Publications

Systemic lupus erythematosus (SLE) is a prototypic, inflammatory autoimmune disease characterized by significant gender bias. Previous studies have established a role for hormones in SLE pathogenesis, including the sex hormone estrogen. Estrogen regulates gene expression by translocating estrogen receptors (ER) α and β into the nucleus where they induce transcription by binding to estrogen response elements (EREs) of target genes. The ZAS3 locus encodes a signaling and transcriptional molecule involved in regulating inflammatory responses. We show that ZAS3 is significantly up-regulated in SLE patients at both the protein and mRNA levels in peripheral blood mononuclear cells (PBMCs). Furthermore, estrogen stimulates the expression of ZAS3 in vitro in several leukocyte and breast cancer cell lines of both human and murine origin. In vivo estrogen treatment mediates induction of tissue specific ZAS3 expression in several lymphoid organs in mice. Estrogen stimulation also significantly up-regulates ZAS3 expression in primary PBMCs, while treatment with testosterone has no effect. Mechanistically, estrogen induces differential ERα binding to putative EREs within the ZAS3 gene and ERα knockdown with siRNA prevents estrogen induced ZAS3 up-regulation. In contrast, siRNA targeting IFNα has no effect. These data demonstrate that ZAS3 expression is directly regulated by estrogen and that ZAS3 is overexpressed in lupus. Since ZAS3 has been shown to regulate inflammatory pathways, its up-regulation by estrogen could play a critical role in female-biased autoimmune disorders.

Legionella pneumophila (L. pneumophila) is an intracellular bacterium of human alveolar macrophages that causes Legionnaires' disease. In contrast to humans, most inbred mouse strains are restrictive to L. pneumophila replication. We demonstrate that autophagy targets L. pneumophila vacuoles to lysosomes and that this process requires ubiquitination of L. pneumophila vacuoles and the subsequent binding of the autophagic adaptor p62/SQSTM1 to ubiquitinated vacuoles. The L. pneumophila legA9 encodes for an ankyrin-containing protein with unknown role. We show that the legA9 mutant replicate in WT mice and their bone marrow-derived macrophages. This is the first L. pneumophila mutant to be found to replicate in WT bone marrow-derived macrophages other than the Fla mutant. Less legA9 mutant-containing vacuoles acquired ubiquitin labeling and p62/SQSTM1 staining, evading autophagy uptake and avoiding lysosomal fusion. Thus, we describe a bacterial protein that targets the L. pneumophila-containing vacuole for autophagy uptake.

The expression of monocyte surface markers was compared between tuberculosis patients with and without type 2 diabetes (DM2). DM2 was associated with increased CCR2 expression, which may restrain monocyte traffic to the lung. Other host factors associated with baseline monocyte changes were older age (associated with lower CD11b) and obesity (associated with higher RAGE). Given that DM2 patients are more likely to be older and obese, their monocytes are predicted to be altered in function in ways that affect their interaction with Mycobacterium tuberculosis.

Tuberculosis (TB) is known to be fueled by HIV as well as social and economic factors. With progression of the diabetes mellitus (DM) pandemic in countries where TB is also endemic, focus is increasing on the potential links between DM and TB. Despite the magnitude of the DM-TB association worldwide, it is striking how little we know about the underlying biology that promotes this association which is a major concern to public health. In this review we summarize current findings regarding the alterations in the innate and adaptive immune responses of DM patients to Mycobacterium tuberculosis (Mtb). Current findings suggest underperforming innate immunity followed by a hyper-reactive cellular response to Mtb, but the contribution of these altered responses to TB susceptibility or to the more adverse clinical outcomes of TB patients with DM remains unclear. Elucidating the basic mechanisms underlying the higher susceptibility of DM patients to TB should lead to a strategy for stratification of the millions of DM patients worldwide into those with the highest TB risk for targeted TB prevention.

Intracellular pathogens present a major health risk because of their innate ability to evade clearance. Their location within host cells and ability to react to the host environment by mutation or transcriptional changes often enables survival mechanisms to resist standard therapies. Host-directed drugs do not target the pathogen, minimizing the potential development of drug resistance; however, they can be difficult to deliver efficiently to intracellular sites. Vehicle delivery of host-mediated response drugs not only improves drug distribution and toxicity profiles, but can reduce the total amount of drug necessary to clear infection. In this article, we will review some host-directed drugs and current drug delivery techniques that can be used to efficiently clear intracellular infections.

Tuberculosis (TB) remains one of the greatest threats to human health. The causative bacterium, Mycobacterium tuberculosis (Mtb), is acquired by the respiratory route. It is exquisitely human adapted and a prototypic intracellular pathogen of macrophages, with alveolar macrophages (AMs) being the primary conduit of infection and disease. The outcome of primary infection is most often a latently infected healthy human host, in whom the bacteria are held in check by the host immune response. Such individuals can develop active TB later in life with impairment in the immune system. In contrast, in a minority of infected individuals, the host immune response fails to control the growth of bacilli, and progressive granulomatous disease develops, facilitating spread of the bacilli via infectious aerosols coughed out into the environment and inhaled by new hosts. The molecular details of the Mtb-macrophage interaction continue to be elucidated. However, it is clear that a number of complex processes are involved at the different stages of infection that may benefit either the bacterium or the host. Macrophages demonstrate tremendous phenotypic heterogeneity and functional plasticity which, depending on the site and stage of infection, facilitate the diverse outcomes. Moreover, host responses vary depending on the specific characteristics of the infecting Mtb strain. In this chapter, we describe a contemporary view of the behavior of AMs and their interaction with various Mtb strains in generating unique immunologic lung-specific responses.

Tuberculosis (TB) is a leading cause worldwide of human mortality attributable to a single infectious agent. Recent studies targeting candidate genes and "case-control" association have revealed numerous polymorphisms implicated in host susceptibility to TB. Here, we review current progress in the understanding of causative polymorphisms in host innate immune genes associated with TB pathogenesis. We discuss genes encoding several types of proteins: macrophage receptors, such as the mannose receptor (MR, CD206), dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN, CD209), Dectin-1, Toll-like receptors (TLRs), complement receptor 3 (CR3, CD11b/CD18), nucleotide oligomerization domain 1 (NOD1) and NOD2, CD14, P2X7, and the vitamin D nuclear receptor (VDR); soluble C-type lectins, such as surfactant protein-A (SP-A), SP-D, and mannose-binding lectin (MBL); phagocyte cytokines, such as tumor necrosis factor (TNF), interleukin-1β (IL-1β), IL-6, IL-10, IL-12, and IL-18; chemokines, such as IL-8, monocyte chemoattractant protein 1 (MCP-1), RANTES, and CXCL10; and other important innate immune molecules, such as inducible nitric oxide synthase (iNOS) and solute carrier protein 11A1 (SLC11A1). Polymorphisms in these genes have been variably associated with susceptibility to TB among different populations. This apparent variability is probably accounted for by evolutionary selection pressure as a result of long-term host-pathogen interactions in certain regions or populations and, in part, by lack of proper study design and limited knowledge of molecular and functional effects of the implicated genetic variants. Finally, we discuss genomic technologies that hold promise for resolving questions regarding the evolutionary paths of the human genome, functional effects of polymorphisms, and corollary impacts of adaptation on human health, ultimately leading to novel approaches to controlling TB.

Alveolar macrophages (AMs) are exposed to frequent challenges from inhaled particulates and microbes and function as a first line of defense with a highly regulated immune response because of their unique biology as prototypic alternatively activated macrophages. Lung collectins, particularly surfactant protein A (SP-A), contribute to this activation state by fine-tuning the macrophage inflammatory response. During short-term (10 min-2 h) exposure, SP-A's regulation of human macrophage responses occurs through decreased activity of kinases required for proinflammatory cytokine production. However, AMs are continuously exposed to surfactant, and the biochemical pathways underlying long-term reduction of proinflammatory cytokine activity are not known. We investigated the molecular mechanism(s) underlying SP-A- and surfactant lipid-mediated suppression of proinflammatory cytokine production in response to Toll-like receptor (TLR) 4 (TLR4) activation over longer time periods. We found that exposure of human macrophages to SP-A for 6-24 h upregulates expression of IL-1 receptor-associated kinase M (IRAK-M), a negative regulator of TLR-mediated NF-κB activation. Exposure to Survanta, a natural bovine lung extract lacking SP-A, also enhances IRAK-M expression, but at lower magnitude and for a shorter duration than SP-A. Surfactant-mediated upregulation of IRAK-M in macrophages suppresses TLR4-mediated TNF-α and IL-6 production in response to LPS, and IRAK-M knockdown by small interfering RNA reverses this suppression. In contrast to TNF-α and IL-6, the surfactant components upregulate LPS-mediated immunoregulatory IL-10 production, an effect reversed by IRAK-M knockdown. In conclusion, these data identify an important signaling regulator in human macrophages that is used by surfactant to control the long-term alveolar inflammatory response, i.e., enhanced IRAK-M activity.

Members of the Mycobacterium avium complex (MAC) are naturally occurring bacteria in the environment. A link has been suggested between M. avium strains in drinking water and clinical isolates from infected individuals. There is a need to develop new screening methodologies that can identify specific virulence properties of M. avium isolates found in water that predict a level of risk to exposed individuals. In this work we have characterized 15 clinical and environmental M. avium spp. isolates provided by the US Environmental Protection Agency (EPA) to improve our understanding of the key processes involved in the binding, uptake and survival of these isolates in primary human macrophages. M. avium serovar 8 was predominant among the isolates studied. Different amounts and exposure of mannose-capped lipoarabinomannan (ManLAM) and glycopeptidolipids (GPLs), both major mycobacterial virulence factors, were found among the isolates studied. Reference clinical isolate 104 serovar 1 and clinical isolates 11 and 14 serovar 8 showed an increased association with macrophages. Serum opsonization increased the cell association and survival at 2 h post infection for all isolates. However, only the clinical isolates 104 and 3 among those tested showed an increased growth in primary human macrophages. The other isolates varied in their survival in these cells. Thus we conclude that the amounts of cell envelope ManLAM and GPL, as well as GPL serovar specificity are not the only important bacterial factors for dictating the early interactions of M. avium with human macrophages.

Pseudomonas aeruginosa causes chronic lung infections in the airways of cystic fibrosis (CF) patients. Psl is an extracellular polysaccharide expressed by non-mucoid P. aeruginosa strains, which are believed to be initial colonizers. We hypothesized that Psl protects P. aeruginosa from host defences within the CF lung prior to their conversion to the mucoid phenotype. We discovered that serum opsonization significantly increased the production of reactive oxygen species (ROS) by neutrophils exposed to a psl-deficient mutant, compared with wild-type (WT) and Psl overexpressing strains (Psl(++)). Psl-deficient P. aeruginosa were internalized and killed by neutrophils and macrophages more efficiently than WT and Psl(++) variants. Deposition of complement components C3, C5 and C7 was significantly higher on psl-deficient strains compared with WT and Psl(++) bacteria. In an in vivo pulmonary competition assay, there was a 4.5-fold fitness advantage for WT over psl-deficient P. aeruginosa. Together, these data show that Psl inhibits efficient opsonization, resulting in reduced neutrophil ROS production, and decreased killing by phagocytes. This provides a survival advantage in vivo. Since phagocytes are critical in early recognition and control of infection, therapies aimed at Psl could improve the quality of life for patients colonized with P. aeruginosa.