Mucosal-Associated Invariant T Cell Levels Are Reduced in the Peripheral Blood and Lungs of Children With Active Pulmonary Tuberculosis

Mucosal associated invariant T (MAIT) cells are unconventional, semi-invariant T lymphocytes that recognize microbial-derived vitamin B2 (riboflavin) biosynthesis precursor derivatives presented by the monomorphic MHC class 1-related (MR1) molecule. Upon microbial infection, MAIT cells rapidly produce cytokines and cytotoxic effectors, and are thus important players in anti-microbial defense. MAIT cells are protective in experimental models of infection and are decreased in the blood of adult patients with bacterial infections, including Mycobacterium tuberculosis (Mtb). In children, the risk of rapid progression to active tuberculosis (TB) following Mtb infection is higher than in adults. Whether MAIT cells influence the outcome of Mtb infection in children is therefore, an important issue. We analyzed MAIT cell numbers and phenotype in 115 children investigated for pulmonary TB and determined their potential correlation with disease progression. MAIT cells were reduced in numbers and activated in the peripheral blood of children with active TB as compared to those with latent TB infection (LTBI) and healthy children. Moreover, MAIT cells did not accumulate and did not proliferate in the lung of children with active TB. These results suggest that MAIT cells may be important in preventing progression of Mtb infection to active TB in children

Organised Genome Dynamics in the Escherichia coli Species Results in Highly Diverse Adaptive Paths

The Escherichia coli species represents one of the best-studied model organisms, but also encompasses a variety of commensal and pathogenic strains that diversify by high rates of genetic change. We uniformly (re-) annotated the genomes of 20 commensal and pathogenic E. coli strains and one strain of E. fergusonii (the closest E. coli related species), including seven that we sequenced to completion. Within the ∼18,000 families of orthologous genes, we found ∼2,000 common to all strains. Although recombination rates are much higher than mutation rates, we show, both theoretically and using phylogenetic inference, that this does not obscure the phylogenetic signal, which places the B2 phylogenetic group and one group D strain at the basal position. Based on this phylogeny, we inferred past evolutionary events of gain and loss of genes, identifying functional classes under opposite selection pressures. We found an important adaptive role for metabolism diversification within group B2 and Shigella strains, but identified few or no extraintestinal virulence-specific genes, which could render difficult the development of a vaccine against extraintestinal infections. Genome flux in E. coli is confined to a small number of conserved positions in the chromosome, which most often are not associated with integrases or tRNA genes. Core genes flanking some of these regions show higher rates of recombination, suggesting that a gene, once acquired by a strain, spreads within the species by homologous recombination at the flanking genes. Finally, the genome's long-scale structure of recombination indicates lower recombination rates, but not higher mutation rates, at the terminus of replication. The ensuing effect of background selection and biased gene conversion may thus explain why this region is A+T-rich and shows high sequence divergence but low sequence polymorphism. Overall, despite a very high gene flow, genes co-exist in an organised genome

Virulence Potential and Genomic Mapping of the Worldwide Clone Escherichia coli ST131

Recently, the worldwide propagation of clonal CTX-M-15-producing Escherichia coli isolates, namely ST131 and O25b:H4, has been reported. Like the majority of extra-intestinal pathogenic E. coli isolates, the pandemic clone ST131 belongs to phylogenetic group B2, and has recently been shown to be highly virulent in a mouse model, even though it lacks several genes encoding key virulence factors (Pap, Cnf1 and HlyA). Using two animal models, Caenorhabditis elegans and zebrafish embryos, we assessed the virulence of three E. coli ST131 strains (2 CTX-M-15- producing urine and 1 non-ESBL-producing faecal isolate), comparing them with five non-ST131 B2 and a group A uropathogenic E. coli (UPEC). In C. elegans, the three ST131 strains showed intermediate virulence between the non virulent group A isolate and the virulent non-ST131 B2 strains. In zebrafish, the CTX-M-15-producing ST131 UPEC isolates were also less virulent than the non-ST131 B2 strains, suggesting that the production of CTX-M-15 is not correlated with enhanced virulence. Amongst the non-ST131 B2 group isolates, variation in pathogenic potential in zebrafish embryos was observed ranging from intermediate to highly virulent. Interestingly, the ST131 strains were equally persistent in surviving embryos as the non-ST131-group B2 strains, suggesting similar mechanisms may account for development of persistent infection. Optical maps of the genome of the ST131 strains were compared with those of 24 reference E. coli strains. Although small differences were seen within the ST131 strains, the tree built on the optical maps showed that these strains belonged to a specific cluster (86% similarity) with only 45% similarity with the other group B2 strains and 25% with strains of group A and D. Thus, the ST131 clone has a genetic composition that differs from other group B2 strains, and appears to be less virulent than previously suspected

Transmission and pathophysiology of HIV infection : lessons learned from the study of CXCR4-using viruses

L'entrée du VIH-1 dans ses cellules cibles nécessite l'interaction de sa glycoprotéine d'enveloppe (Env) à la surface du virus avec le récepteur, CD4, puis avec un corécepteur, CCR5 ou CXCR4, selon le tropisme viral. Les virus qui utilisent CCR5 (virus R5) sont majoritairement représentés chez les patients. Ceux qui utilisent CXCR4, seuls (virus X4) ou en sus de CCR5 (virus R5X4) ne sont que rarement transmis mais leur fréquence s'accroit au cours de l'infection. Un lien entre tropisme viral et pathogénicité de l'infection a été proposé. Les virus utilisant CXCR4 sont plus fréquemment que les virus R5 associés à une accélération du déclin des LTCD4. Des contre-exemples à ce paradigme général de la pathogénicité accrue des virus X4 sont cependant nombreux, suggérant qu'il existe une hétérogénéité dans la pathogénicité des virus X4 dont les bases moléculaires ne sont pas connues. Lors de ma thèse, j'ai étudié le rôle du ligand naturel de CXCR4, i.e. la chimiokine CXCL12, comme force motrice de l'évolution de ces virus au cours de l'infection. La liaison de la chimiokine bloque en effet l'entrée virale in vitro, suggérant qu'elle représente une contrainte aux virus X4 in vivo. Nous avons émis l'hypothèse que ceux-ci évolueraient dans le sens d'un échappement à cette contrainte, conduisant à façonner leurs propriétés phénotypiques, en particulier leur pathogénicité. Pour étudier cette hypothèse, nous avons mesuré la sensibilité à l'inhibition par CXCL12 d'une trentaine de virus isolés de patients à différents stades de l'infection. Ces expériences ont permis d'identifier que des virus résistants (RES) à l'inhibition par CXCL12 sont fréquents chez les patients présentant des taux bas de LTCD4, fréquemment dans les phases tardives de l'infection, plus rarement au cours de la primo-infection. Ces résultats ont orienté mes travaux vers deux axes, l'un ayant trait aux mécanismes de la résistance des virus à CXCL12 et son incidence sur la pathogenèse de l'infection, l'autre aux mécanismes de la contre-sélection des virus X4 au cours de la transmission. J'ai montré que la résistance à CXCL12 est associée à des modifications des propriétés antigéniques d'Env et résulte de l'exploitation par les virus de molécules de CXCR4 faiblement affines pour CXCL12. Mes résultats suggèrent aussi que la résistance à CXCL12 accroit la capacité des virus à dépléter les LTCD4, notamment les LTCD4 effecteurs mémoires activés du sang périphérique. Mes résultats suggèrent que dans des sites anatomiques où la chimiokine CXCL12 est exprimée (e.g. tissus lymphoïdes), les virus RES infecteront plus efficacement les LTCD4, en particulier les cellules naïves. Ils seront donc plus enclins à altérer l'homéostasie des LTCD4, expliquant leur association avec des taux de LTCD4 bas chez les patients. Le fait que ces virus RES soient rares lors de la phase de primo-infection m'a aussi amenée dans le cadre d'un second axe à m'interroger sur les mécanismes qui conduisent les virus X4 à être contre-sélectionnés lors de la transmission. Ces résultats suggéraient que contrairement à l'idée prévalente, CXCL12 n'est pas le facteur majeur de la contre-sélection des virus X4 et que d'autres déterminants sont impliqués. Mes travaux suggèrent que des différences de tropisme cellulaire, d'antigénicité des Envs ou encore de sensibilité aux interférons de type I n'expliquent pas entièrement pourquoi les virus X4 sont moins efficacement transmis que les virus R5. D'autres hypothèses seront donc proposées à cet égard à la lumière de données récentes de la littérature. Dans son ensemble ce travail améliore notre compréhension de la physiopathologie et de la transmission du VIH-1. Aussi, l'identification que les virus RES utilisent des formes de CXCR4 différentes de celles utilisées par CXCL12 permet d'envisager de contrecarrer ces virus pour prévenir la déplétion des LTCD4 chez les patients VIH+ sous cART tout en préservant les fonctions homéostatiques essentielles de la chimiokine.The entry of HIV-1 into its target cells requires the interaction of its envelope glycoprotein (Env) on the surface of the virus with the receptor, CD4, and then with a co-receptor, CCR5 or CXCR4, depending on the viral tropism. Viruses that use CCR5 (R5 viruses) are predominantly represented in patients. Those using CXCR4, alone (X4 viruses) or in addition to CCR5 (R5X4 viruses) are rarely transmitted but their frequency increases during the course of the infection. A link between viral tropism and pathogenicity of the infection has been proposed. CXCR4-using viruses are more frequently associated with accelerated CD4TL decline than R5 viruses. Counterexamples to this general paradigm of increased pathogenicity of X4 viruses are, however, numerous, suggesting that there is heterogeneity in the pathogenicity of X4 viruses for which the molecular basis is not known. In my thesis, I studied the role of the natural ligand of CXCR4, i.e. the chemokine CXCL12, as a driving force of the evolution of these viruses during infection. Chemokine binding indeed blocks viral entry in vitro, suggesting that it represents a constraint to X4 viruses in vivo. We hypothesized that the viruses would evolve in the direction of escape from this constraint, leading to the shaping of their phenotypic properties, in particular their pathogenicity. To study this hypothesis, we measured the sensitivity to CXCL12 inhibition of about 30 viruses isolated from patients at different stages of infection. These experiments allowed us to identify that viruses resistant (RES) to CXCL12 inhibition are frequent in patients with low levels of CD4TL, frequently in the late stages of infection, more rarely during the primary infection. These results directed my work towards two axes, one dealing with the mechanisms of virus resistance to CXCL12 and its impact on the pathogenesis of the infection, the other with the mechanisms of X4 virus counter-selection during transmission. I showed that CXCL12 resistance is associated with changes in Env antigenic properties and results from viruses exploiting CXCR4 molecules that are weakly affine for CXCL12. My results also suggest that resistance to CXCL12 increases the ability of viruses to deplete CD4TL, including activated memory effector CD4TL from the peripheral blood. My results suggest that in anatomical sites where CXCL12 chemokine is expressed (e.g. lymphoid tissues), RES viruses will more efficiently infect CD4TL, especially naive cells. They will therefore be more likely to alter LTCD4 homeostasis, explaining their association with low LTCD4 levels in patients. The fact that these RES viruses are rare during the primary infection phase also led me in a second axis to question the mechanisms that lead X4 viruses to be counter-selected during transmission. These results suggested that contrary to the prevalent idea, CXCL12 is not the major factor in the counter-selection of X4 viruses and that other determinants are involved. My work suggests that differences in cell tropism, Envs antigenicity or type I interferon sensitivity do not fully explain why X4 viruses are less efficiently transmitted than R5 viruses. Other hypotheses will therefore be proposed in this respect in the light of recent data from the literature. Overall, this work improves our understanding of the pathophysiology and transmission of HIV-1. Also, the identification that RES viruses use different forms of CXCR4 than those used by CXCL12 allows us to consider counteracting these viruses to prevent CD4TL depletion in HIV+ patients on cART while preserving the essential homeostatic functions of the chemokine

Transmission et physiopathologie de l'infection VIH : leçons tirées de l'étude des virus utilisant CXCR4 comme corécepteur

L'entrée du VIH-1 dans ses cellules cibles nécessite l'interaction de sa glycoprotéine d'enveloppe (Env) à la surface du virus avec le récepteur, CD4, puis avec un corécepteur, CCR5 ou CXCR4, selon le tropisme viral. Les virus qui utilisent CCR5 (virus R5) sont majoritairement représentés chez les patients. Ceux qui utilisent CXCR4, seuls (virus X4) ou en sus de CCR5 (virus R5X4) ne sont que rarement transmis mais leur fréquence s'accroit au cours de l'infection. Un lien entre tropisme viral et pathogénicité de l'infection a été proposé. Les virus utilisant CXCR4 sont plus fréquemment que les virus R5 associés à une accélération du déclin des LTCD4. Des contre-exemples à ce paradigme général de la pathogénicité accrue des virus X4 sont cependant nombreux, suggérant qu'il existe une hétérogénéité dans la pathogénicité des virus X4 dont les bases moléculaires ne sont pas connues. Lors de ma thèse, j'ai étudié le rôle du ligand naturel de CXCR4, i.e. la chimiokine CXCL12, comme force motrice de l'évolution de ces virus au cours de l'infection. La liaison de la chimiokine bloque en effet l'entrée virale in vitro, suggérant qu'elle représente une contrainte aux virus X4 in vivo. Nous avons émis l'hypothèse que ceux-ci évolueraient dans le sens d'un échappement à cette contrainte, conduisant à façonner leurs propriétés phénotypiques, en particulier leur pathogénicité. Pour étudier cette hypothèse, nous avons mesuré la sensibilité à l'inhibition par CXCL12 d'une trentaine de virus isolés de patients à différents stades de l'infection. Ces expériences ont permis d'identifier que des virus résistants (RES) à l'inhibition par CXCL12 sont fréquents chez les patients présentant des taux bas de LTCD4, fréquemment dans les phases tardives de l'infection, plus rarement au cours de la primo-infection. Ces résultats ont orienté mes travaux vers deux axes, l'un ayant trait aux mécanismes de la résistance des virus à CXCL12 et son incidence sur la pathogenèse de l'infection, l'autre aux mécanismes de la contre-sélection des virus X4 au cours de la transmission. J'ai montré que la résistance à CXCL12 est associée à des modifications des propriétés antigéniques d'Env et résulte de l'exploitation par les virus de molécules de CXCR4 faiblement affines pour CXCL12. Mes résultats suggèrent aussi que la résistance à CXCL12 accroit la capacité des virus à dépléter les LTCD4, notamment les LTCD4 effecteurs mémoires activés du sang périphérique. Mes résultats suggèrent que dans des sites anatomiques où la chimiokine CXCL12 est exprimée (e.g. tissus lymphoïdes), les virus RES infecteront plus efficacement les LTCD4, en particulier les cellules naïves. Ils seront donc plus enclins à altérer l'homéostasie des LTCD4, expliquant leur association avec des taux de LTCD4 bas chez les patients. Le fait que ces virus RES soient rares lors de la phase de primo-infection m'a aussi amenée dans le cadre d'un second axe à m'interroger sur les mécanismes qui conduisent les virus X4 à être contre-sélectionnés lors de la transmission. Ces résultats suggéraient que contrairement à l'idée prévalente, CXCL12 n'est pas le facteur majeur de la contre-sélection des virus X4 et que d'autres déterminants sont impliqués. Mes travaux suggèrent que des différences de tropisme cellulaire, d'antigénicité des Envs ou encore de sensibilité aux interférons de type I n'expliquent pas entièrement pourquoi les virus X4 sont moins efficacement transmis que les virus R5. D'autres hypothèses seront donc proposées à cet égard à la lumière de données récentes de la littérature. Dans son ensemble ce travail améliore notre compréhension de la physiopathologie et de la transmission du VIH-1. Aussi, l'identification que les virus RES utilisent des formes de CXCR4 différentes de celles utilisées par CXCL12 permet d'envisager de contrecarrer ces virus pour prévenir la déplétion des LTCD4 chez les patients VIH+ sous cART tout en préservant les fonctions homéostatiques essentielles de la chimiokine.The entry of HIV-1 into its target cells requires the interaction of its envelope glycoprotein (Env) on the surface of the virus with the receptor, CD4, and then with a co-receptor, CCR5 or CXCR4, depending on the viral tropism. Viruses that use CCR5 (R5 viruses) are predominantly represented in patients. Those using CXCR4, alone (X4 viruses) or in addition to CCR5 (R5X4 viruses) are rarely transmitted but their frequency increases during the course of the infection. A link between viral tropism and pathogenicity of the infection has been proposed. CXCR4-using viruses are more frequently associated with accelerated CD4TL decline than R5 viruses. Counterexamples to this general paradigm of increased pathogenicity of X4 viruses are, however, numerous, suggesting that there is heterogeneity in the pathogenicity of X4 viruses for which the molecular basis is not known. In my thesis, I studied the role of the natural ligand of CXCR4, i.e. the chemokine CXCL12, as a driving force of the evolution of these viruses during infection. Chemokine binding indeed blocks viral entry in vitro, suggesting that it represents a constraint to X4 viruses in vivo. We hypothesized that the viruses would evolve in the direction of escape from this constraint, leading to the shaping of their phenotypic properties, in particular their pathogenicity. To study this hypothesis, we measured the sensitivity to CXCL12 inhibition of about 30 viruses isolated from patients at different stages of infection. These experiments allowed us to identify that viruses resistant (RES) to CXCL12 inhibition are frequent in patients with low levels of CD4TL, frequently in the late stages of infection, more rarely during the primary infection. These results directed my work towards two axes, one dealing with the mechanisms of virus resistance to CXCL12 and its impact on the pathogenesis of the infection, the other with the mechanisms of X4 virus counter-selection during transmission. I showed that CXCL12 resistance is associated with changes in Env antigenic properties and results from viruses exploiting CXCR4 molecules that are weakly affine for CXCL12. My results also suggest that resistance to CXCL12 increases the ability of viruses to deplete CD4TL, including activated memory effector CD4TL from the peripheral blood. My results suggest that in anatomical sites where CXCL12 chemokine is expressed (e.g. lymphoid tissues), RES viruses will more efficiently infect CD4TL, especially naive cells. They will therefore be more likely to alter LTCD4 homeostasis, explaining their association with low LTCD4 levels in patients. The fact that these RES viruses are rare during the primary infection phase also led me in a second axis to question the mechanisms that lead X4 viruses to be counter-selected during transmission. These results suggested that contrary to the prevalent idea, CXCL12 is not the major factor in the counter-selection of X4 viruses and that other determinants are involved. My work suggests that differences in cell tropism, Envs antigenicity or type I interferon sensitivity do not fully explain why X4 viruses are less efficiently transmitted than R5 viruses. Other hypotheses will therefore be proposed in this respect in the light of recent data from the literature. Overall, this work improves our understanding of the pathophysiology and transmission of HIV-1. Also, the identification that RES viruses use different forms of CXCR4 than those used by CXCL12 allows us to consider counteracting these viruses to prevent CD4TL depletion in HIV+ patients on cART while preserving the essential homeostatic functions of the chemokine

Etude du dialogue entre les cellules dendritiques et les lymphocytes T CD4+

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

HIV-1 cell-to-cell spread overcomes the virus entry block of non-macrophage-tropic strains in macrophages

International audienceMacrophages (MΦ) are increasingly recognized as HIV-1 target cells involved in the pathogenesis and persistence of infection. Paradoxically, in vitro infection assays suggest that virus isolates are mostly T-cell-tropic and rarely MΦ-tropic. The latter are assumed to emerge under CD4+ T-cell paucity in tissues such as the brain or at late stage when the CD4 T-cell count declines. However, assays to qualify HIV-1 tropism use cell-free viral particles and may not fully reflect the conditions of in vivo MΦ infection through cell-to-cell viral transfer. Here, we investigated the capacity of viruses expressing primary envelope glycoproteins (Envs) with CCR5 and/or CXCR4 usage from different stages of infection, including transmitted/founder Envs, to infect MΦ by a cell-free mode and through cell-to-cell transfer from infected CD4+ T cells. The results show that most viruses were unable to enter MΦ as cell-free particles, in agreement with the current view that non-M-tropic viruses inefficiently use CD4 and/or CCR5 or CXCR4 entry receptors on MΦ. In contrast, all viruses could be effectively cell-to-cell transferred to MΦ from infected CD4+ T cells. We further showed that viral transfer proceeded through Env-dependent cell-cell fusion of infected T cells with MΦ targets, leading to the formation of productively infected multinucleated giant cells. Compared to cell-free infection, infected T-cell/MΦ contacts showed enhanced interactions of R5 M-and non-M-tropic Envs with CD4 and CCR5, resulting in a reduced dependence on receptor expression levels on MΦ for viral entry. Altogether, our results show that virus cellto-cell transfer overcomes the entry block of isolates initially defined as non-macrophagetropic, indicating that HIV-1 has a more prevalent tropism for MΦ than initially suggested. This sheds light into the role of this route of virus cell-to-cell transfer to MΦ in CD4+ T cell rich tissues for HIV-1 transmission, dissemination and formation of tissue viral reservoirs

Gut microbiota composition alterations are associated with the onset of diabetes in kidney transplant recipients.

MethodsPatients transplanted at our institution provided fecal samples before, and 3-9 months after KT. Fecal bacterial DNA was extracted and 9 bacteria or bacterial groups were quantified by qPCR.Results50 patients (19 controls without diabetes, 15 who developed New Onset Diabetes After Transplantation, NODAT, and 16 with type 2 diabetes before KT) were included. Before KT, Lactobacillus sp. tended to be less frequently detected in controls than in those who would become diabetic following KT (NODAT) and in initially diabetic patients (60%, 87.5%, and 100%, respectively, p = 0.08). The relative abundance of Faecalibacterium prausnitzii was 30 times lower in initially diabetic patients than in controls (p = 0.002). The relative abundance of F. prausnitzii of NODAT patients was statistically indistinguishable from controls and from diabetic patients. The relative abundance of Lactobacillus sp. increased following KT in NODAT and in initially diabetic patients (20-fold, p = 0.06, and 25-fold, p = 0.02, respectively). In contrast, the proportion of Akkermansia muciniphila decreased following KT in NODAT and in initially diabetic patients (2,500-fold, p = 0.04, and 50,000-fold, pConclusionAn alteration of the gut microbiota composition involving Lactobacillus sp., A. muciniphila and F. prausnitzii is associated with the glycemic status in KT recipients, raising the question of their role in the genesis of NODAT