Prominent role for T cell-derived Tumour Necrosis Factor for sustained control of Mycobacterium tuberculosis infection

Tumour Necrosis Factor (TNF) is critical for host control of M. tuberculosis, but the relative contribution of TNF from innate and adaptive immune responses during tuberculosis infection is unclear. Myeloid versus T-cell-derived TNF function in tuberculosis was investigated using cell type-specific TNF deletion. Mice deficient for TNF expression in macrophages/neutrophils displayed early, transient susceptibility to M. tuberculosis but recruited activated, TNF-producing CD4+ and CD8+ T-cells and controlled chronic infection. Strikingly, deficient TNF expression in T-cells resulted in early control but susceptibility and eventual mortality during chronic infection with increased pulmonary pathology. TNF inactivation in both myeloid and T-cells rendered mice critically susceptible to infection with a phenotype resembling complete TNF deficient mice, indicating that myeloid and T-cells are the primary TNF sources collaborating for host control of tuberculosis. Thus, while TNF from myeloid cells mediates early immune function, T-cell derived TNF is essential to sustain protection during chronic tuberculosis infection

Mycobacterial PIMs Inhibit Host Inflammatory Responses through CD14-Dependent and CD14-Independent Mechanisms

Mycobacteria develop strategies to evade the host immune system. Among them, mycobacterial LAM or PIMs inhibit the expression of pro-inflammatory cytokines by activated macrophages. Here, using synthetic PIM analogues, we analyzed the mode of action of PIM anti-inflammatory effects. Synthetic PIM1 isomer and PIM2 mimetic potently inhibit TNF and IL-12 p40 expression induced by TLR2 or TLR4 pathways, but not by TLR9, in murine macrophages. We show inhibition of LPS binding to TLR4/MD2/CD14 expressing HEK cells by PIM1 and PIM2 analogues. More specifically, the binding of LPS to CD14 was inhibited by PIM1 and PIM2 analogues. CD14 was dispensable for PIM1 and PIM2 analogues functional inhibition of TLR2 agonists induced TNF, as shown in CD14-deficient macrophages. The use of rough-LPS, that stimulates TLR4 pathway independently of CD14, allowed to discriminate between CD14-dependent and CD14-independent anti-inflammatory effects of PIMs on LPS-induced macrophage responses. PIM1 and PIM2 analogues inhibited LPS-induced TNF release by a CD14-dependent pathway, while IL-12 p40 inhibition was CD14-independent, suggesting that PIMs have multifold inhibitory effects on the TLR4 signalling pathway

Tumor necrosis factor neutralization combined with chemotherapy enhances Mycobacterium tuberculosis clearance and reduces lung pathology

Tuberculosis (TB) is a major health problem requiring sustained immunity to inhibit Mycobacterium tuberculosis growth and appropriate antimicrobial therapy to prevent dissemination and drug resistance. Cell-mediated immune responses to M. tuberculosis involve the activation of cytokines such as Tumor Necrosis Factor (TNF) which is critical for granuloma formation and host resistance against TB. TNF inhibition, used as therapy for the treatment of inflammatory diseases, disrupts granuloma allowing replication of mycobacteria which may increase the efficacy of TB chemotherapy. To test this hypothesis mice infected with M. tuberculosis were treated with isoniazid (INH) and rifampicin (RMP) in the presence or absence of Enbrel, a soluble TNF receptor antagonist during three phases of M. tuberculosis infection. Inhibition of TNF with Enbrel augmented the efficacy of TB chemotherapy as shown by enhanced mycobacterial clearance from the lung of acute and established infection as well as in chronically infected mice. Furthermore, TNF inhibition significantly reduced lung pathology as compared to TB chemotherapy alone. Therefore, the experimental data suggest that TB chemotherapy may be more effective in the presence of a TNF inhibitor, which may be relevant to eradicate mycobacteria during chronic M. tuberculosis infection or reactivation

Synthesis and Biological Investigation of PIM Mimics Carrying Biotin or a Fluorescent Label for Cellular Imaging

Phosphatidyl inositol mannosides (PIMs) are constituents of the mycobacterial cell wall; these glycolipids are known to exhibit potent inhibitory activity toward the LPS-induced production of cytokines by macrophages, and therefore have potential as anti-inflammatory agents. Recently, heterocyclic analogues of PIMs in which the inositol is replaced by a piperidine (aza-PIM mimics) or a tetrahydropyran moiety (oxa-PIM mimics) have been prepared by short synthetic sequences and shown to retain the biological activity of the parent PIM structures. In this investigation, the aza-PIM analogue was used as a convenient scaffold to link biotin or a fluorescent label (tetramethyl-rhodamine) by way of an aminocaproyl spacer, with the goal of using these conjugates for intracellular localization and for the study of the mechanism of their antiinflammatory action. The synthesis of these compounds is reported, as well as the evaluation of their activities as inhibitors of LPS-induced cytokine production by macrophages (TNFα, IL12p40); preliminary investigations by FACS and confocal microscopy indicated that PIM–biotin conjugate binds to macrophage membranes with rapid kinetics

Protein kinase C-theta is required for development of experimental cerebral malaria.

International audienceCerebral malaria is the most severe neurologic complication in children and young adults infected with Plasmodium falciparum. T-cell activation is required for development of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (CM). To characterize the T-cell activation pathway involved, the role of protein kinase C-theta (PKC-θ) in experimental CM development was examined. PKC-θ-deficient mice are resistant to CM development. In the absence of PKC-θ, no neurologic sign of CM developed after blood stage PbA infection. Resistance of PKC-θ-deficient mice correlated with unaltered cerebral microcirculation and absence of ischemia, as documented by magnetic resonance imaging and magnetic resonance angiography, whereas wild-type mice developed distinct microvascular pathology. Recruitment and activation of CD8(+) T cells, and ICAM-1 and CD69 expression were reduced in the brain of resistant mice; however, the pulmonary inflammation and edema associated with PbA infection were still present in the absence of functional PKC-θ. Resistant PKC-θ-deficient mice developed high parasitemia, and died at 3 weeks with severe anemia. Therefore, PKC-θ signaling is crucial for recruitment of CD8(+) T cells and development of brain microvascular pathology resulting in fatal experimental CM, and may represent a novel therapeutic target of CM

Limited Contribution of IL-36 versus IL-1 and TNF Pathways in Host Response to Mycobacterial Infection

IL-36 cytokines are members of the IL-1 family of cytokines that stimulate dendritic cells and T cells leading to enhanced T helper 1 responses in vitro and in vivo; however, their role in host defense has not been fully addressed thus far. The objective of this study was to examine the role of IL-36R signaling in the control of mycobacterial infection, using models of systemic attenuated M. bovis BCG infection and virulent aerogenic M. tuberculosis infection. IL-36γ expression was increased in the lung of M. bovis BCG infected mice. However, IL-36R deficient mice infected with M. bovis BCG showed similar survival and control of the infection as compared to wild-type mice, although their lung pathology and CXCL1 response were transiently different. While highly susceptible TNF-α deficient mice succumbed with overwhelming M. tuberculosis infection, and IL-1RI deficient mice showed intermediate susceptibility, IL-36R-deficient mice controlled the infection, with bacterial burden, lung inflammation and pathology, similar to wild-type controls. Therefore, IL-36R signaling has only limited influence in the control of mycobacterial infection

Relative contribution of IL-1a, IL-1b and TNF to the host response to Mycobacterium tuberculosis and attenuated M. bovis BCG

TNF and IL-1 are major mediators involved in severe inflammatory diseases against which therapeutic neutralizing antibodies are developed. However, both TNF and IL-1 receptor pathways are essential for the control of Mycobacterium tuberculosis infection, and it is critical to assess the respective role of IL-1α, IL-1β, and TNF. Using gene-targeted mice we show that absence of both IL-1α and IL-1β recapitulates the uncontrolled M. tuberculosis infection with increased bacterial burden, exacerbated lung inflammation, high IFNγ, reduced IL-23 p19 and rapid death seen in IL-1R1-deficient mice. However, presence of either IL-1α or IL-1β in single-deficient mice is sufficient to control acute M. tuberculosis infection, with restrained bacterial burden and lung pathology, in conditions where TNF deficient mice succumbed within 4 weeks with overwhelming infection. Systemic infection by attenuated M. bovis BCG was controlled in the absence of functional IL-1 pathway, but not in the absence of TNF. Therefore, although both IL-1α and IL-1β are required for a full host response to virulent M. tuberculosis, the presence of either IL-1α or IL-1β allows some control of acute M. tuberculosis infection, and IL-1 pathway is dispensable for controlling M. bovis BCG acute infection. This is in sharp contrast with TNF, which is essential for host response to both attenuated and virulent mycobacteria and may have implications for anti-inflammatory therapy with IL-1β neutralizing antibodies

Biocontrol of Biofilm Formation: Jamming of Sessile-Associated Rhizobial Communication by Rhodococcal Quorum-Quenching

International audienceBiofilms are complex structures formed by a community of microbes adhering to a surface and/or to each other through the secretion of an adhesive and protective matrix. The establishment of these structures requires a coordination of action between microorganisms through powerful communication systems such as quorum-sensing. Therefore, auxiliary bacteria capable of interfering with these means of communication could be used to prevent biofilm formation and development. The phytopathogen Rhizobium rhizogenes, which causes hairy root disease and forms large biofilms in hydroponic crops, and the biocontrol agent Rhodococcus erythropolis R138 were used for this study. Changes in biofilm biovolume and structure, as well as interactions between rhizobia and rhodococci, were monitored by confocal laser scanning microscopy with appropriate fluorescent biosensors. We obtained direct visual evidence of an exchange of signals between rhizobia and the jamming of this communication by Rhodococcus within the biofilm. Signaling molecules were characterized as long chain (C14) N-acyl-homoserine lactones. The role of the Qsd quorum-quenching pathway in biofilm alteration was confirmed with an R. erythropolis mutant unable to produce the QsdA lactonase, and by expression of the qsdA gene in a heterologous host, Escherichia coli. Finally, Rhizobium biofilm formation was similarly inhibited by a purified extract of QsdA enzyme

IL-12Rβ2 Is Essential for the Development of Experimental Cerebral Malaria

International audienceA Th1 response is required for the development of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM). The role of pro-Th1 IL-12 in malaria is complex and controversial. In this study, we addressed the role of IL-12Rβ2 in ECM development. C57BL/6 mice deficient for IL-12Rβ2, IL-12p40, or IL-12p35 were analyzed for ECM development after blood-stage PbA infection in terms of ischemia and blood flow by noninvasive magnetic resonance imaging and angiography, T cell recruitment, and gene expression. Without IL-12Rβ2, no neurologic sign of ECM developed upon PbA infection. Although wild-type mice developed distinct brain microvascular pathology, ECM-resistant, IL-12Rβ2-deficient mice showed unaltered cerebral microcirculation and the absence of ischemia after PbA infection. In contrast, mice deficient for IL-12p40 or IL-12p35 were sensitive to ECM development. The resistance of IL-12Rβ2-deficient mice to ECM correlated with reduced recruitment of activated T cells and impaired overexpression of lymphotoxin-α, TNF-α, and IFN-γ in the brain after PbA infection. Therefore, IL-12Rβ2 signaling is essential for ECM development but independent from IL-12p40 and IL-12p35. We document a novel link between IL-12Rβ2 and lymphotoxin-α, TNF-α, and IFN-γ expression, key cytokines for ECM pathogenesis

Controlled acute M. tuberculosis infection in mice under treatment with IL-17A or IL-17F antibodies, in contrast to TNFα neutralization

Antibodies targeting IL-17A or its receptor IL-17RA show unprecedented efficacy in the treatment of autoimmune diseases such as psoriasis. These therapies, by neutralizing critical mediators of immunity, may increase susceptibility to infections and Mycobacterium tuberculosis reactivation. The effect of antibodies neutralizing IL-17A, IL-17F or TNFα- on host response to M. tuberculosis infection was evaluated by lung transcriptomic, microbiological and histological analyses. Gene array analyses of infected lungs revealed major changes of inflammatory and immune gene expression signatures 4 weeks post-infection. Specifically, gene expression associated with host-pathogen interactions, macrophage recruitment, activation and polarization, host-antimycobacterial activities, immunomodularory responses, as well as extracellular matrix metallopeptidases, were markedly modulated by TNFα blockade. This coincided with a significant increase of mycobacterial burden, whereas, IL-17A or IL-17F blockade had no major effect on gene expression nor on host resistance to acute TB infection. Further, the absence of both IL-17RA and IL-22 pathways in genetically deficient mice did not profoundly compromise host control of M. tuberculosis over a 6-months period, ruling out potential compensation between these two pathways, while TNFα-deficient mice succumbed rapidly. These data provide experimental confirmation of the low clinical risk of mycobacterial infection under anti-IL-17A therapy, in contrast to anti-TNFα treatment