Publications

PURPOSE OF REVIEW: Mycobacterium tuberculosis (M.tb), the etiologic agent of tuberculosis, is a prominent global health threat because of the enormous reservoir of subclinical latent tuberculosis infection (LTBI). Current diagnostic approaches are limited in their ability to predict reactivation risk and LTBI is recalcitrant to antibiotic treatment. The present review summarizes recent advances in our ability to detect, treat and model LTBI as well as our understanding of bacterial physiology during latency.

RECENT FINDINGS: T-cell subsets and circulating proteins have been identified which could serve as biomarkers for LTBI or indicators of reactivation risk. In addition, experimental and in-silico models have enabled discoveries regarding bacterial physiology during latency and the host immune response following infection with latent M.tb.

SUMMARY: Despite recent advances, much more research is needed to bolster our ability to detect, implement treatment and model LTBI. The present work is crucial for the eradication of this global problem.

Much progress has been made in the identification of specific human gene variants that contribute to enhanced susceptibility or resistance to viral diseases. Herein we review multiple discoveries made with genome-wide or candidate gene approaches that have revealed significant insights into virus-host interactions. Genetic factors that have been identified include genes encoding virus receptors, receptor-modifying enzymes, and a wide variety of innate and adaptive immunity-related proteins. We discuss a range of pathogenic viruses, including influenza virus, respiratory syncytial virus, human immunodeficiency virus, human T cell leukemia virus, human papilloma virus, hepatitis B and C viruses, herpes simplex virus, norovirus, rotavirus, parvovirus, and Epstein-Barr virus. Understanding the genetic underpinnings that affect infectious disease outcomes should allow tailored treatment and prevention approaches in the future.

Cystic fibrosis (CF) is a fatal, genetic disorder that critically affects the lungs and is directly caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective CFTR function. Macroautophagy/autophagy is a highly regulated biological process that provides energy during periods of stress and starvation. Autophagy clears pathogens and dysfunctional protein aggregates within macrophages. However, this process is impaired in CF patients and CF mice, as their macrophages exhibit limited autophagy activity. The study of microRNAs (Mirs), and other noncoding RNAs, continues to offer new therapeutic targets. The objective of this study was to elucidate the role of Mirs in dysregulated autophagy-related genes in CF macrophages, and then target them to restore this host-defense function and improve CFTR channel function. We identified the Mirc1/Mir17-92 cluster as a potential negative regulator of autophagy as CF macrophages exhibit decreased autophagy protein expression and increased cluster expression when compared to wild-type (WT) counterparts. The absence or reduced expression of the cluster increases autophagy protein expression, suggesting the canonical inverse relationship between Mirc1/Mir17-92 and autophagy gene expression. An in silico study for targets of Mirs that comprise the cluster suggested that the majority of the Mirs target autophagy mRNAs. Those targets were validated by luciferase assays. Notably, the ability of macrophages expressing mutant F508del CFTR to transport halide through their membranes is compromised and can be restored by downregulation of these inherently elevated Mirs, via restoration of autophagy. In vivo, downregulation of Mir17 and Mir20a partially restored autophagy expression and hence improved the clearance of Burkholderia cenocepacia. Thus, these data advance our understanding of mechanisms underlying the pathobiology of CF and provide a new therapeutic platform for restoring CFTR function and autophagy in patients with CF.

BACKGROUND: Sepsis patients with cardiac dysfunction have significantly higher mortality. Although several pathways are associated with myocardial damage in sepsis, the precise cause(s) remains unclear and treatment options are limited. This study was designed to develop a new model to investigate the early events of cardiac damage during sepsis progression.

METHODS AND RESULTS: Francisella tularensis subspecies novicida (Ft.n) is a Gram-negative intracellular pathogen causing severe sepsis syndrome in mice. BALB/c mice (N=12) were sham treated or infected with Ft.n through the intranasal route. Serial electrocardiograms were recorded at multiple time points until 96 hours. Hearts were then harvested for histology and gene expression studies. Similar to septic patients, we illustrate both cardiac electrical and structural phenotypes in our murine Ft.n infection model, including prominent R' wave formation, prolonged QRS intervals, and significant left ventricular dysfunction. Notably, in infected animals, we detected numerous microlesions in the myocardium, previously observed following nosocomial Streptococcus infection and in sepsis patients. We show that Ft.n-mediated microlesions are attributed to cardiomyocyte apoptosis, increased immune cell infiltration, and expression of inflammatory mediators (tumor necrosis factor, interleukin [IL]-1β, IL-8, and superoxide dismutase 2). Finally, we identify increased expression of microRNA-155 and rapid degradation of heat shock factor 1 following cardiac Ft.n infection as a primary cause of myocardial inflammation and apoptosis.

CONCLUSIONS: We have developed and characterized an Ft.n infection model to understand the pathogenesis of cardiac dysregulation in sepsis. Our findings illustrate novel in vivo phenotypes underlying cardiac dysfunction during Ft.n infection with significant translational impact on our understanding of sepsis pathophysiology.

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.

Francisella tularensiscauses tularemia and is a potential biothreat. Given the limited antibiotics for treating tularemia and the possible use of antibiotic-resistant strains as a biowarfare agent, new antibacterial agents are needed. AR-12 is an FDA-approved investigational new drug (IND) compound that induces autophagy and has shown host-directed, broad-spectrum activityin vitroagainstSalmonella entericaserovar Typhimurium andF. tularensis We have shown that AR-12 encapsulated within acetalated dextran (Ace-DEX) microparticles (AR-12/MPs) significantly reduces host cell cytotoxicity compared to that with free AR-12, while retaining the ability to controlS.Typhimurium within infected human macrophages. In the present study, the toxicity and efficacy of AR-12/MPs in controlling virulent type AF. tularensisSchuS4 infection were examinedin vitroandin vivo No significant toxicity of blank MPs or AR-12/MPs was observed in lung histology sections when the formulations were given intranasally to uninfected mice. In histology sections from the lungs of intranasally infected mice treated with the formulations, increased macrophage infiltration was observed for AR-12/MPs, with or without suboptimal gentamicin treatment, but not for blank MPs, soluble AR-12, or suboptimal gentamicin alone. AR-12/MPs dramatically reduced the burden ofF. tularensisin infected human macrophages, in a manner similar to that of free AR-12. However,in vivo, AR-12/MPs significantly enhanced the survival ofF. tularensisSchuS4-infected mice compared to that seen with free AR-12. In combination with suboptimal gentamicin treatment, AR-12/MPs further improved the survival ofF. tularensisSchuS4-infected mice. These studies provide support for Ace-DEX-encapsulated AR-12 as a promising new therapeutic agent for tularemia.

Disseminated candidiasis has become one of the leading causes of hospital-acquired blood stream infections with high mobility and mortality. However, the molecular basis of host defense against disseminated candidiasis remains elusive, and treatment options are limited. Here we report that the E3 ubiquitin ligase CBLB directs polyubiquitination of dectin-1 and dectin-2, two key pattern-recognition receptors for sensing Candida albicans, and their downstream kinase SYK, thus inhibiting dectin-1- and dectin-2-mediated innate immune responses. CBLB deficiency or inactivation protects mice from systemic infection with a lethal dose of C. albicans, and deficiency of dectin-1, dectin-2, or both in Cblb(-/-) mice abrogates this protection. Notably, silencing the Cblb gene in vivo protects mice from lethal systemic C. albicans infection. Our data reveal that CBLB is crucial for homeostatic control of innate immune responses mediated by dectin-1 and dectin-2. Our data also indicate that CBLB represents a potential therapeutic target for protection from disseminated candidiasis.

Accumulating evidence from experimental animal models suggests that antibodies play a protective role against tuberculosis (TB). However, little is known about the antibodies generated upon Mycobacterium tuberculosis (MTB) exposure in humans. Here, we performed a molecular and functional characterization of the human B-cell response to MTB by generating recombinant monoclonal antibodies from single isolated B cells of untreated adult patients with acute pulmonary TB and from MTB-exposed healthcare workers. The data suggest that the acute plasmablast response to MTB originates from reactivated memory B cells and indicates a mucosal origin. Through functional analyses, we identified MTB inhibitory antibodies against mycobacterial antigens including virulence factors that play important roles in host cell infection. The inhibitory activity of anti-MTB antibodies was directly linked to their isotype. Monoclonal as well as purified serum IgA antibodies showed MTB blocking activity independently of Fc alpha receptor expression, whereas IgG antibodies promoted the host cell infection. Together, the data provide molecular insights into the human antibody response to MTB and may thereby facilitate the design of protective vaccination strategies.