Claudia Carranza

Expression of Cathelicidin LL-37 during Mycobacterium tuberculosis Infection in Human Alveolar Macrophages, Monocytes, Neutrophils, and Epithelial Cells

ABSTRACT The innate immune response in human tuberculosis is not completely understood. To improve our knowledge regarding the role of cathelicidin hCAP-18/LL37 in the innate immune response to tuberculosis infection, we used immunohistochemistry, immunoelectron microscopy, and gene expression to study the induction and production of the antimicrobial peptide in A549 epithelial cells, alveolar macrophages (AM), neutrophils, and monocyte-derived macrophages (MDM) after infection with Mycobacterium tuberculosis. We demonstrated that mycobacterial infection induced the expression and production of LL-37 in all cells studied, with AM being the most efficient. We did not detect peptide expression in tuberculous granulomas, suggesting that LL-37 participates only during early infection. Through the study of Toll-like receptors (TLR) in MDM, we showed that LL-37 can be induced by stimulation through TLR-2, TLR-4, and TLR-9. This last TLR was strongly stimulated by M. tuberculosis DNA. We concluded that LL-37 may have an important role in the innate immune response against M. tuberculosis.

Enhanced Responses to Mycobacterium tuberculosis Antigens by Human Alveolar Lymphocytes during Active Pulmonary Tuberculosis

Responses to mycobacterial and nonmycobacterial antigens were examined in bronchoalveolar cells (BAC) and peripheral blood mononuclear cells (PBMC) from patients with active pulmonary tuberculosis (n=16) and healthy subjects (n=23). DNA synthesis in BAC (but not PBMC) from tuberculosis patients was significantly increased in response to the mycobacterial antigens purified protein derivative (PPD), antigen 85, and mannose-capped lipoarabinomannan but not to nonmycobacterial antigens. The response to PPD was also increased in enriched alveolar lymphocytes from tuberculosis patients (P<.05). The frequency of interferon-gamma but not interleukin-4- or -10-producing cells by ELISAspot was increased in PPD-stimulated BAC from patients with tuberculosis (P<.05). Accessory function of alveolar macrophages for T lymphocyte responses was similar and suppressive activity was variably decreased in tuberculosis patients. Thus, there is compartmentalization of mycobacterial antigen-specific lymphocytes to the lungs during active tuberculosis that on challenge produce a Th1-type cytokine host response.

NOD2 enhances the innate response of alveolar macrophages to Mycobacterium tuberculosis in humans

A role for the nucleotide‐binding oligomerization domain 2 (NOD2) receptor in pulmonary innate immune responses has recently been explored. In the present study, we investigated the role that NOD2 plays in human alveolar macrophage innate responses and determined its involvement in the response to infection with virulent Mycobacterium tuberculosis. Our results showed that NOD2 was expressed in human alveolar macrophages, and significant amounts of IL‐1β, IL‐6, and TNF‐α were produced upon ligand recognition with muramyldipeptide (MDP). NOD2 ligation induced the transcription and protein expression of the antimicrobial peptide LL37 and the autophagy enzyme IRGM in alveolar macrophages, demonstrating a novel function for this receptor in these cells. MDP treatment of alveolar macrophages improved the intracellular growth control of virulent M. tuberculosis; this was associated with a significant release of TNF‐α and IL‐6 and overexpression of bactericidal LL37. In addition, the autophagy proteins IRGM, LC3 and ATG16L1 were recruited to the bacteria‐containing autophagosome after treatment with MDP. In conclusion, our results suggest that NOD2 can modulate the innate immune response of alveolar macrophages and play a role in the initial control of respiratory M. tuberculosis infections.

Nucleotide-oligomerizing domain-1 (NOD1) receptor activation induces pro-inflammatory responses and autophagy in human alveolar macrophages

BackgroundNucleotide-binding oligomerizing domain-1 (NOD1) is a cytoplasmic receptor involved in recognizing bacterial peptidoglycan fragments that localize to the cytosol. NOD1 activation triggers inflammation, antimicrobial mechanisms and autophagy in both epithelial cells and murine macrophages. NOD1 mediates intracellular pathogen clearance in the lungs of mice; however, little is known about NOD1’s role in human alveolar macrophages (AMs) or its involvement in Mycobacterium tuberculosis (Mtb) infection.MethodsAMs, monocytes (MNs), and monocyte-derived macrophages (MDMs) from healthy subjects were assayed for NOD1 expression. Cells were stimulated with the NOD1 ligand Tri-DAP and cytokine production and autophagy were assessed. Cells were infected with Mtb and treated with Tri-DAP post-infection. CFUs counting determined growth control, and autophagy protein recruitment to pathogen localization sites was analyzed by immunoelectron microscopy.ResultsNOD1 was expressed in AMs, MDMs and to a lesser extent MNs. Tri-DAP stimulation induced NOD1 up-regulation and a significant production of IL1β, IL6, IL8, and TNFα in AMs and MDMs; however, the level of NOD1-dependent response in MNs was limited. Autophagy activity determined by expression of proteins Atg9, LC3, IRGM and p62 degradation was induced in a NOD1-dependent manner in AMs and MDMs but not in MNs. Infected AMs could be activated by stimulation with Tri-DAP to control the intracellular growth of Mtb. In addition, recruitment of NOD1 and the autophagy proteins IRGM and LC3 to the Mtb localization site was observed in infected AMs after treatment with Tri-DAP.ConclusionsNOD1 is involved in AM and MDM innate responses, which include proinflammatory cytokines and autophagy, with potential implications in the killing of Mtb in humans.

Modulation of Human Macrophage Responses to Mycobacterium tuberculosis by Silver Nanoparticles of Different Size and Surface Modification

Exposure to silver nanoparticles (AgNP) used in consumer products carries potential health risks including increased susceptibility to infectious pathogens. Systematic assessments of antimicrobial macrophage immune responses in the context of AgNP exposure are important because uptake of AgNP by macrophages may lead to alterations of innate immune cell functions. In this study we examined the effects of exposure to AgNP with different particle sizes (20 and 110 nm diameters) and surface chemistry (citrate or polyvinlypyrrolidone capping) on cellular toxicity and innate immune responses against Mycobacterium tuberculosis (M.tb) by human monocyte-derived macrophages (MDM). Exposures of MDM to AgNP significantly reduced cellular viability, increased IL8 and decreased IL10 mRNA expression. Exposure of M.tb-infected MDM to AgNP suppressed M.tb-induced expression of IL1B, IL10, and TNFA mRNA. Furthermore, M.tb-induced IL-1β, a cytokine critical for host resistance to M.tb, was inhibited by AgNP but not by carbon black particles indicating that the observed immunosuppressive effects of AgNP are particle specific. Suppressive effects of AgNP on the M.tb-induced host immune responses were in part due to AgNP-mediated interferences with the TLR signaling pathways that culminate in the activation of the transcription factor NF-κB. AgNP exposure suppressed M.tb-induced expression of a subset of NF-κB mediated genes (CSF2, CSF3, IFNG, IL1A, IL1B, IL6, IL10, TNFA, NFKB1A). In addition, AgNP exposure increased the expression of HSPA1A mRNA and the corresponding stress-induced Hsp72 protein. Up-regulation of Hsp72 by AgNP can suppress M.tb-induced NF-κB activation and host immune responses. The observed ability of AgNP to modulate infectious pathogen-induced immune responses has important public health implications.

Diminished effector and memory CD8+ circulating T lymphocytes in patients with severe influenza caused by the AH1N1 pdm09 virus.

The T cell immune response to viral infection includes the expansion of naïve T cells, effector cell differentiation and the induction of long-lived memory cells. We compared the differentiation of CD8+ T cells in patients with severe or mild pneumonia induced by influenza infection occurring during the 2009 influenza outbreak and compared their T cell subsets with those in blood samples obtained from healthy volunteers before the AH1N1 influenza outbreak in Mexico. Patients with severe influenza exhibited significantly lower numbers of effector memory CD8+CD26 high CD45RO+CCR7+ phenotype and lower numbers of central memory CD8+CD26high CD62L+CCR7+, CD26 high CD62L+CD127+ or CD26 high CD45RO+CD57 low phenotypes than patients with mild influenza or unexposed healthy subjects. Effector T cells with CD8+CD26CD62L low CD57+ phenotype were significantly diminished in severe influenza patients compared to those in patients with mild influenza or unexposed healthy subjects. These results suggest that low levels of circulating CD8+ T effector and central memory cells are associated with influenza severity.

Effect of inhaled air pollution particulate matter in alveolar macrophages on local pro-inflammatory cytokine and peripheral interferon γ production in response to Mycobacterium tuberculosis

Abstract Background Human alveolar macrophages are first-line cellular responders to inhaled particulate matter (PM) and microbial pathogens in the lungs. We examined whether PM exposure affects human bronchoalveolar and peripheral blood mononuclear cell responses to Mycobacterium tuberculosis . Methods Bronchoalveolar and peripheral blood mononuclear cells were obtained from 30 healthy adult volunteers living in Mexico City, Mexico. Proportions of alveolar macrophages with PM and areas of cytoplasm occupied by inhaled PM within alveolar macrophages were evaluated in 50 bronchoalveolar cells from each donor by ImageJ (version 1.49) on 1000× digital bright field microscopy cytospin images. Ambient PM 2.5 (aerodynamic diameters M tuberculosis (strain H37Ra at multiplicities of infection [MOI] of 1 and 10), lipopolysaccharide, 6 kDa early secretory antigenic target (ESAT-6), purified protein derivative, and PM 2.5 were enumerated by ELISPOT assays. Findings While 26–60% of bronchoalveolar cells harboured inhaled PM, percentages of alveolar macrophage areas covered by PM were between 0·9% (±0·16) and 5·49% (±0·78). In unstimulated bronchoalveolar cells and bronchoalveolar cells exposed in vitro to PM (PM 2.5 ; 0·1, 1·0, 10 μg/mL), frequencies of interleukin-1β and TNF-α-producing cells decreased with higher percentages of alveolar macrophages containing inhaled PM (r=–0·78, p=0·0078 and r=–0·63, p=0·038, respectively). By contrast, frequencies of interferon-γ-producing cells increased in bronchoalveolar cells with alveolar macrophages containing greater areas of inhaled PM (r=0·59, p=0·049). In bronchoalveolar cells infected in vitro with M tuberculosis , frequencies of cells producing interleukin 1β significantly decreased in volunteers with higher percentages of alveolar macrophages containing inhaled PM. Frequencies of interferon-γ-producing cells were significantly decreased in peripheral blood mononuclear cells stimulated with purified protein derivative from volunteers with higher percentages of inhaled PM-containing alveolar macrophages (r=–0·7167, p=0·0184). Interpretation The presence of inhaled PM in alveolar macrophages significantly decreases interleukin 1β and TNF-α production in bronchoalveolar cells in response to M tuberculosis infection as well as interferon-γ-production in peripheral blood mononuclear cells in response to purified protein derivative. These findings provide important insights into the potential mechanisms underlying the epidemiological observations of increased risk of tuberculosis development in air pollution exposure settings and indicate that air pollution may have an adverse impact on global tuberculosis control efforts. Funding NIEHS 5RO1ES020382-05.