Immunosuppressive Features of the Microenvironment in Lymph Nodes Granulomas from Tuberculosis and HIV-Co-Infected Patients.

Tuberculosis (TB) and HIV co-infection claims many lives every year. This study assessed immune responses in Mycobacterium tuberculosis-infected lymph node tissues from HIV-negative and HIV-positive patients compared with the peripheral circulation with a focus on myeloid cells and the cell-signaling enzymes, inducible nitric oxide synthase, and arginase (Arg)-1. Methods included immunohistochemistry or confocal microscopy and computerized image analyses, quantitative real-time PCR, multiplex Luminex, and flow cytometry. These findings indicate enhanced chronic inflammation and immune activation in TB/HIV co-infection but also enhanced immunosuppressive responses. Poorly formed necrotic TB granulomas with a high expression of M. tuberculosis antigens were elevated in TB/HIV-co-infected lymph nodes, and inducible nitric oxide synthase and Arg-1 expression was significantly higher in TB/HIV-co-infected compared with HIV-negative TB or control tissues. High Arg-1 expression was found in myeloid cells with a phenotype characteristic of myeloid-derived suppressor cells (MDCS) that were particularly abundant in TB/HIV-co-infected tissues. Accordingly, Lin-/HLA-DRlow/int/CD33+/CD11b+/CD15+ granulocytic myeloid-derived suppressor cells were significantly elevated in blood samples from TB/HIV-co-infected patients. CD15+ myeloid-derived suppressor cells correlated with plasma HIV viral load and M. tuberculosis antigen load in tissue but were inversely associated with peripheral CD4 T-cells counts. Enhanced chronic inflammation driven by M. tuberculosis and HIV co-infection may promote Arg-1-expressing MDSCs at the site of infection thereby advancing TB disease progression.

IL-7δ5 protein is expressed in human tissues and induces expression of the oxidized low density lipoprotein receptor 1 (OLR1) in CD14+ monocytes.

OBJECTIVES: The 6-exon-spanning 'canonical' Interleukin-7 (IL-7c) is a non-redundant cytokine in human T-cell homeostasis that undergoes extensive alternative pre-mRNA splicing. The IL-7 gene variant lacking, exon 5 (IL-7δ5), exhibits agonistic effects as compared to IL-7c. We studied in this report for the first time the protein expression of IL-7δ5 variant in tissues and its role in monocyte activation. METHODS: We visualized the expression of IL-7δ5 protein by immunohistochemistry in both healthy and malignant (human) tissues and investigated the impact of IL-7δ5 stimulation on CD14+ monocytes using gene expression analysis and flow cytometry. RESULTS: IL-7δ5 is largely expressed by human epithelial cells, yet also by stromal cells in malignant lesions. Gene expression analysis in CD14+ monocytes, induced by the 6-exon spanning IL-7 or IL-7δ5 showed similar changes resulting in a pro-inflammatory phenotype and increased expression of genes involved in lipid metabolism. IL7δ5 was superior in inducing upregulation of the oxidised low density lipoprotein receptor (OLR), measured by flow cytometry, in CD14+ cells. CONCLUSION: IL-7δ5, produced from non-transformed and transformed cells, may contribute to chronic inflammatory responses and development of 'foamy' cells by increased OLR1 expression that mediates increased oxLDL uptake.

Peptide microarray-based characterization of antibody responses to host proteins after bacille Calmette-Guérin vaccination.

BACKGROUND: Bacille Calmette-Guérin (BCG) is the world's most widely distributed vaccine, used against tuberculosis (TB), in cancer immunotherapy, and in autoimmune diseases due to its immunomodulatory properties. To date, the effect of BCG vaccination on antibody responses to host proteins has not been reported. High-content peptide microarrays (HCPM) offer a unique opportunity to gauge specific humoral immune responses. METHODS: The sera of BCG-vaccinated healthy adults were tested on a human HCPM platform (4953 randomly selected epitopes of human proteins) to detect specific immunoglobulin gamma (IgG) responses. Samples were obtained at 56, 112, and 252 days after vaccination. Immunohistology was performed on lymph node tissue from patients with TB lymphadenitis. Results were analysed with a combination of existing and novel statistical methods. RESULTS: IgG recognition of host peptides exhibited a peak at day 56 post BCG vaccination in all study subjects tested, which diminished over time. Primarily, IgG responses exhibited increased reactivity to ion transporters (sodium, calcium channels), cytokine receptors (interleukin 2 receptor ß (IL2Rß), fibroblast growth factor receptor 1 (FGFR1)), other cell surface receptors (inositol, somatostatin, angiopoeitin), ribonucleoprotein, and enzymes (tyrosine kinases, phospholipase) on day 56. There was decreased IgG reactivity to transforming growth factor-beta type 1 receptor (TGFßR1) and, in agreement with the peptide microarray findings, immunohistochemical analysis of TB-infected lymph node samples revealed an overexpression of TGFßR in granulomatous lesions. Moreover, the vesicular monoamine transporter (VMAT2) showed increased reactivity on days 112 and 252, but not on day 56 post-vaccination. IgG to interleukin 4 receptor (IL4R) showed increased reactivity at 112 days post-vaccination, while IgG to IL2Rß and FGFR1 showed decreased reactivity on days 112 and 252 as compared to day 56 post BCG vaccination. CONCLUSIONS: BCG vaccination modifies the host's immune landscape after 56 days, but this imprint changes over time. This may influence the establishment of immunological memory in BCG-vaccinated individuals.

Pulmonary tuberculosis patients with a vitamin D deficiency demonstrate low local expression of the antimicrobial peptide LL-37 but enhanced FoxP3+ regulatory T cells and IgG-secreting cells.

Control of human tuberculosis (TB) requires induction and maintenance of both macrophage and T cell effector functions. We demonstrate that pulmonary TB patients with a vitamin D deficiency had significantly reduced local levels of the vitamin D-inducible antimicrobial peptide LL-37 in granulomatous lesions compared to distal parenchyma from the infected lung. Instead, TB lesions were abundant in CD3(+) T cells and FoxP3(+) regulatory T cells as well as IgG-secreting CD20(+) B cells, particularly in sputum-smear positive patients with cavitary TB. Mycobacteria-specific serum IgG titers were also elevated in patients with active TB. An up-regulation of the B cell stimulatory cytokine IL-21 correlated with mRNA expression of CD20, total IgG and also IL-10 in the TB lesions. Altogether, vitamin D-deficient TB patients expressed a weak antimicrobial response but an IL-21 associated expansion of IgG-secreting B cells combined with a rise in FoxP3(+) regulatory T cells at the local site of infection.

Prime-boost vaccination with rBCG/rAd35 enhances CD8⁺ cytolytic T-cell responses in lesions from Mycobacterium tuberculosis-infected primates.

To prevent the global spread of tuberculosis (TB) infection, a novel vaccine that triggers potent and long-lived immunity is urgently required. A plasmid-based vaccine has been developed to enhance activation of major histocompatibility complex (MHC) class I-restricted CD8⁺ cytolytic T cells using a recombinant Bacille Calmette-Guérin (rBCG) expressing a pore-forming toxin and the Mycobacterium tuberculosis (Mtb) antigens Ag85A, 85B and TB10.4 followed by a booster with a nonreplicating adenovirus 35 (rAd35) vaccine vector encoding the same Mtb antigens. Here, the capacity of the rBCG/rAd35 vaccine to induce protective and biologically relevant CD8⁺ T-cell responses in a nonhuman primate model of TB was investigated. After prime/boost immunizations and challenge with virulent Mtb in rhesus macaques, quantification of immune responses at the single-cell level in cryopreserved tissue specimen from infected organs was performed using in situ computerized image analysis as a technological platform. Significantly elevated levels of CD3⁺ and CD8⁺ T cells as well as cells expressing interleukin (IL)-7, perforin and granulysin were found in TB lung lesions and spleen from rBCG/rAd35-vaccinated animals compared with BCG/rAd35-vaccinated or unvaccinated animals. The local increase in CD8⁺ cytolytic T cells correlated with reduced expression of the Mtb antigen MPT64 and also with prolonged survival after the challenge. Our observations suggest that a protective immune response in rBCG/rAd35-vaccinated nonhuman primates was associated with enhanced MHC class I antigen presentation and activation of CD8⁺ effector T-cell responses at the local site of infection in Mtb-challenged animals.

Compartmentalization of immune responses in human tuberculosis: few CD8+ effector T cells but elevated levels of FoxP3+ regulatory t cells in the granulomatous lesions.

Immune responses were assessed at the single-cell level in lymph nodes from children with tuberculous lymphadenitis. Tuberculosis infection was associated with tissue remodeling of lymph nodes as well as altered cellular composition. Granulomas were significantly enriched with CD68+ macrophages expressing the M. tuberculosis complex-specific protein antigen MPT64 and inducible nitric oxide synthase. There was a significant increase in CD8+ cytolytic T cells surrounding the granuloma; however, CD8+ T cells expressed low levels of the cytolytic and antimicrobial effector molecules perforin and granulysin in the granulomatous lesions. Quantitative real-time mRNA analysis revealed that interferon-gamma, tumor necrosis factor-alpha, and interleukin-17 were not up-regulated in infected lymph nodes, but there was a significant induction of both transforming growth factor-beta and interleukin-13. In addition, granulomas contained an increased number of CD4+FoxP3+ T cells co-expressing the immunoregulatory cytotoxic T-lymphocyte antigen-4 and glucocorticoid-induced tumor necrosis factor receptor molecules. Low numbers of CD8+ T cells in the lesions correlated with high levels of transforming growth factor-beta and FoxP3+ regulatory T cells, suggesting active immunosuppression at the local infection site. Compartmentalization and skewing of the immune response toward a regulatory phenotype may result in an uncoordinated effector T-cell response that reduces granule-mediated killing of M. tuberculosis-infected cells and subsequent disease control.

Mycobacterium tuberculosis-induced apoptotic neutrophils trigger a pro-inflammatory response in macrophages through release of heat shock protein 72, acting in synergy with the bacteria.

Mycobacterium tuberculosis (Mtb) survive inside macrophages by manipulating microbicidal functions such as phago-lysosome fusion, production of reactive oxygen species and nitric oxide, and by rendering macrophages non-responsive to IFN-gamma. Mtb-infected lung tissue does however not only contain macrophages, but also significant numbers of infiltrating polymorphonuclear neutrophils (PMN). These are able to phagocytose and kill ingested Mtb, but are short-lived cells that constantly need to be removed from tissues to avoid tissue damage. Phagocytosis of aged or UV-induced apoptotic PMN by macrophages induce an anti-inflammatory response in macrophages. However, in the present study, we show that engulfment of Mtb-induced apoptotic PMN by macrophages initiates secretion of TNF-alpha from the macrophages, reflecting a pro-inflammatory response. Moreover, Mtb-induced apoptotic PMN up-regulate heat shock proteins 60 and 72 (Hsp60, Hsp72) intracellularly and also release Hsp72 extracellularly. We found that both recombinant Hsp72 and released Hsp72 enhanced the pro-inflammatory response to both Mtb-induced apoptotic PMN and Mtb. This stimulatory effect of the supernatant was abrogated by depleting the Hsp72 with immunoprecipitation. These findings indicate that released Hsp72 from Mtb-infected PMN can trigger macrophage activation during the early stage of Mtb infections, thereby creating a link between innate and adaptive immunity.

Impaired expression of perforin and granulysin in CD8+ T cells at the site of infection in human chronic pulmonary tuberculosis.

Protective immunity in tuberculosis is dependent on the coordinated release of cytolytic effector molecules from effector T cells and the subsequent granule-associated killing of infected target cells. In this study, we investigated the expression of cytolytic (perforin and granzyme A) and antimicrobial (granulysin) molecules at the single-cell level in cryopreserved lung tissue from patients with chronic, progressive tuberculosis disease. Quantification of protein-expressing cells was performed by in situ imaging, while mRNA levels in the infected tissue were analyzed by real-time PCR. Persistent inflammation, including excessive expression of inducible nitric oxide synthase in CD68+ macrophages and significant infiltration of CD3+, CD8+ and CD4+ T cells, was evident in tuberculosis lesions in all patients. However, despite the accumulation of CD3+ T cells, perforin- and granulysin-expressing CD3+ T cells were detected at two- to threefold-lower ratios in the tuberculosis lesions than in distal lung parenchyma and uninfected control lungs, respectively. This was evident at both the protein and mRNA levels. Moreover, perforin- and granulysin-expressing CD8+ T cells were scarce in individual granulomas within the tuberculosis lesions. In contrast, significant up-regulation of granzyme A-expressing CD3+ T cells was evident in the lesions from all patients. Confocal microscopy revealed coexpression of perforin and granulysin, primarily in CD8+ T cells; however, this expression was lower in the tuberculosis lesions. These findings suggest that symptomatic, chronic tuberculosis disease is associated with insufficient up-regulation of perforin and granulysin coexpression in CD8+ T cells at the local site of infection.

Satellite-based Flood Modeling Using TRMM-based Rainfall Products.

Increasingly available and a virtually uninterrupted supply of satellite-estimatedrainfall data is gradually becoming a cost-effective source of input for flood predictionunder a variety of circumstances. However, most real-time and quasi-global satelliterainfall products are currently available at spatial scales ranging from 0.25o to 0.50o andhence, are considered somewhat coarse for dynamic hydrologic modeling of basin-scaleflood events. This study assesses the question: what are the hydrologic implications ofuncertainty of satellite rainfall data at the coarse scale? We investigated this question onthe 970 km² Upper Cumberland river basin of Kentucky. The satellite rainfall productassessed was NASA's Tropical Rainfall Measuring Mission (TRMM) Multi-satellitePrecipitation Analysis (TMPA) product called 3B41RT that is available in pseudo real timewith a latency of 6-10 hours. We observed that bias adjustment of satellite rainfall data canimprove application in flood prediction to some extent with the trade-off of more falsealarms in peak flow. However, a more rational and regime-based adjustment procedureneeds to be identified before the use of satellite data can be institutionalized among floodmodelers.