Inhibition of caspase pathways limits CD4+ T cell loss and restores host anti-retroviral function in HIV-1 infected humanized mice with augmented lymphoid tissue.

The study of HIV infection and pathogenicity in physical reservoirs requires a biologically relevant model. The human immune system (HIS) mouse is an established model of HIV infection, but defects in immune tissue reconstitution remain a challenge for examining pathology in tissues. We utilized exogenous injection of the human recombinant FMS-like tyrosine kinase 3 ligand (rFLT-3 L) into the hematopoietic stem cell (HSC) cord blood HIS mouse model to significantly expand the total area of lymph node (LN) and the number of circulating human T cells. The results enabled visualization and quantification of HIV infectivity, CD4 T cell depletion and other measures of pathogenesis in the secondary lymphoid tissues of the spleen and LN. Treatment with the Caspase-1/4 inhibitor VX-765 limited CD4+ T cell loss in the spleen and reduced viral load in both the spleen and axillary LN. In situ hybridization further demonstrated a decrease in viral RNA in both the spleen and LN. Transcriptomic analysis revealed that in vivo inhibition of caspase-1/4 led to an upregulation in host HIV restriction factors including SAMHD1 and APOBEC3A. These findings highlight the use of rFLT-3 L to augment human immune system characteristics in HIS mice to support investigations of HIV pathogenesis and test host directed therapies, though further refinements are needed to further augment LN architecture and cellular populations. The results further provide in vivo evidence of the potential to target inflammasome pathways as an avenue of host-directed therapy to limit immune dysfunction and virus replication in tissue compartments of HIV+ persons.

HIV infection impairs the host response to Mycobacterium tuberculosis infection by altering surfactant protein D function in the human lung alveolar mucosa.

Tuberculosis is the leading cause of death for people living with HIV (PLWH). We hypothesized that altered functions of innate immune components in the human alveolar lining fluid of PLWH (HIV-ALF) drive susceptibility to Mycobacterium tuberculosis (M.tb) infection. Our results indicate a significant increase in oxidation of innate proteins and chemokine levels and significantly lower levels and function of complement components and Th1/Th2/Th17 cytokines in HIV-ALF versus control-ALF (non-HIV-infected people). We further found a deficiency of surfactant protein D (SP-D) and reduced binding of SP-D to M.tb that had been exposed to HIV-ALF. Primary human macrophages infected with M.tb exposed to HIV-ALF were significantly less capable of controlling the infection, which was reversed by SP-D replenishment in HIV-ALF. Thus, based on the limited number of participants in this study, our data suggest that PLWH without antiretroviral therapy (ART) have declining host innate defense function in their lung mucosa, thereby favoring M.tb and potentially other pulmonary infections.

Novel Role for Macrophage Galactose-Type Lectin-1 to Regulate Innate Immunity against Mycobacterium tuberculosis.

Tuberculosis (TB) caused by infection with Mycobacterium tuberculosis is characterized by inflammatory pathology and poorly understood mechanisms of innate immunity. Pattern recognition receptors, expressed on the surface of macrophages, determine the balance of inflammatory and antimicrobial functions that influence disease outcome. Carbohydrate moieties displayed by mycobacteria can serve as pattern recognition receptor ligands for some members of the C-type lectin receptor (CLR) family, interactions that mediate a variety of incompletely understood immune outcomes. This work identifies a novel role for the CLR macrophage galactose-type lectin (MGL)-1 in a mouse model (C57BL/6 and MGL-1-/-) of experimental TB. Murine macrophages upregulated MGL-1 following in vitro exposure to M. tuberculosis, whereas MGL+ cells accumulated at sites of mycobacteria-driven inflammation in the lung. Pulmonary macrophages from MGL-1-deficient mice displayed increased production of proinflammatory cytokines (IL-1ß, IL-6, and IFN-γ) that were associated with greater lipid accumulation, following M. tuberculosis infection. Surprisingly, for a CLR, we also observed MGL-1-dependent antimycobacterial activity as evidenced by greater M. tuberculosis proliferation in bone marrow-derived macrophages, and the lung, of MGL-1-deficient mice. Differential transcriptome analysis further revealed that loss of MGL-1 perturbs the activation of various genes involved in the regulation of inflammation and lipid metabolism in the setting of M. tuberculosis infection. These results identify MGL-1 signaling as an important mechanism that regulates innate immunity against M. tuberculosis and indicates the potential for the MGL pathway as a novel therapeutic target for anti-TB immunity.

Small Animal Model of Post-chemotherapy Tuberculosis Relapse in the Setting of HIV Co-infection.

Tuberculosis relapse following drug treatment of active disease is an important global public health problem due to the poorer clinical outcomes and increased risk of drug resistance development. Concurrent infection with HIV, including in those receiving anti-retroviral therapy (ART), is an important risk factor for relapse and expansion of drug resistant Mycobacterium tuberculosis (Mtb) isolates. A greater understanding of the HIV-associated factors driving TB relapse is important for development of interventions that support immune containment and complement drug therapy. We employed the humanized mouse to develop a new model of post-chemotherapy TB relapse in the setting of HIV infection. Paucibacillary TB infection was observed following treatment with Rifampin and Isoniazid and subsequent infection with HIV-1 was associated with increased Mtb burden in the post-drug phase. Organized granulomas were observed during development of acute TB and appeared to resolve following TB drug therapy. At relapse, granulomatous pathology in the lung was infrequent and mycobacteria were most often observed in the interstitium and at sites of diffuse inflammation. Compared to animals with HIV mono-infection, higher viral replication was observed in the lung and liver, but not in the periphery, of animals with post-drug TB relapse. The results demonstrate a potential role for the humanized mouse as an experimental model of TB relapse in the setting of HIV. Long term, the model could facilitate discovery of disease mechanisms and development of clinical interventions.

Hacking the host: exploitation of macrophage polarization by intracellular bacterial pathogens.

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.

Dual activity of niclosamide to suppress replication of integrated HIV-1 and Mycobacterium tuberculosis (Beijing).

The human immunodeficiency virus (HIV) pandemic is driving the re-emergence of tuberculosis (TB) as a global health threat, both by increasing the susceptibility of HIV-infected people to infection with Mycobacterium tuberculosis (Mtb), and increasing the rate of emergence of drug-resistant Mtb. There are several other clinical challenges for treatment of co-infected patients including: expense, pill burden, toxicity, and malabsorption that further necessitate the search for new drugs that may be effective against both pathogens simultaneously. The anti-helminthic niclosamide has been shown to have activity against a laboratory strain of Mtb in liquid culture while bacteriostatic activity against non-replicating M. abscessus was also recently described. Here we extend these findings to further demonstrate that niclosamide inhibits mycobacterial growth in infected human macrophages and mediates potent bacteriostatic activity against the virulent Mtb Beijing strain. Importantly, we provide the first evidence that niclosamide inhibits HIV replication in human macrophages and Jurkat T cells through post-integration effects on pro-virus transcription. The dual antiviral and anti-mycobacterial activity was further observed in an in vitro model of HIV and Mtb co-infection using human primary monocyte-derived macrophages. These results support further investigation of niclosamide and derivatives as anti-retroviral/anti-mycobacterial agents that may reduce clinical challenges associated with multi-drug regimens and drug resistance.

Nanoscale Peptide Self-assemblies Boost BCG-primed Cellular Immunity Against Mycobacterium tuberculosis.

Bacillus Calmette-Guerin (BCG) is the only vaccine against TB and has limited protection efficacy, which wanes past adolescence. Multifunctional CD8+ T cells (IFN-γ+/TNF-α+/IL-2+) are associated with lower reactivation risk and enhanced control of active Mtb infection. Since boosting with BCG is contraindicated, booster vaccines that augment T cell immunity in the lungs of BCG-vaccinated individuals are urgently needed. We developed a vaccination strategy based on self-assembling peptide nanofibers presenting Mtb-specific CD8+ or CD4+ T cell epitopes that induce high frequency and antigen-specific effector memory T cells producing IFN-γ and IL-2. Intranasal immunization with peptide nanofibers was well tolerated in mice leading to increased antigen-specific CD8+ T cell population in the lungs. Co-assembled nanofibers of CD8+ T cell epitopes and toll-like receptor 2 (TLR2) agonists induced a 8-fold expansion in multifunctional CD8+ T cell populations in the lungs of vaccinated mice. Aerosol challenge with Mtb in BCG-primed and nanofiber-boosted mice provided an additional 0.5-log CFU reduction in lung bacterial load and indicating enhanced protection compared to BCG alone. Together, these data suggest that heterologous prime-boost with BCG and peptide nanofiber vaccines induces cell mediated immunity in the lung, reduces bacterial burden, and is a potentially safer alternative for boosting BCG-primed immunity.

Pulmonary Tuberculosis in Humanized Mice Infected with HIV-1.

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-1ß, 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.

Further characterization of a highly attenuated Yersinia pestis CO92 mutant deleted for the genes encoding Braun lipoprotein and plasminogen activator protease in murine alveolar and primary human macrophages.

We recently characterized the Δlpp Δpla double in-frame deletion mutant of Yersinia pestis CO92 molecularly, biologically, and immunologically. While Braun lipoprotein (Lpp) activates toll-like receptor-2 to initiate an inflammatory cascade, plasminogen activator (Pla) protease facilitates bacterial dissemination in the host. The Δlpp Δpla double mutant was highly attenuated in evoking bubonic and pneumonic plague, was rapidly cleared from mouse organs, and generated humoral and cell-mediated immune responses to provide subsequent protection to mice against a lethal challenge dose of wild-type (WT) CO92. Here, we further characterized the Δlpp Δpla double mutant in two murine macrophage cell lines as well as in primary human monocyte-derived macrophages to gauge its potential as a live-attenuated vaccine candidate. We first demonstrated that the Δpla single and the Δlpp Δpla double mutant were unable to survive efficiently in murine and human macrophages, unlike WT CO92. We observed that the levels of Pla and its associated protease activity were not affected in the Δlpp single mutant, and, likewise, deletion of the pla gene from WT CO92 did not alter Lpp levels. Further, our study revealed that both Lpp and Pla contributed to the intracellular survival of WT CO92 via different mechanisms. Importantly, the ability of the Δlpp Δpla double mutant to be phagocytized by macrophages, to stimulate production of tumor necrosis factor-α and interleukin-6, and to activate the nitric oxide killing pathways of the host cells remained unaltered when compared to the WT CO92-infected macrophages. Finally, macrophages infected with either the WT CO92 or the Δlpp Δpla double mutant were equally efficient in their uptake of zymosan particles as determined by flow cytometric analysis. Overall, our data indicated that although the Δlpp Δpla double mutant of Y. pestis CO92 was highly attenuated, it retained the ability to elicit innate and subsequent acquired immune responses in the host similar to that of WT CO92, which are highly desirable in a live-attenuated vaccine candidate.

Caspase-1/ASC inflammasome-mediated activation of IL-1ß-ROS-NF-κB pathway for control of Trypanosoma cruzi replication and survival is dispensable in NLRP3-/- macrophages.

In this study, we have utilized wild-type (WT), ASC-/-, and NLRP3-/- macrophages and inhibition approaches to investigate the mechanisms of inflammasome activation and their role in Trypanosoma cruzi infection. We also probed human macrophages and analyzed published microarray datasets from human fibroblasts, and endothelial and smooth muscle cells for T. cruzi-induced changes in the expression genes included in the RT Profiler Human Inflammasome arrays. T. cruzi infection elicited a subdued and delayed activation of inflammasome-related gene expression and IL-1ß production in mφs in comparison to LPS-treated controls. When WT and ASC-/- macrophages were treated with inhibitors of caspase-1, IL-1ß, or NADPH oxidase, we found that IL-1ß production by caspase-1/ASC inflammasome required reactive oxygen species (ROS) as a secondary signal. Moreover, IL-1ß regulated NF-κB signaling of inflammatory cytokine gene expression and, subsequently, intracellular parasite replication in macrophages. NLRP3-/- macrophages, despite an inability to elicit IL-1ß activation and inflammatory cytokine gene expression, exhibited a 4-fold decline in intracellular parasites in comparison to that noted in matched WT controls. NLRP3-/- macrophages were not refractory to T. cruzi, and instead exhibited a very high basal level of ROS (>100-fold higher than WT controls) that was maintained after infection in an IL-1ß-independent manner and contributed to efficient parasite killing. We conclude that caspase-1/ASC inflammasomes play a significant role in the activation of IL-1ß/ROS and NF-κB signaling of cytokine gene expression for T. cruzi control in human and mouse macrophages. However, NLRP3-mediated IL-1ß/NFκB activation is dispensable and compensated for by ROS-mediated control of T. cruzi replication and survival in macrophages.

A humanized mouse model of tuberculosis.

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/γc(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.

Mycobacterium tuberculosis-specific CD8+ T cell recall in convalescing TB subjects with HIV co-infection.

Memory T cell populations recover following phase I chemotherapy for tuberculosis (TB) and augment the effectiveness of antibiotics during the continuation phase of treatment. For those with human immunodeficiency virus (HIV), the CD8(+)T cells may have an especially important role in host defense to Mycobacterium tuberculosis (M.tb) as CD4(+)T cell function and/or numbers decline. Here we performed a preliminary study to investigate the impact of HIV infection status on CD8(+)T cell effector function during the convalescent TB period. Peripheral blood samples from convalescent HIV(+) and HIV(-) TB subjects were used to determine CD4(+)T cell count and monitor antigen-specific CD8(+) T cell activation of effector function (lymphoproliferation, IFN-γ, granulysin) in response to M.tb antigen. Our preliminary results suggest that HIV co-infection is associated with moderate suppression of the M.tb-specific memory CD8(+)T cell compartment in many subjects convalescent for TB. Interestingly, highly activated CD8(+)T cells were observed in recall experiments using peripheral blood from several HIV+ subjects that had low (<200 cells/mm(3)) CD4(+)T cell counts. Further investigation may provide important information for development of novel approaches to target M.tb-specific CD8(+)T cell memory to protect against TB in HIV-endemic regions.

CXCL10 production by human monocytes in response to Leishmania braziliensis infection.

Leishmania (subgenus Viannia) braziliensis is the causative agent of mucocutaneous leishmaniasis (ML) in South America, and ML is characterized by excessive T- and B-cell responses to the parasite. We speculate that the unbalanced production of inflammatory mediators in response to L. braziliensis infection contributes to cell recruitment and disease severity. To test this hypothesis, we first examined the response of peripheral blood mononuclear cells (PBMCs) from healthy volunteers to L. braziliensis infection. We observed that while L. braziliensis infection induced the production of chemokine (C-X-C motif) ligand 10 (CXCL10) and interleukin-10 (IL-10) in human PBMCs and macrophages (MPhis), enhanced expression of CXCL10 and its receptor, chemokine CXC receptor (CXCR3), was predominantly detected in CD14(+) monocytes. The chemoattractant factors secreted by L. braziliensis-infected cells were highly efficient in recruiting uninfected PBMCs (predominantly CD14(+) cells) through Transwell membranes. Serum samples from American tegumentary leishmaniasis (ATL) patients (especially the ML cases) had significantly higher levels of CXCL10, CCL4, and soluble tumor necrosis factor (TNF) receptor II (sTNFRII) than did those of control subjects. Our results suggest that, following L. braziliensis infection, the production of multiple inflammatory mediators by the host may contribute to disease severity by increasing cellular recruitment.

Analysis of the differential host cell nuclear proteome induced by attenuated and virulent hemorrhagic arenavirus infection.

Arenaviruses are important emerging pathogens and include a number of hemorrhagic fever viruses classified as NIAID category A priority pathogens and CDC potential biothreat agents. Infection of guinea pigs with the New World arenavirus Pichindé virus (PICV) has been used as a biosafety level 2 model for the Lassa virus. Despite continuing research, little is known about the molecular basis of pathogenesis, and this has hindered the design of novel antiviral therapeutics. Modulation of the host response is a potential strategy for the treatment of infectious diseases. We have previously investigated the global host response to attenuated and lethal arenavirus infections by using high-throughput immunoblotting and kinomics approaches. In this report, we describe the differential nuclear proteomes of a murine cell line induced by mock infection and infection with attenuated and lethal variants of PICV, investigated by using two-dimensional gel electrophoresis. Spot identification using tandem mass spectrometry revealed the involvement of a number of proteins that regulate inflammation via potential modulation of NF-kappaB activity and of several heterogeneous nuclear ribonuclear proteins. Pathway analysis revealed a potential role for transcription factor XBP-1, a transcription factor involved in major histocompatibility complex II (MHC-II) expression; differential DNA-binding activity was revealed by electrophoretic mobility shift assay, and differences in surface MHC-II expression were seen following PICV infection. These data are consistent with the results of several previous studies and highlight potential differences between transcriptional and translational regulation. This study provides a number of differentially expressed targets for further research and suggests that key events in pathogenesis may be established early in infection.

Mycobacterium bovis BCG vaccination induces memory CD4+ T cells characterized by effector biomarker expression and anti-mycobacterial activity.

The effector mechanisms used by CD4+ T cells to control mycobacteria differ between humans and rodent models of TB and should be investigated in additional animal models. In these studies, the bovine model was used to characterize the mycobactericidal CD4+ T cell response induced by vaccination with the attenuated Mycobacterium bovis bacillus Calmette-Guérin (BCG). Antigenic stimulation of peripheral blood CD4+ T cells from BCG-vaccinated cattle enhanced expression of perforin and IFNgamma in cells expressing a CD45RA-CD45RO+CD62L+ cell surface phenotype, enhanced transcription of granulysin, IFNgamma, perforin, IL-4, IL-13, and IL-21, and enhanced anti-mycobacterial activity of CD4+ T cells against BCG-infected macrophages.

Bovine natural killer cells acquire cytotoxic/effector activity following activation with IL-12/15 and reduce Mycobacterium bovis BCG in infected macrophages.

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 CD25(lo)CD62(hi) when isolated and becomes CD25hiCD62L(lo) following cytokine stimulation. Activated bovine NK cells increase expression of granulysin, interferon-gamma, 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.