Intrinsic antibacterial activity of nanoparticles made of β-cyclodextrins potentiates their effect as drug nanocarriers against tuberculosis

Multi-drug-resistant tuberculosis (TB) is a major public health problem, concerning about half a million cases each year. Patients hardly adhere to the current strict treatment consisting of more than 10 000 tablets over a 2-year period. There is a clear need for efficient and better formulated medications. We have previously shown that nanoparticles made of cross-linked poly-β-cyclodextrins (pβCD) are efficient vehicles for pulmonary delivery of powerful combinations of anti-TB drugs. Here, we report that in addition to being efficient drug carriers, pβCD nanoparticles are endowed with intrinsic antibacterial properties. Empty pβCD nanoparticles are able to impair Mycobacterium tuberculosis (Mtb) establishment after pulmonary administration in mice. pβCD hamper colonization of macrophages by Mtb by interfering with lipid rafts, without inducing toxicity. Moreover, pβCD provoke macrophage apoptosis, leading to depletion of infected cells, thus creating a lung microenvironment detrimental to Mtb persistence. Taken together, our results suggest that pβCD nanoparticles loaded or not with antibiotics have an antibacterial action on their own and could be used as a carrier in drug regimen formulations effective against TB.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Rôle de Tirap/Mal, un adaptateur des TLRs, dans le contrôle de l'infection à Mycobacterium tuberculosis

The innate immune response against infectious agents involves a multitude of genes encoding various molecular actors that are essential for the establishment of a protective response. Among these essential actors, receptors that recognize different patterns associated with pathogens are a key step in host defense. Toll-like receptors (TLRs) represent the most evolutionarily conserved class of receptors. TLRs signaling pathways are significantly regulated to i) respond efficiently to different pathogens, ii) prevent loss of tolerance to commensal organisms, and iii) prevent damage caused by prolonged inflammation. This evidence is provided by human genetic polymorphisms conferring resistance or susceptibility to different pathogens. In the case of downstream signaling of TLRs, the study of genetic variants of Tirap (Toll/Interleukin-1 Receptor domain containing Adaptor Protein), an adaptor protein essential for the signaling of some of these receptors, has revealed different effects on protection against infectious agents. For example, the loss-of-function heterozygous S180L mutation in Tirap confers protection against pneumococcal, malaria and HIV infection. However, in the case of tuberculosis, several studies have associated this mutation with opposite effects on protection against infection.The main objective of this thesis is to study how a heterozygous or homozygous genetic deficiency for Tirap, could affect the infection with Mycobacterium tuberculosis (Mtb), etiological agent of tuberculosis.The worsening of the pathology associated with tuberculosis is closely related to the imbalance of the immune balance, resulting in the host's inability to control the infection. A loss of function of Tirap could have a significant impact on the inflammatory response induced following Mtb infection. In addition, through several virulence factors, Mtb can interact with host factors to interfere with the bactericidal responses mounted by the cell to allow elimination of the infectious agent. In this thesis, we showed that a heterozygous deficiency for Tirap induced an intermediate inflammation in the Mtb-infected macrophage correlating with low replication of the bacterium. These macrophages heterozygous for Tirap prevent the bacteria from establishing a favorable niche for replication by inducing efficient maturation of the intracellular compartments that contain them. In non-deficient cells, we also showed that infection induces the expression of Tirap which induces through STAT5-mediated signaling the expression of Cish, a protein controlling the acidification of the vacuole containing the bacteria. In mice, heterozygosity for Tirap is also associated with 5resistance to Mtb infection. This result can be correlated with other human studies showing that the polymorphism associated with a loss of Tirap function confers resistance to TB.With these results, we provide new insights into one of the mechanisms that Mtb uses to manipulate the host cell and ensure its survival. Ultimately, a better understanding at the molecular and cellular level of these host-pathogen interactions should lead to better control of TB.La réponse immunitaire innée contre les agents infectieux fait appel à une multitude de gènes codant pour divers acteurs moléculaires indispensables à l’établissement d’une réponse protectrice. Parmi ces acteurs essentiels, les récepteurs reconnaissants les différents motifs associés aux pathogènes, constituent une étape clé dans la défense de l’hôte. Les récepteurs Toll-Like (TLRs) représentent la classe de récepteurs la plus conservée au cours de l’évolution. Les voies de signalisation des TLRs sont sensiblement régulées pour i) répondre de manière efficace aux différents pathogènes, ii) prévenir la perte de tolérance envers les organismes commensaux et iii) prévenir les dommages causés par une inflammation prolongée. Cette preuve est apportée par des polymorphismes génétiques humains conférant une résistance ou une susceptibilité à différents agents pathogènes. Dans le cas de signalisation en aval des TLRs, l’étude des variants génétiques de Tirap (Toll/Interleukin-1 Receptor domain containing Adaptor Protein), protéine adaptatrice essentielle à la signalisation de certain de ces récepteurs, a révélé différents effets sur la protection contre les agents infectieux. Par exemple, la mutation hétérozygote S180L de Tirap, mutation associée à une perte de fonction, confère une protection contre l’infection à pneumocoque, à la malaria et au VIH. Cependant, dans le cas de la tuberculose, plusieurs études ont associé cette mutation à des effets opposés quant à la protection contre l’infection.L’objectif principal de cette thèse est d’étudier comment une déficience génétique hétérozygote ou homozygote pour Tirap, pourrait affecter l'infection à Mycobacterium tuberculosis (Mtb), agent étiologique de la tuberculose.L’aggravation de la pathologie associée à la tuberculose est étroitement liée au déséquilibre de la balance immunitaire, entraînant l'incapacité de l'hôte à contrôler l'infection. Une perte de fonction de Tirap pourrait avoir un impact important sur la réponse inflammatoire induite suite à l’infection par Mtb. De plus, grâce à plusieurs facteurs de virulence, Mtb peut interagir avec des facteurs de l’hôte pour interférer avec les réponses bactéricides mises en place par la cellule pour permettre l’élimination de l’agent infectieux. Au cours de cette thèse, nous avons montré qu’une déficience hétérozygote pour Tirap induisait une inflammation intermédiaire dans le macrophage infecté par Mtb corrélant avec une faible réplication de la bactérie. Ces macrophages hétérozygotes pour Tirap empêchent les bactéries d’établir une niche favorable à leur réplication en induisant une maturation efficace des compartiments intracellulaires qui les contiennent. Dans les cellules non déficientes, nous avons également montré que l'infection induit l'expression de Tirap qui induit au travers d’une signalisation médiée par STAT5 et l’expression de Cish, une protéine contrôlant l'acidification de la vacuole contenant la bactérie. Chez la souris, l’hétérozygotie pour Tirap est également associée avec une résistance à l'infection par Mtb. Ce résultat peut être corrélé avec d'autres études humaines montrant que le polymorphisme associé à une perte de fonction Tirap confère une résistance à la tuberculose.Grâce à ces résultats, nous apportons de nouvelles connaissances sur l’un des mécanismes que Mtb emploie pour manipuler la cellule hôte et assurer sa survie. A terme, une meilleure compréhension à l’échelle moléculaire et cellulaire de ces interactions hôte-agents pathogènes devrait permettre une meilleure lutte contre la tuberculose

Tirap controls Mycobacterium tuberculosis phagosomal acidification

International audienceProgression of tuberculosis is tightly linked to a disordered immune balance, resulting in inability of the host to restrict intracellular bacterial replication and its subsequent dissemination. The immune response is mainly characterized by an orchestrated recruitment of inflammatory cells secreting cytokines. This response results from the activation of innate immunity receptors that trigger downstream intracellular signaling pathways involving adaptor proteins such as the TIR-containing adaptor protein (Tirap). In humans, resistance to tuberculosis is associated with a loss-of-function in Tirap. Here, we explore how genetic deficiency in Tirap impacts resistance to Mycobacterium tuberculosis (Mtb) infection in a mouse model and ex vivo . Interestingly, compared to wild type littermates, Tirap heterozygous mice were more resistant to Mtb infection. Upon investigation at the cellular level, we observed that mycobacteria were not able to replicate in Tirap-deficient macrophages compared to wild type counterparts. We next showed that Mtb infection induced Tirap expression which prevented phagosomal acidification and rupture. We further demonstrate that the Tirap-mediated anti-tuberculosis effect occurs through a Cish-dependent signaling pathway. Our findings provide new molecular evidence about how Mtb manipulates innate immune signaling to enable intracellular replication and survival of the pathogen, thus paving the way for host-directed approaches to treat tuberculosis

Tirap controls Mycobacterium tuberculosis phagosomal acidification.

Progression of tuberculosis is tightly linked to a disordered immune balance, resulting in inability of the host to restrict intracellular bacterial replication and its subsequent dissemination. The immune response is mainly characterized by an orchestrated recruitment of inflammatory cells secreting cytokines. This response results from the activation of innate immunity receptors that trigger downstream intracellular signaling pathways involving adaptor proteins such as the TIR-containing adaptor protein (Tirap). In humans, resistance to tuberculosis is associated with a loss-of-function in Tirap. Here, we explore how genetic deficiency in Tirap impacts resistance to Mycobacterium tuberculosis (Mtb) infection in a mouse model and ex vivo. Interestingly, compared to wild type littermates, Tirap heterozygous mice were more resistant to Mtb infection. Upon investigation at the cellular level, we observed that mycobacteria were not able to replicate in Tirap-deficient macrophages compared to wild type counterparts. We next showed that Mtb infection induced Tirap expression which prevented phagosomal acidification and rupture. We further demonstrate that the Tirap-mediated anti-tuberculosis effect occurs through a Cish-dependent signaling pathway. Our findings provide new molecular evidence about how Mtb manipulates innate immune signaling to enable intracellular replication and survival of the pathogen, thus paving the way for host-directed approaches to treat tuberculosis