Anca Dorhoi

The Mycobacterium tuberculosis regulatory network and hypoxia

We have taken the first steps towards a complete reconstruction of the Mycobacterium tuberculosis regulatory network based on ChIP-Seq and combined this reconstruction with system-wide profiling of messenger RNAs, proteins, metabolites and lipids during hypoxia and re-aeration. Adaptations to hypoxia are thought to have a prominent role in M. tuberculosis pathogenesis. Using ChIP-Seq combined with expression data from the induction of the same factors, we have reconstructed a draft regulatory network based on 50 transcription factors. This network model revealed a direct interconnection between the hypoxic response, lipid catabolism, lipid anabolism and the production of cell wall lipids. As a validation of this model, in response to oxygen availability we observe substantial alterations in lipid content and changes in gene expression and metabolites in corresponding metabolic pathways. The regulatory network reveals transcription factors underlying these changes, allows us to computationally predict expression changes, and indicates that Rv0081 is a regulatory hub.

The NLRP3 Inflammasome Is Differentially Activated by Pneumolysin Variants and Contributes to Host Defense in Pneumococcal Pneumonia

Streptococcus pneumoniae is a leading cause of pneumonia, meningitis, and sepsis. Pneumococci can be divided into >90 serotypes that show differences in the pathogenicity and invasiveness. We tested the hypotheses that the innate immune inflammasome pathway is involved in fighting pneumococcal pneumonia and that some invasive pneumococcal types are not recognized by this pathway. We show that human and murine mononuclear cells responded to S. pneumoniae expressing hemolytic pneumolysin by producing IL-1β. This IL-1β production depended on the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. Some serotype 1, serotype 8, and serotype 7F bacteria, which have previously been associated with increased invasiveness and with production of toxins with reduced hemolytic activity, or bacterial mutants lacking pneumolysin did not stimulate notable IL-1β production. We further found that NLRP3 was beneficial for mice during pneumonia caused by pneumococci expressing hemolytic pneumolysin and was involved in cytokine production and maintenance of the pulmonary microvascular barrier. Overall, the inflammasome pathway is protective in pneumonia caused by pneumococci expressing hemolytic toxin but is not activated by clinically important pneumococcal sequence types causing invasive disease. The study indicates that a virulence factor polymorphism may substantially affect the recognition of bacteria by the innate immune system.

The adaptor molecule CARD9 is essential for tuberculosis control

The cross talk between host and pathogen starts with recognition of bacterial signatures through pattern recognition receptors (PRRs), which mobilize downstream signaling cascades. We investigated the role of the cytosolic adaptor caspase recruitment domain family, member 9 (CARD9) in tuberculosis. This adaptor was critical for full activation of innate immunity by converging signals downstream of multiple PRRs. Card9−/− mice succumbed early after aerosol infection, with higher mycobacterial burden, pyogranulomatous pneumonia, accelerated granulocyte recruitment, and higher abundance of proinflammatory cytokines and granulocyte colony-stimulating factor (G-CSF) in serum and lung. Neutralization of G-CSF and neutrophil depletion significantly prolonged survival, indicating that an exacerbated systemic inflammatory disease triggered lethality of Card9−/− mice. CARD9 deficiency had no apparent effect on T cell responses, but a marked impact on the hematopoietic compartment. Card9−/− granulocytes failed to produce IL-10 after Mycobaterium tuberculosis infection, suggesting that an absent antiinflammatory feedback loop accounted for granulocyte-dominated pathology, uncontrolled bacterial replication, and, ultimately, death of infected Card9−/− mice. Our data provide evidence that deregulated innate responses trigger excessive lung inflammation and demonstrate a pivotal role of CARD9 signaling in autonomous innate host defense against tuberculosis.

AhR sensing of bacterial pigments regulates antibacterial defence

The aryl hydrocarbon receptor (AhR) is a highly conserved ligand-dependent transcription factor that senses environmental toxins and endogenous ligands, thereby inducing detoxifying enzymes and modulating immune cell differentiation and responses. We hypothesized that AhR evolved to sense not only environmental pollutants but also microbial insults. We characterized bacterial pigmented virulence factors, namely the phenazines from Pseudomonas aeruginosa and the naphthoquinone phthiocol from Mycobacterium tuberculosis, as ligands of AhR. Upon ligand binding, AhR activation leads to virulence factor degradation and regulated cytokine and chemokine production. The relevance of AhR to host defence is underlined by heightened susceptibility of AhR-deficient mice to both P. aeruginosa and M. tuberculosis. Thus, we demonstrate that AhR senses distinct bacterial virulence factors and controls antibacterial responses, supporting a previously unidentified role for AhR as an intracellular pattern recognition receptor, and identify bacterial pigments as a new class of pathogen-associated molecular patterns.

For better or for worse: the immune response against Mycobacterium tuberculosis balances pathology and protection

Summary:  Tuberculosis (TB) is a complex disease, and the success of the bacterium as an intracellular pathogen is the outcome of its close and longstanding coevolution with the mammalian host. The dialogue between Mycobacterium tuberculosis and the host is becoming understandable at the molecular, cellular, and tissue level. This has led to the elucidation of the (i) interaction between pattern recognition receptors and pathogen‐associated molecular patterns, (ii) cross‐talk between immune cells, and (iii) mechanisms underlying granuloma development. Disease as an eventual but not a necessary consequence of infection results from a sensitive balance between protective immunity and destructive pathology. Early events, governed largely by conserved mechanisms of host recognition, impact not only on type and course of adaptive immunity but also on lung parenchymal function. New interpretations of how these responses shape the lung environment and direct granuloma development emphasize that the disease results from pathologic consequences of non‐resolving inflammation. We review recent advances in TB research within the context of this ambitious view of TB.

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.

Recombinant BCG ΔureC hly+ Induces Superior Protection Over Parental BCG by Stimulating a Balanced Combination of Type 1 and Type 17 Cytokine Responses

Background. New vaccines against tuberculosis (TB) are urgently needed because the only available vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), fails to protect against pulmonary TB in adults. The recombinant ΔureC hly+ BCG (rBCG) is more efficient than parental BCG (pBCG) against pulmonary TB in preclinical studies and has proven safe and immunogenic in phase I clinical trials. Methods. In an attempt to identify the mechanisms underlying the superior protection of rBCG, we compared the immune responses elicited after vaccination and subsequent aerosol infection with Mycobacterium tuberculosis (MTB) in mice. Results. We demonstrate that both rBCG and pBCG induce marked type 1 cytokine responses, whereas only rBCG elicits a profound type 17 cytokine response in addition. We observed earlier recruitment of antigen-specific T lymphocytes to the lung upon MTB infection of rBCG-vaccinated mice. These T cells produced abundant type 1 cytokines after restimulation, resulting in 10-fold reduced bacterial burden 90 days after infection. Conclusions. Our findings identify a general immunologic pathway for improved vaccination strategies against TB that can also be harnessed by other vaccine candidates.

Activation of the NLRP3 inflammasome by Mycobacterium tuberculosis is uncoupled from susceptibility to active tuberculosis

As a hallmark of tuberculosis (TB), Mycobacterium tuberculosis (MTB) induces granulomatous lung lesions and systemic inflammatory responses during active disease. Molecular regulation of inflammation is associated with inflammasome assembly. We determined the extent to which MTB triggers inflammasome activation and how this impacts on the severity of TB in a mouse model. MTB stimulated release of mature IL‐1β in macrophages while attenuated M. bovis BCG failed to do so. Tubercle bacilli specifically activated the NLRP3 inflammasome and this propensity was strictly controlled by the virulence‐associated RD1 locus of MTB. However, Nlrp3‐deficient mice controlled pulmonary TB, a feature correlated with NLRP3‐independent production of IL‐1β in infected lungs. Our studies demonstrate that MTB activates the NLRP3 inflammasome in macrophages in an ESX‐1‐dependent manner. However, during TB, MTB promotes NLRP3‐ and caspase‐1‐independent IL‐1β release in myeloid cells recruited to lung parenchyma and thus overcomes NLRP3 deficiency in vivo in experimental models.

CXCL5-secreting pulmonary epithelial cells drive destructive neutrophilic inflammation in tuberculosis

Successful host defense against numerous pulmonary infections depends on bacterial clearance by polymorphonuclear leukocytes (PMNs); however, excessive PMN accumulation can result in life-threatening lung injury. Local expression of CXC chemokines is critical for PMN recruitment. The impact of chemokine-dependent PMN recruitment during pulmonary Mycobacterium tuberculosis infection is not fully understood. Here, we analyzed expression of genes encoding CXC chemokines in M. tuberculosis-infected murine lung tissue and found that M. tuberculosis infection promotes upregulation of Cxcr2 and its ligand Cxcl5. To determine the contribution of CXCL5 in pulmonary PMN recruitment, we generated Cxcl5(-/-) mice and analyzed their immune response against M. tuberculosis. Both Cxcr2(-/-) mice and Cxcl5(-/-) mice, which are deficient for only one of numerous CXCR2 ligands, exhibited enhanced survival compared with that of WT mice following high-dose M. tuberculosis infection. The resistance of Cxcl5(-/-) mice to M. tuberculosis infection was not due to heightened M. tuberculosis clearance but was the result of impaired PMN recruitment, which reduced pulmonary inflammation. Lung epithelial cells were the main source of CXCL5 upon M. tuberculosis infection, and secretion of CXCL5 was reduced by blocking TLR2 signaling. Together, our data indicate that TLR2-induced epithelial-derived CXCL5 is critical for PMN-driven destructive inflammation in pulmonary tuberculosis.

Type I IFN signaling triggers immunopathology in tuberculosis-susceptible mice by modulating lung phagocyte dynamics.

General interest in the biological functions of IFN type I in Mycobacterium tuberculosis (Mtb) infection increased after the recent identification of a distinct IFN gene expression signature in tuberculosis (TB) patients. Here, we demonstrate that TB‐susceptible mice lacking the receptor for IFN I (IFNAR1) were protected from death upon aerogenic infection with Mtb. Using this experimental model to mimic primary progressive pulmonary TB, we dissected the immune processes affected by IFN I. IFNAR1 signaling did not affect T‐cell responses, but markedly altered migration of inflammatory monocytes and neutrophils to the lung. This process was orchestrated by IFNAR1 expressed on both immune and tissue‐resident radioresistant cells. IFNAR1‐driven TB susceptibility was initiated by augmented Mtb replication and in situ death events, along with CXCL5/CXCL1‐driven accumulation of neutrophils in alveoli, followed by the discrete compartmentalization of Mtb in lung phagocytes. Early depletion of neutrophils rescued TB‐susceptible mice to levels observed in mice lacking IFNAR1. We conclude that IFN I alters early innate events at the site of Mtb invasion leading to fatal immunopathology. These data furnish a mechanistic explanation for the detrimental role of IFN I in pulmonary TB and form a basis for understanding the complex roles of IFN I in chronic inflammation.