Tracy L. Keiser

Characterization of Host and Microbial Determinants in Individuals with Latent Tuberculosis Infection Using a Human Granuloma Model

ABSTRACT Granulomas sit at the center of tuberculosis (TB) immunopathogenesis. Progress in biomarkers and treatment specific to the human granuloma environment is hindered by the lack of a relevant and tractable infection model that better accounts for the complexity of the host immune response as well as pathogen counterresponses that subvert host immunity in granulomas. Here we developed and characterized an in vitro granuloma model derived from human peripheral blood mononuclear cells (PBMCs) and autologous serum. Importantly, we interrogated this model for its ability to discriminate between host and bacterial determinants in individuals with and without latent TB infection (LTBI). By the use of this model, we provide the first evidence that granuloma formation, bacterial survival, lymphocyte proliferation, pro- and anti-inflammatory cytokines, and lipid body accumulation are significantly altered in LTBI individuals. Moreover, we show a specific transcriptional signature of Mycobacterium tuberculosis associated with survival within human granuloma structures depending on the host immune status. Our report provides fundamentally new information on how the human host immune status and bacterial transcriptional signature may dictate early granuloma formation and outcome and provides evidence for the validity of the granuloma model and its potential applications. IMPORTANCE In 2012, approximately 1.3 million people died from tuberculosis (TB), the highest rate for any single bacterial pathogen. The long-term control of TB requires a better understanding of Mycobacterium tuberculosis pathogenesis in appropriate research models. Granulomas represent the characteristic host tissue response to TB, controlling the bacilli while concentrating the immune response to a limited area. However, complete eradication of bacteria does not occur, since M. tuberculosis has its own strategies to adapt and persist. Thus, the M. tuberculosis-containing granuloma represents a unique environment for dictating both the host immune response and the bacterial response. Here we developed and characterized an in vitro granuloma model derived from blood cells of individuals with latent TB infection that more accurately defines the human immune response and metabolic profiles of M. tuberculosis within this uniquely regulated immune environment. This model may also prove beneficial for understanding other granulomatous diseases. In 2012, approximately 1.3 million people died from tuberculosis (TB), the highest rate for any single bacterial pathogen. The long-term control of TB requires a better understanding of Mycobacterium tuberculosis pathogenesis in appropriate research models. Granulomas represent the characteristic host tissue response to TB, controlling the bacilli while concentrating the immune response to a limited area. However, complete eradication of bacteria does not occur, since M. tuberculosis has its own strategies to adapt and persist. Thus, the M. tuberculosis-containing granuloma represents a unique environment for dictating both the host immune response and the bacterial response. Here we developed and characterized an in vitro granuloma model derived from blood cells of individuals with latent TB infection that more accurately defines the human immune response and metabolic profiles of M. tuberculosis within this uniquely regulated immune environment. This model may also prove beneficial for understanding other granulomatous diseases.

Cutting Edge: STAT1 and T-bet Play Distinct Roles in Determining Outcome of Visceral Leishmaniasis Caused by Leishmania donovani

T-bet and STAT1 regulate IFN-γ gene transcription in CD4+ T cells, which mediate protection against Leishmania. Here we show that T-bet and STAT1 are required for the induction of an efficient Th1 response during Leishmania donovani infection, but they play distinct roles in determining disease outcome. Both STAT1−/− and T-bet−/− mice failed to mount a Th1 response, but STAT1−/− mice were highly resistant to L. donovani and developed less immunopathology, whereas T-bet−/− mice were highly susceptible and eventually developed liver inflammation. Adoptive cell transfer studies showed that RAG2−/− recipients receiving STAT1+/+ or STAT1−/− T cells developed comparable liver pathology, but those receiving STAT1−/− T cells were significantly more susceptible to infection. These unexpected findings reveal distinct roles for T-bet and STAT1 in mediating host immunity and liver pathology during visceral leishmaniasis.

Development of protective immunity against cutaneous leishmaniasis is dependent on STAT1-mediated IFN signaling pathway.

Although STAT1‐dependent signaling mediates biological functions of IFN‐α/β and IFN‐γ, recent reports indicate that STAT1‐independent IFN signaling also regulates expression of several genes. To determine the roles of STAT1‐dependent and ‐independent IFN signaling in the regulation of immunity during cutaneous leishmaniasis, we studied the course of Leishmania major infection in resistant C57BL/6 mice lacking the STAT1 gene. While L. major‐infected STAT1+/+ mice resolved their lesions, STAT1–/– mice developed large lesions containing significantly more parasites. Moreover, the inability of STAT1–/– mice to control L. major infection was due to the lack of Th1 development associated with reduced production of IL‐12, IFN‐γ and nitric oxide. Although STAT1–/– mice produced more IL‐4 and total IgE than STAT1+/+ mice later during infection, these differences were not significant. Nevertheless, at these time points lymph node cells from STAT1–/– mice produced significantly more IL‐10. Finally, STAT1–/– mice were also susceptible to low dose L. major infection. Thesefindings demonstrate that STAT1‐mediated IFN signaling is indispensable for the development of protective immunity against cutaneous L. major infection. Moreover, they also suggest that the protective role of STAT1‐mediated signaling is due to its ability to induce Th1 development during infection with this parasite.

Macrophage migration inhibitory factor (MIF) plays a critical role in pathogenesis of ultraviolet-B (UVB) -induced nonmelanoma skin cancer (NMSC)

Mounting evidence suggests that macrophage migration inhibitory factor (MIF) may serve as an important link between chronic inflammation and cancer development. The proinflammatory and proangiogenic activities of MIF position it as a potentially important player in the development and progression of nonmelanoma skin cancer (NMSC). To assess the role of MIF in the development and progression of NMSC, we exposed MIF–/– BALB/c mice to acute and chronic ultraviolet B (UVB) irradiation. Our studies demonstrate that MIF–/– BALB/c mice have a significantly diminished acute inflammatory response to UVB exposure compared to wild‐type mice, as measured by myeloperoxidase activity, dermal neutrophil infiltration, and edematous response. Relative to wild‐type mice, MIF–/– mice also show significantly lower vascular endothelial growth factor (VEGF) concentrations in whole skin and significantly lower 8‐oxo‐dG adduct concentrations in epidermal DNA following UVB exposure. Furthermore, MIF–/– mice showed significant increases in p53 activity, epidermal thickness, and epidermal cell proliferation following acute UVB insult. In response to chronic UVB exposure, MIF–/– mice showed a 45% reduction in tumor incidence, significantly less angiogenesis, and delayed tumor progression when compared to their wild‐type counterparts. These data indicate that MIF plays an important role in UVB‐induced NMSC development and progression.— Martin, J., Duncan, F. J., Keiser, T., Shin, S., Kusewitt, D. F., Oberyszyn, T., Satoskar, A. R., VanBus‐kirk, A. M. Macrophage migration inhibitory factor (MIF) plays a critical role in pathogenesis of ultraviolet‐B (UVB) ‐induced nonmelanoma skin cancer (NMSC). FASEB J. 23, 720–730 (2009)

Critical role for phosphoinositide 3-kinase gamma in parasite invasion and disease progression of cutaneous leishmaniasis

Obligate intracellular pathogens such as Leishmania specifically target host phagocytes for survival and replication. Phosphoinositide 3-kinase γ (PI3Kγ), a member of the class I PI3Ks that is highly expressed by leukocytes, controls cell migration by initiating actin polymerization and cytoskeletal reorganization, which are processes also critical for phagocytosis. In this study, we demonstrate that class IB PI3K, PI3Kγ, plays a critical role in pathogenesis of chronic cutaneous leishmaniasis caused by L. mexicana. Using the isoform-selective PI3Kγ inhibitor, AS-605240 and PI3Kγ gene-deficient mice, we show that selective blockade or deficiency of PI3Kγ significantly enhances resistance against L. mexicana that is associated with a significant suppression of parasite entry into phagocytes and reduction in recruitment of host phagocytes as well as regulatory T cells to the site of infection. Furthermore, we demonstrate that AS-605240 is as effective as the standard antileishmanial drug sodium stibogluconate in treatment of cutaneous leishmaniasis caused by L. mexicana. These findings reveal a unique role for PI3Kγ in Leishmania invasion and establishment of chronic infection, and demonstrate that therapeutic targeting of host pathways involved in establishment of infection may be a viable strategy for treating infections caused by obligate intracellular pathogens such as Leishmania.

Changes in the major cell envelope components of Mycobacterium tuberculosis during in vitro growth

One-third of the worlds population is infected with Mycobacterium tuberculosis (M.tb), which causes tuberculosis. Mycobacterium tuberculosis cell envelope components such as glycolipids, lipoglycans and polysaccharides play important roles in bacteria-host cell interactions that dictate the host immune response. However, little is known about the changes in the amounts and types of these cell envelope components as the bacillus divides during in vitro culture. To shed light on these phenomena, we examined growth-dependent changes over time in major cell envelope components of virulent M.tb by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, thin-layer chromatography, mass spectrometry, immunoblotting and flow cytometry. Our studies provide evidence that major mannosylated glycoconjugates on the M.tb cell envelope change as M.tb grows in vitro on the widely used Middlebrook 7H11 agar. In particular, our compositional analyses show that from Day 9 to 28 the amounts of mannose-containing molecules, such as mannose-capped lipoarabinomannan, lipomannan and phosphatidyl-myo-inositol mannosides, change continuously in both the cell envelope and outer cell surface. Along with these changes, mannan levels on the outer cell surface also increase significantly over time. The implications of these differences in terms of how M.tb is grown for studies performed in vitro and in vivo for assessing M.tb-host recognition and establishment of infection are discussed.

Comparative Transcriptional Study of the Putative Mannose Donor Biosynthesis Genes in Virulent Mycobacterium tuberculosis and Attenuated Mycobacterium bovis BCG Strains

ABSTRACT Mycobacterium tuberculosis contains mannosylated cell wall components which are important in macrophage recognition and response. The building block for the mannosyl constituents of these components is GDP-mannose, which is synthesized through a series of enzymes involved in the mannose donor biosynthesis pathway. Nothing is known about the expression levels of the genes encoding these enzymes during the course of infection. To generate transcriptional profiles for the mannose donor biosynthesis genes from virulent M. tuberculosis and attenuated Mycobacterium bovis BCG, bacteria were grown in broth culture and within human macrophages. Our results with broth-grown bacteria show that there are differences in expression of the selected genes between M. tuberculosis and BCG, with increased expression of manC in M. tuberculosis and manA in BCG during stationary-phase growth. Results for M. tuberculosis extracted from within macrophages show that whiB2 is highly expressed and manB and manC are moderately expressed during infection. Rv3256c, Rv3258c, and ppm1 have high expression levels early and decreased expression as the infection progresses. Results with BCG show that, as in M. tuberculosis, whiB2 is highly expressed throughout infection, whereas there is either low expression or little change in expression of the remaining genes studied. Overall, our results show that there is differential regulation of expression of several genes in the mannose donor biosynthesis pathway of M. tuberculosis and BCG grown in broth and within macrophages, raising the possibility that the level of mannose donors may vary during the course of infection and thereby impact the biosynthesis of mannose-containing cell wall molecules.