Hans J. Mollenkopf

Early granuloma formation after aerosol Mycobacterium tuberculosis infection is regulated by neutrophils via CXCR3-signaling chemokines.

Among the first cells to invade a site of infection, polymorphonuclear neutrophils (PMN) play an important role in the control of numerous infections. While PMN are considered critical for control of acute infections, their role in chronic infections remains less well understood. Here we report that PMN are essential for accurate early granuloma formation during chronic M. tuberculosis infection without influencing mycobacterial growth restriction. The PMN‐mediated regulation of granuloma formation depended on chemokines signaling through CXCR3, in particular MIG, as indicated by immune histochemical analysis of lung sections from C57BL/6 wild‐type and CXCR3–/– mutant mice and supported by microarray transcriptome analysis. Hence, PMN play a central role in regulating the focal granulomatous response in the lung, and this early granuloma formation can be segregated from long‐term protection against pulmonary M. tuberculosis infection.

Unique Transcriptome Signature of Mycobacterium tuberculosis in Pulmonary Tuberculosis

ABSTRACT Although tuberculosis remains a substantial global threat, the mechanisms that enable mycobacterial persistence and replication within the human host are ill defined. This study represents the first genome-wide expression analysis of Mycobacterium tuberculosis from clinical lung samples, which has enabled the identification of M. tuberculosis genes actively expressed during pulmonary tuberculosis. To obtain optimal information from our DNA array analyses, we analyzed the differentially expressed genes within the context of computationally inferred protein networks. Protein networks were constructed using functional linkages established by the Rosetta stone, phylogenetic profile, conserved gene neighbor, and operon computational methods. This combined approach revealed that during pulmonary tuberculosis, M. tuberculosis actively transcribes a number of genes involved in active fortification and evasion from host defense systems. These genes may provide targets for novel intervention strategies.

Candidate biomarkers for discrimination between infection and disease caused by Mycobacterium tuberculosis.

Infection with Mycobacterium tuberculosis is controlled by an efficacious immune response in about 90% of infected individuals who do not develop disease. Although essential mediators of protection, e.g., interferon-γ, have been identified, these factors are insufficient to predict the outcome of M. tuberculosis infection. As a first step to determine additional biomarkers, we compared gene expression profiles of peripheral blood mononuclear cells from tuberculosis patients and M. tuberculosis-infected healthy donors by microarray analysis. Differentially expressed candidate genes were predominantly derived from monocytes and comprised molecules involved in the antimicrobial defense, inflammation, chemotaxis, and intracellular trafficking. We verified differential expression for alpha-defensin 1, alpha-defensin 4, lactoferrin, Fcγ receptor 1A (cluster of differentiation 64 [CD64]), bactericidal permeability-increasing protein, and formyl peptide receptor 1 by quantitative polymerase chain reaction analysis. Moreover, we identified increased protein expression of CD64 on monocytes from tuberculosis patients. Candidate biomarkers were then assessed for optimal study group discrimination. Using a linear discriminant analysis, a minimal group of genes comprising lactoferrin, CD64, and the Ras-associated GTPase 33A was sufficient for classification of (1) tuberculosis patients, (2) M. tuberculosis-infected healthy donors, and (3) noninfected healthy donors.

Differential T cell responses to Mycobacterium tuberculosis ESAT6 in tuberculosis patients and healthy donors

Vaccination against and diagnosis of tuberculosis are still insufficient. Proteins secreted by Mycobacterium tuberculosis induce strong immune responses in tuberculosis and constitute prime candidates for development of novel vaccines against tuberculosis as well as for immunodiagnostic assays. We investigated the role of the secreted proteins MPT63, MPT64 and ESAT6 from M. tuberculosis in healthy individuals and tuberculosis patients. None of the secreted proteins stimulated peripheral blood mononuclear cells from healthy donors. In contrast, CD4+ T cells from many tuberculosis patients were stimulated in an MHC class II‐restricted fashion by ESAT6, but not by MPT63 or MPT64. T cell reactivities of tuberculosis patients were focused on the N‐terminal region of ESAT6. The ESAT6 T cell epitopes were presented by different HLA‐DR phenotypes. Cell cultures responding to either ESAT6 or synthetic peptides thereof showed mRNA transcripts for macrophage inflammatory protein (MIP)‐1α, monocyte chemotactic protein (MCP)‐1 or IL‐8 and production of IFN‐γ and MIP‐1α. Our results suggest that the secreted M. tuberculosis proteins MPT63, MPT64 or ESAT6 do not stimulate unprimed T cells, and that ESAT6 may be a potential candidate antigen for detection of clinical disease.

Alternative activation deprives macrophages of a coordinated defense program to Mycobacterium tuberculosis

A potent Th1 immune response is critical to the control of tuberculosis. The impact of an additive Th2 response on the course of disease has so far been insufficiently characterized, despite increased morbidity after co‐infection with Mycobacterium tuberculosis and Th2‐eliciting helminths and possible involvement of Th2 polarization in reactivation of latent tuberculosis. Here, we describe the gene expression profile of murine bone marrow‐derived macrophages alternatively activated by IL‐4 in response to infection with M. tuberculosis. Comparison of transcriptional profiles of infected IL‐4‐ and IFN‐γ‐activated macrophages revealed delayed and partially diminished responses to intracellular bacteria in alternatively activated macrophages, characterized by reduced exposure to nitrosative stress and increased iron availability, respectively. Alternative activation of host macrophages correlated with elevated expression of the M. tuberculosis iron storage protein bacterioferritin as well as reduced expression of the mycobactin synthesis genes mbtI and mbtJ. The extracellular matrix‐remodeling enzyme matrix metalloproteinase (MMP)‐12 was induced in alternatively activated macrophages in vitro, and MMP‐12‐expressing macrophages were abundant at late, but not early, stages of tuberculosis in murine lungs. Our findings emphasize that alternative activation deprives macrophages of control mechanisms that limit mycobacterial growth in vivo, thus supporting intracellular persistence of M. tuberculosis.

MicroRNA-223 controls susceptibility to tuberculosis by regulating lung neutrophil recruitment

The molecular mechanisms that control innate immune cell trafficking during chronic infection and inflammation, such as in tuberculosis (TB), are incompletely understood. During active TB, myeloid cells infiltrate the lung and sustain local inflammation. While the chemoattractants that orchestrate these processes are increasingly recognized, the posttranscriptional events that dictate their availability are unclear. We identified microRNA-223 (miR-223) as an upregulated small noncoding RNA in blood and lung parenchyma of TB patients and during murine TB. Deletion of miR-223 rendered TB-resistant mice highly susceptible to acute lung infection. The lethality of miR-223(–/–) mice was apparently not due to defects in antimycobacterial T cell responses. Exacerbated TB in miR-223(–/–) animals could be partially reversed by neutralization of CXCL2, CCL3, and IL-6, by mAb depletion of neutrophils, and by genetic deletion of Cxcr2. We found that miR-223 controlled lung recruitment of myeloid cells, and consequently, neutrophil-driven lethal inflammation. We conclude that miR-223 directly targets the chemoattractants CXCL2, CCL3, and IL-6 in myeloid cells. Our study not only reveals an essential role for a single miRNA in TB, it also identifies new targets for, and assigns biological functions to, miR-223. By regulating leukocyte chemotaxis via chemoattractants, miR-223 is critical for the control of TB and potentially other chronic inflammatory diseases.

Characterization of the ArsRS Regulon of Helicobacter pylori, Involved in Acid Adaptation

The human gastric pathogen Helicobacter pylori is extremely well adapted to the highly acidic conditions encountered in the stomach. The pronounced acid resistance of H. pylori relies mainly on the ammonia-producing enzyme urease; however, urease-independent mechanisms are likely to contribute to acid adaptation. Acid-responsive gene regulation is mediated at least in part by the ArsRS two-component system consisting of the essential OmpR-like response regulator ArsR and the nonessential cognate histidine kinase ArsS, whose autophosphorylation is triggered in response to low pH. In this study, by global transcriptional profiling of an ArsS-deficient H. pylori mutant grown at pH 5.0, we define the ArsR approximately P-dependent regulon consisting of 109 genes, including the urease gene cluster, the genes encoding the aliphatic amidases AmiE and AmiF, and the rocF gene encoding arginase. We show that ArsR approximately P controls the acid-induced transcription of amiE and amiF by binding to extended regions located upstream of the -10 box of the respective promoters. In contrast, transcription of rocF is repressed by ArsR approximately P at neutral, acidic, and mildly alkaline pH via high-affinity binding of the response regulator to a site overlapping the promoter of the rocF gene.

Functional correlations of pathogenesis-driven gene expression signatures in tuberculosis.

Tuberculosis remains a major health threat and its control depends on improved measures of prevention, diagnosis and treatment. Biosignatures can play a significant role in the development of novel intervention measures against TB and blood transcriptional profiling is increasingly exploited for their rational design. Such profiles also reveal fundamental biological mechanisms associated with the pathology of the disease. We have compared whole blood gene expression in TB patients, as well as in healthy infected and uninfected individuals in a cohort in The Gambia, West Africa and validated previously identified signatures showing high similarities of expression profiles among different cohorts. In this study, we applied a unique combination of classical gene expression analysis with pathway and functional association analysis integrated with intra-individual expression correlations. These analyses were employed for identification of new disease-associated gene signatures, identifying a network of Fc gamma receptor 1 signaling with correlating transcriptional activity as hallmark of gene expression in TB. Remarkable similarities to characteristic signatures in the autoimmune disease systemic lupus erythematosus (SLE) were observed. Functional gene clusters of immunoregulatory interactions involving the JAK-STAT pathway; sensing of microbial patterns by Toll-like receptors and IFN-signaling provide detailed insights into the dysregulation of critical immune processes in TB, involving active expression of both pro-inflammatory and immunoregulatory systems. We conclude that transcriptomics (i) provides a robust system for identification and validation of biosignatures for TB and (ii) application of integrated analysis tools yields novel insights into functional networks underlying TB pathogenesis.

Targeting the proteasome: partial inhibition of the proteasome by bortezomib or deletion of the immunosubunit LMP7 attenuates experimental colitis

Background and aims Inflammatory bowel disease (IBD), comprising Crohn´s disease and ulcerative colitis, is characterised by chronic relapsing inflammation of the gut. Increased proteasome activity, associated with the expression of immunoproteasomes, was found to enhance proinflammatory signalling and thus promotes inflammation in patients with IBD. The aim of this study was to explore whether modulation of the proteasomal activity is a suitable therapeutic approach to limit inflammation in colitis. Methods This concept was assessed in two different experimental set-ups. Development of dextran sodium sulfate (DSS)-induced colitis was tested (1) in lmp7−/− mice lacking the immunoproteasome subunit LMP7 and (2) in wild-type (WT) mice treated with the proteasome inhibitor bortezomib. Results Compared with WT mice, lmp7−/− mice develop significantly attenuated colitis due to reduced nuclear factor-κB (NF-κB) signalling in the absence of LMP7. Further, treatment with bortezomib revealed dose-dependent amelioration of DSS-induced inflammation. In both approaches modulation of the proteasome activity limited the secretion of proinflammatory cytokines and chemokines. Consequently, infiltration of the colon by neutrophils and expansion of inflammatory T helper 1 (Th1) and Th17 T cells was diminished and thus prevented excessive tissue damage. Conclusions It was demonstrated that modulation of the proteasome activity is effective in attenuating experimental colitis. The results reveal that reduction of the proteasome activity either by partial inhibition with bortezomib or by specifically targeting the immunoproteasome subunit LMP7 is a suitable treatment of intestinal inflammation.

Application of Mycobacterial Proteomics to Vaccine Design: Improved Protection by Mycobacterium bovis BCG Prime-Rv3407 DNA Boost Vaccination against Tuberculosis

ABSTRACT Information from comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guérin (BCG) principally allows prediction of potential vaccine candidates. Thirty-six M. tuberculosis DNA vaccine candidates identified by comparative proteome analysis were evaluated in the mouse model for protection against low-dose aerosol M. tuberculosis infection. We identified the DNA vaccine candidate Rv3407 as a protective antigen and analyzed putative major histocompatibility complex class I epitopes by computational predictions and gamma interferon Elispot assays. Importantly, we discovered that the DNA vaccine Rv3407 improved the efficacy of BCG vaccination in a heterologous prime-boost vaccination protocol. Our data demonstrate the rationale of a combination of proteomics, epitope prediction, and broad screening of putative antigens for identification of novel DNA vaccine candidates. Furthermore, our experiments show that heterologous prime-boost vaccination with a defined antigen boost “on top” of a BCG primer provides superior protection against tuberculosis over vaccination with BCG alone.