophage responses to M.tb, including influence on macrophage phenotype, cell signaling, and cellular metabolism. We pay particular attention to PPARγ since it is required for differentiation of alveolar macrophages, an important niche for M.tb, and its role during M.tb infection is becoming increasingly appreciated. Research into NRs and M.tb is still in its early stages, therefore continuing to advance our understanding of the complex interactions between M.tb and macrophage NRs may reveal the potential of NRs as pharmacological targets for the treatment of tuberculosis.

Nuclear receptors (NRs) are ligand-activated transcription factors that are expressed in a variety of cells, including macrophages. For decades, NRs have been therapeutic targets because their activity can be pharmacologically modulated by specific ligands and small molecule inhibitors. NRs regulate a variety of processes, including those intersecting metabolic and immune functions, and have been studied in regard to various autoimmune diseases. However, the complex roles of NRs in host response to infection are only recently being investigated. The NRs peroxisome proliferator-activated receptor γ (PPARγ) and liver X receptors (LXRs) have been most studied in the context of infectious diseases; however, recent work has also linked xenobiotic pregnane X receptors (PXRs), vitamin D receptor (VDR), REV-ERBα, the nuclear receptor 4A (NR4A) family, farnesoid X receptors (FXRs), and estrogen-related receptors (ERRs) to macrophage responses to pathogens. Pharmacological inhibition or antagonism of certain NRs can greatly influence overall disease outcome, and NRs that are protective against some diseases can lead to susceptibility to others. Targeting NRs as a novel host-directed treatment approach to infectious diseases appears to be a viable option, considering that these transcription factors play a pivotal role in macrophage lipid metabolism, cholesterol efflux, inflammatory responses, apoptosis, and production of antimicrobial byproducts. In the current review, we discuss recent findings concerning the role of NRs in infectious diseases with an emphasis on PPARγ and LXR, the two most studied. We also highlight newer work on the activity of emerging NRs during infection.

BACKGROUND: There are no effective treatments for Burkholderia cenocepacia in patients with cystic fibrosis (CF) due to bacterial multi-drug resistance and defective host killing. We demonstrated that decreased bacterial killing in CF is caused by reduced macrophage autophagy due to defective cystic fibrosis transmembrane conductance regulator (CFTR) function. AR-12 is a small molecule autophagy inducer that kills intracellular pathogens such as Francisella. We evaluated the efficacy of AR-12 and a new analogue AR-13 in reducing bacterial burden in CF phagocytes.

METHODS: Human CF and non-CF peripheral blood monocyte-derived macrophages, neutrophils, and nasal epithelial cells were exposed to CF bacterial strains in conjunction with treatment with antibiotics and/or AR compounds.

RESULTS: AR-13 and not AR-12 had growth inhibition on B. cenocepacia and methicillin-resistantStaphylococcus aureus (MRSA) in media alone. There was a 99% reduction in MRSA in CF macrophages, 71% reduction in Pseudomonas aeruginosa in CF neutrophils, and 70% reduction in non-CF neutrophils using AR-13. Conversely, there was no reduction in B. cenocepacia in infected CF and non-CF macrophages using AR-13 alone, but AR-13 and antibiotics synergistically reduced B. cenocepacia in CF macrophages. AR-13 improved autophagy in CF macrophages and CF patient-derived epithelial cells, and increased CFTR protein expression and channel function in CF epithelial cells.

CONCLUSIONS: The novel AR-12 analogue AR-13, in combination with antibiotics, reduced antibiotic-resistant bacterial burden in CF phagocytes, which correlated with increased autophagy and CFTR expression. AR-13 is a novel therapeutic for patients infected with B. cenocepacia and other resistant organisms that lack effective therapies.

Immunonutrition as a therapeutic approach is rapidly gaining interest in the fight against infection. Targeting l-arginine metabolism is intriguing, considering this amino acid is the substrate for antimicrobial NO production by macrophages. The importance of l-arginine during infection is supported by the finding that inhibiting its synthesis from its precursor l-citrulline blunts host defense. During the first few weeks following pulmonary mycobacterial infection, we found a drastic increase in l-citrulline in the lung, even though serum concentrations were unaltered. This correlated with increased gene expression of the l-citrulline-generating (i.e., iNOS) and l-citrulline-using (i.e., Ass1) enzymes in key myeloid populations. Eliminating l-arginine synthesis from l-citrulline in myeloid cells via conditional deletion of either Ass1 or Asl resulted in increased Mycobacterium bovis bacillus Calmette-Guérin and Mycobacterium tuberculosis H37Rv burden in the lungs compared with controls. Our data illustrate the necessity of l-citrulline metabolism for myeloid defense against mycobacterial infection and highlight the potential for host-directed therapy against mycobacterial disease targeting this nutrient and/or its metabolic pathway.

The mechanisms underlying abnormal granuloma formation in patients with sarcoidosis are complex and remain poorly understood. A novel in vitro human granuloma model was used to determine the molecular mechanisms of granuloma genesis in patients with sarcoidosis in response to putative disease-causing mycobacterial antigens. Peripheral blood mononuclear cells (PBMCs) from patients with active sarcoidosis and from normal, disease-free control subjects were incubated for 7 days with purified protein derivative-coated polystyrene beads. Molecular responses, as reflected by differential expression of genes, extracellular cytokine patterns, and cell surface receptor expression, were analyzed. Unbiased systems biology approaches were used to identify signaling pathways engaged during granuloma formation. Model findings were compared with human lung and mediastinal lymph node gene expression profiles. Compared with identically treated PBMCs of control subjects (n = 5), purified protein derivative-treated sarcoidosis PBMCs (n = 6) were distinguished by the formation of cellular aggregates resembling granulomas. Ingenuity Pathway Analysis of differential expression gene patterns identified molecular pathways that are primarily regulated by IL-13, which promotes alternatively activated (M2) macrophage polarization. M2 polarization was further demonstrated by immunohistochemistry performed on the in vitro sarcoidosis granuloma-like structures. IL-13-regulated gene pathways were confirmed in human sarcoidosis lung and mediastinal lymph node tissues. The in vitro human sarcoidosis granuloma model provides novel insights into early granuloma formation, particularly IL-13 regulation of molecular networks that regulate M2 macrophage polarization. M2 macrophages are predisposed to aggregation and multinucleated giant cell formation, which are characteristic features of sarcoidosis granulomas. Clinical trial registered with www.clinicaltrials.gov (NCT01857401).

As we age, there is an increased risk for the development of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection. Few studies consider that age-associated changes in the alveolar lining fluid (ALF) may increase susceptibility by altering soluble mediators of innate immunity. We assessed the impact of adult or elderly human ALF during Mtb infection in vitro and in vivo. We identified amplification of pro-oxidative and proinflammatory pathways in elderly ALF and decreased binding capability of surfactant-associated surfactant protein A (SP-A) and surfactant protein D (SP-D) to Mtb. Human macrophages infected with elderly ALF-exposed Mtb had reduced control and fewer phagosome-lysosome fusion events, which was reversed when elderly ALF was replenished with functional SP-A/SP-D. In vivo, exposure to elderly ALF exacerbated Mtb infection in young mice. Our studies demonstrate how the pulmonary environment changes as we age and suggest that Mtb may benefit from declining host defenses in the lung mucosa of the elderly.

Despite intensive research efforts, several fundamental disease processes for tuberculosis (TB) remain poorly understood. A central enigma is that host immunity is necessary to control disease yet promotes transmission by causing lung immunopathology. Our inability to distinguish these processes makes it challenging to design rational novel interventions. Elucidating basic immune mechanisms likely requires both in vivo and in vitro analyses, since Mycobacterium tuberculosis is a highly specialized human pathogen. The classic immune response is the TB granuloma organized in three dimensions within extracellular matrix. Several groups are developing cell culture granuloma models. In January 2018, NIAID convened a workshop, entitled "3-D Human in vitro TB Granuloma Model" to advance the field. Here, we summarize the arguments for developing advanced TB cell culture models and critically review those currently available. We discuss how integrating complementary approaches, specifically organoids and mathematical modeling, can maximize progress, and conclude by discussing future challenges and opportunities.

Vaccine development against tuberculosis (TB) is based on the induction of adaptive immune responses endowed with long-term memory against mycobacterial antigens. Memory B and T cells initiate a rapid and robust immune response upon encounter with Mycobacterium tuberculosis, thus achieving long-lasting protection against infection. Recent studies have shown, however, that innate immune cell populations such as myeloid cells and NK cells also undergo functional adaptation after infection or vaccination, a de facto innate immune memory that is also termed trained immunity. Experimental and epidemiological data have shown that induction of trained immunity contributes to the beneficial heterologous effects of vaccines such as bacille Calmette-Guérin (BCG), the licensed TB vaccine. Moreover, increasing evidence argues that trained immunity also contributes to the anti-TB effects of BCG vaccination. An interaction among immunological signals, metabolic rewiring, and epigenetic reprogramming underlies the molecular mechanisms mediating trained immunity in myeloid cells and their bone marrow progenitors. Future studies are warranted to explore the untapped potential of trained immunity to develop a future generation of TB vaccines that would combine innate and adaptive immune memory induction.