The zebrafish as a new model for the in vivo study of Shigella flexneri interaction with phagocytes and bacterial autophagy.

Autophagy, an ancient and highly conserved intracellular degradation process, is viewed as a critical component of innate immunity because of its ability to deliver cytosolic bacteria to the lysosome. However, the role of bacterial autophagy in vivo remains poorly understood. The zebrafish (Danio rerio) has emerged as a vertebrate model for the study of infections because it is optically accessible at the larval stages when the innate immune system is already functional. Here, we have characterized the susceptibility of zebrafish larvae to Shigella flexneri, a paradigm for bacterial autophagy, and have used this model to study Shigella-phagocyte interactions in vivo. Depending on the dose, S. flexneri injected in zebrafish larvae were either cleared in a few days or resulted in a progressive and ultimately fatal infection. Using high resolution live imaging, we found that S. flexneri were rapidly engulfed by macrophages and neutrophils; moreover we discovered a scavenger role for neutrophils in eliminating infected dead macrophages and non-immune cell types that failed to control Shigella infection. We observed that intracellular S. flexneri could escape to the cytosol, induce septin caging and be targeted to autophagy in vivo. Depletion of p62 (sequestosome 1 or SQSTM1), an adaptor protein critical for bacterial autophagy in vitro, significantly increased bacterial burden and host susceptibility to infection. These results show the zebrafish larva as a new model for the study of S. flexneri interaction with phagocytes, and the manipulation of autophagy for anti-bacterial therapy in vivo

Septins restrict inflammation and protect zebrafish larvae from Shigella infection

Shigella flexneri, a Gram-negative enteroinvasive pathogen, causes inflammatory destruction of the human intestinal epithelium. Infection by S. flexneri has been well-studied in vitro and is a paradigm for bacterial interactions with the host immune system. Recent work has revealed that components of the cytoskeleton have important functions in innate immunity and inflammation control. Septins, highly conserved cytoskeletal proteins, have emerged as key players in innate immunity to bacterial infection, yet septin function in vivo is poorly understood. Here, we use S. flexneri infection of zebrafish (Danio rerio) larvae to study in vivo the role of septins in inflammation and infection control. We found that depletion of Sept15 or Sept7b, zebrafish orthologs of human SEPT7, significantly increased host susceptibility to bacterial infection. Live-cell imaging of Sept15-depleted larvae revealed increasing bacterial burdens and a failure of neutrophils to control infection. Strikingly, Sept15-depleted larvae present significantly increased activity of Caspase-1 and more cell death upon S. flexneri infection. Dampening of the inflammatory response with anakinra, an antagonist of interleukin-1 receptor (IL-1R), counteracts Sept15 deficiency in vivo by protecting zebrafish from hyper-inflammation and S. flexneri infection. These findings highlight a new role for septins in host defence against bacterial infection, and suggest that septin dysfunction may be an underlying factor in cases of hyper-inflammation

Stochastic modeling of T cell receptor gamma gene rearrangement.

The mechanisms controlling the recombination process of the gamma genes that encode the gamma chain of the antigen receptor of the gammadelta T lymphocytes are unclear. Based on experimental data on the recombination status of the two major TCR gamma genes expressed in V(gamma)4+ and V(gamma)1+ thymocytes, we tested the plausibility of three possible rearrangement mechanisms: (1) a time window mechanism according to which the two chromosomes are accessible to the recombination machinery during a defined period of time; (2) a feedback mechanism in which recombination stops shortly after the first in-frame rearrangement event anywhere in both chromosomes; and (3) a feedback mechanism with asynchronous chromosome accessibility, in which there is a first period when only one chromosome is accessible for recombination, followed by a second period when both chromosomes are accessible; shortly after the first in-frame rearrangement event, during any of these two periods, recombination will definitely stop. We model the time window mechanism using a pure probabilistic approach and the two feedback mechanisms using a continuous-time Markov chain formalism. We used maximum likelihood methodology to infer the goodness-of-fit of the models showing evidence for the last model, which best fits the data. Further analysis of this model suggests an evolutionary tradeoff between allelic and isotypic exclusion and the probability that a precursor differentiates into a mature gammadelta T lymphocyte

Mechanisms controlling termination of V-J recombination at the TCRgamma locus: implications for allelic and isotypic exclusion of TCRgamma chains.

Analyses of Vgamma-Jgamma rearrangements producing the most commonly expressed TCRgamma chains in over 200 gammadelta TCR(+) thymocytes showed that assembly of TCRgamma V-region genes display properties of allelic exclusion. Moreover, introduction of functionally rearranged TCRgamma and delta transgenes results in a profound inhibition of endogenous TCRgamma rearrangements in progenitor cells. The extent of TCRgamma rearrangements in these cells is best explained by a model in which initiation of TCRgamma rearrangements at both alleles is asymmetric, occurs at different frequencies depending on the V or J segments involved, and is terminated upon production of a functional gammadelta TCR. Approximately 10% of the cells studied contained two functional TCRgamma chains involving different V and Jgamma gene segments, thus defining a certain degree of isotypic inclusion. However, these cells are isotypically excluded at the level of cell surface expression possibly due to pairing restrictions between different TCRgamma and delta chains

Critical role of TCR specificity in the development of Vγ1Vδ6.3+ innate NKTγδ cells.

International audienceA large fraction of innate NKTγδ T cells uses TCRs composed of a semi-invariant Vδ6.3/6.4-Dδ2-Jδ1 chain together with more diverse Vγ1-Jγ4 chains. To address the role of γδTCR specificity in their generation, we analyzed their development in mice transgenic (Tg) for a Vγ1-Jγ4 chain frequently expressed by NKTγδ cells (Tg-γ) and in mice Tg for the same Vγ1-Jγ4 chain together with a Vδ6BDδ2Jδ1 chain not usually found among NKTγδ cells (Tg-γδ). Surprisingly, both promyelocytic leukemia zinc finger (PLZF)(+) and NK1.1(+) NKTγδ cells were found in the thymus of Tg-γδ albeit at lower numbers than in Tg-γ mice, and virtually all of them expressed the Tg TCR. However, the PLZF(+) subset, but not the NK1.1(+) subset, also expressed an endogenous Vδ6.3/6.4 chain, and its size was severely reduced in TCRδ(-/-) Tg-γδ mice. These results could suggest that the PLZF(+) and the NK1.1(+) subsets are developmentally unrelated. However, PLZF(+) and NK1.1(+) NKTγδ cells express identical Vδ6.3/6.4 chains, and NK1.1(+) cells can be obtained upon intrathymic injection of sorted PLZF(+) cells, thus indicating their developmental relationship. In fact, the NK1.1(+) γδ thymocytes present in Tg-γδ mice correspond to a small subset of NK1.1(+) γδ thymocytes in wild-type animals, which express a more diverse repertoire of TCRs and can be recognized by the expression of the CD62L Ag. Collectively, our data demonstrated that TCR specificity is essential for the development of most NKTγδ T cells and revealed a developmental heterogeneity in γδ T cells expressing the NK1.1 marker

Temporal Predisposition to αβ and γδ T Cell Fates in the Thymus.

International audienceHow T cell progenitors engage into the γδ or αβ T cell lineages is a matter of intense debate. In this study, we analyzed the differentiation potential of single thymocytes from wild-type and TCRγδ-transgenic mice at two sequential early developmental stages. Double-negative (DN) 3 progenitors from both wild-type and transgenic mice retain the capacity to engage into both pathways, indicating that full commitment is only completed after this stage. More importantly, DN2 and DN3 progenitors from TCRγδ transgenic mice have strong biases for opposite fates, indicating that developmentally regulated changes, other than the production of a functional TCR, altered their likelihood to become a γδ or an αβ T cell. Thus, unlike the differentiation in other hematopoietic lineages, T cell progenitors did not restrict, but rather switch their differentiation potential as they developed