Mycobacterium tuberculosis ( M.tb ) is the second leading infectious cause of death worldwide and the primary cause of death in people living with HIV/AIDS. There are several excellent animal models employed to study tuberculosis (TB), but many have limitations for reproducing human pathology and none are amenable to the direct study of HIV/ M.tb co-infection. The humanized mouse has been increasingly employed to explore HIV infection and other pathogens where animal models are limiting. Our goal was to develop a small animal model of M.tb infection using the bone marrow, liver, thymus (BLT) humanized mouse. NOD-SCID/γ c null mice were engrafted with human fetal liver and thymus tissue, and supplemented with CD34 + fetal liver cells. Excellent reconstitution, as measured by expression of the human CD45 pan leukocyte marker by peripheral blood populations, was observed at 12 weeks after engraftment. Human T cells (CD3, CD4, CD8), as well as natural killer cells and monocyte/macrophages were all observed within the human leukocyte (CD45 + ) population. Importantly, human T cells were functionally competent as determined by proliferative capacity and effector molecule (e.g. IFN-γ, granulysin, perforin) expression in response to positive stimuli. Animals infected intranasally with M.tb had progressive bacterial infection in the lung and dissemination to spleen and liver from 2–8 weeks post infection. Sites of infection in the lung were characterized by the formation of organized granulomatous lesions, caseous necrosis, bronchial obstruction, and crystallization of cholesterol deposits. Human T cells were distributed throughout the lung, liver, and spleen at sites of inflammation and bacterial growth and were organized to the periphery of granulomas. These preliminary results demonstrate the potential to use the humanized mouse as a model of experimental TB.

Macrophages play an integral role in host defenses against intracellular bacterial pathogens. A remarkable plasticity allows for adaptation to the needs of the host to orchestrate versatile innate immune responses to a variety of microbial threats. Several bacterial pathogens have adapted to macrophage plasticity and modulate the classical (M1) or alternative (M2) activation bias towards a polarization state that increases fitness for intracellular survival. Here, we summarize the current understanding of the host macrophage and intracellular bacterial interface; highlighting the roles of M1/M2 polarization in host defense and the mechanisms employed by several important intracellular pathogens to modulate macrophage polarization to favor persistence or proliferation. Understanding macrophage polarization in the context of disease caused by different bacterial pathogens is important for the identification of targets for therapeutic intervention.

Co-infection with HIV increases the morbidity and mortality associated with tuberculosis due to multiple factors including a poorly understood microbial synergy. We developed a novel small animal model of co-infection in the humanized mouse to investigate how HIV infection disrupts pulmonary containment of Mtb. Following dual infection, HIV-infected cells were localized to sites of Mtb-driven inflammation and mycobacterial replication in the lung. Consistent with disease in human subjects, we observed increased mycobacterial burden, loss of granuloma structure and increased progression of TB disease, due to HIV co-infection. Importantly, we observed an HIV-dependent pro-inflammatory cytokine signature (IL-1β, IL-6, TNFα and IL-8), neutrophil accumulation and greater lung pathology in the Mtb-co-infected lung. These results suggest that in the early stages of acute co-infection in the humanized mouse, infection with HIV exacerbates the pro-inflammatory response to pulmonary Mtb, leading to poorly formed granulomas, more severe lung pathology and increased mycobacterial burden and dissemination.

Bovine natural killer (NK) cells were recently identified by positive selection of a NK cell-activating receptor p46 (NKp46)+ CD3– lymphocyte population, which expresses CD25 and CD8 and lyses tumor cell lines following stimulation with recombinant interleukin-2. In the current work, we characterize the cytotoxic/effector potential of a CD3–CD8–CD11b– population isolated through negative selection of bovine peripheral blood leukocytes. This population is CD25loCD62hi when isolated and becomes CD25hiCD62Llo following cytokine stimulation. Activated bovine NK cells increase expression of granulysin, interferon-γ, and perforin and have cytotoxic activity against human tumor cells and Mycobacterium bovis bacillus Calmette-Guerin-infected alveolar and monocyte-derived macrophages. Expression of a bovine homologue of the CD56 neural adhesion molecule expressed by human NK cells was detected in mRNA from brain tissue but was not detected in peripheral blood mononuclear cells or purified NK cell mRNA. Analysis of mRNA from nonstimulated peripheral blood NK cells demonstrates the constitutive expression of homologues of human NK receptors NKp46, CD244, and CD94 and the granule proteins granulysin and perforin. Phorbol ester-stimulated CD8+ T cells also expressed CD244 and CD94, and CD4+ T cells expressed CD94. These NK cell receptors bearing T lymphocytes may represent memory subsets characterized in humans. The results of these studies demonstrate that bovine NK cells may play an important role in the innate immune responses of cattle.

PBMC infected with HIV-1 inhibit cytokine signaling and antimicrobial protein expression in human CD8+ T cells. Immunosuppression following infection with HIV-1 predisposes patients to a myriad of opportunistic pathogens, one of the most important of which is Mtb. Granulysin, expressed by NK cells and CTL, exhibits potent antimicrobial activity against Mtb and several other opportunistic pathogens associated with HIV-1 infection. The immune signals that promote granulysin expression in human CTL are not fully understood. Using primary human CD8+ T cells, in this study, we identify IL-21 as a strong inducer of granulysin, demonstrate that IL-21 and IL-15 activate granulysin expression within CD8+ CD45RO+ T cells, and establish a role for Jak/STAT signaling in the regulation of granulysin within CD8+ T cells. We show that infection of PBMC from healthy donors in vitro with HIV-1 suppresses granulysin expression by CD8+ T cells, concomitant with reduced p-STAT3 and p-STAT5, following activation with IL-15 and IL-21. Of note, simultaneous signaling through IL-15 and IL-21 could partially overcome the immunosuppressive effects of HIV-1 on granulysin expression by CD8+ T cells. These results suggest that HIV-1 infection of PBMC may reduce the antimicrobial profile of activated CD8+ T cells by disrupting signaling events that are critical for the induction of granulysin. Understanding the effects of HIV-1 on CD8+ T cell activation is essential to understanding the physiological basis for inadequate cytotoxic lymphocyte activity in HIV+ patients and for informed guidance of cytokine-based therapy to restore T cell function.

Natural killer (NK) cells have innate antibacterial activity that could be targeted for clinical interventions for infectious disease caused by naturally occurring or weaponized bacterial pathogens. To determine a potential role for NK cells in immunity toBacillus anthracis, we utilized primary human and murine NK cells,in vitroassays, andin vivoNK cell depletion in a murine model of inhalational anthrax. Our results demonstrate potent antibacterial activity by human NK cells againstB. anthracisbacilli within infected autologous monocytes. Surprisingly, NK cells also mediate moderate antibacterial effects on extracellular vegetative bacilli but do not have activity against extracellular or intracellular spores. The immunosuppressive anthrax lethal toxin impairs NK gamma interferon (IFN-γ) expression, but neither lethal nor edema toxin significantly alters the viability or cytotoxic effector function of NK cells. Compared to human NK cells, murine NK cells have a similar, though less potent, activity against intracellular and extracellularB. anthracis. Thein vivodepletion of murine NK cells does not alter animal survival following intranasal infection withB. anthracisspores in our studies but significantly increases the bacterial load in the blood of infected animals. Our studies demonstrate that NK cells participate in the innate immune response againstB. anthracisand suggest that immune modulation to augment NK cell function in early stages of anthrax should be further explored in animal models as a clinical intervention strategy.