Hepatic fibrosis is a major consequence of chronic liver disease such as non-alcoholic steatohepatitis which is undergoing a dramatic evolution given the obesity progression worldwide, and has no treatment to date. Hepatic stellate cells (HSCs) play a key role in the fibrosis process, because in chronic liver damage, they transdifferentiate from a “quiescent” to an “activated” phenotype responsible for most the collagen deposition in liver tissue. Here, using a diet-induced liver fibrosis murine model (choline-deficient amino acid-defined, high fat diet), we characterized a specific population of HSCs organized as clusters presenting simultaneously hypertrophy of retinoid droplets, quiescent and activated HSC markers. We showed that hypertrophied HSCs co-localized with fibrosis areas in space and time. Importantly, we reported the existence of this phenotype and its association with collagen deposition in three other mouse fibrosis models, including CCl 4 -induced fibrosis model. Moreover, we have also shown its relevance in human liver fibrosis associated with different etiologies (obesity, non-alcoholic steatohepatitis, viral hepatitis C and alcoholism). In particular, we have demonstrated a significant positive correlation between the stage of liver fibrosis and HSC hypertrophy in a cohort of obese patients with hepatic fibrosis. These results lead us to conclude that hypertrophied HSCs are closely associated with hepatic fibrosis in a metabolic disease context and may represent a new marker of metabolic liver disease progression.

Background: Microglial cells are tissue-resident macrophages of the central nervous system. They are extremely dynamic, sensitive to their microenvironment and present a characteristic complex and heterogeneous morphology and distribution within the brain tissue. Many experimental clues highlight a strong link between their morphology and their function in response to aggression. However, due to their complex “dendritic-like” aspect that constitutes the major pool of murine microglial cells and their dense network, precise and powerful morphological studies are not easy to realize and complicate correlation with molecular or clinical parameters. Methods: Using the knock-in mouse model CX3CR1, we developed a 3D automated confocal tissue imaging system coupled with morphological modelling of many thousands of microglial cells revealing precise and quantitative assessment of major cell features: cell density, cell body area, cytoplasm area and number of primary, secondary and tertiary processes. We determined two morphological criteria that are the complexity index (CI) and the covered environment area (CEA) allowing an innovative approach lying in (i) an accurate and objective study of morphological changes in healthy or pathological condition, (ii) an in situ mapping of the microglial distribution in different neuroanatomical regions and (iii) a study of the clustering of numerous cells, allowing us to discriminate different sub-populations. Results: Our results on more than 20,000 cells by condition confirm at baseline a regional heterogeneity of the microglial distribution and phenotype that persists after induction of neuroinflammation by systemic injection of lipopolysaccharide (LPS). Using clustering analysis, we highlight that, at resting state, microglial cells are distributed in four microglial sub-populations defined by their CI and CEA with a regional pattern and a specific behaviour after challenge. Conclusions: Our results counteract the classical view of a homogenous regional resting state of the microglial cells within the brain. Microglial cells are distributed in different defined sub-populations that present specific behaviour after pathological challenge, allowing postulating for a cellular and functional specialization. Moreover, this new experimental approach will provide a support not only to neuropathological diagnosis but also to study microglial function in various disease models while reducing the number of animals needed to approach the international ethical statements.

Background: Skeletal muscle has the capacity to adapt to environmental changes and regenerate upon injury. To study these processes, most experimental methods use quantification of parameters obtained from images of immunostained skeletal muscle. Muscle cross-sectional area, fiber typing, localization of nuclei within the muscle fiber, the number of vessels, and fiber-associated stem cells are used to assess muscle physiology. Manual quantification of these parameters is time consuming and only poorly reproducible. While current state-of-the-art software tools are unable to analyze all these parameters simultaneously, we have developed MuscleJ, a new bioinformatics tool to do so. Methods: Running on the popular open source Fiji software platform, MuscleJ simultaneously analyzes parameters from immunofluorescent staining, imaged by different acquisition systems in a completely automated manner. Results: After segmentation of muscle fibers, up to three other channels can be analyzed simultaneously. Dialog boxes make MuscleJ easy-to-use for biologists. In addition, we have implemented color in situ cartographies of results, allowing the user to directly visualize results on reconstituted muscle sections. Conclusion: We report here that MuscleJ results were comparable to manual observations made by five experts. MuscleJ markedly enhances statistical analysis by allowing reliable comparison of skeletal muscle physiology-pathology results obtained from different laboratories using different acquisition systems. Providing fast robust multi-parameter analyses of skeletal muscle physiology-pathology, MuscleJ is available as a free tool for the skeletal muscle community.

We consider in review current state-of-the-art fluorescence microscopy for investigating the host-pathogen interface. Our perspective is honed from years with literally thousands of microbiologists using the variety of imaging technologies available within our dedicated BSL2/BSL3 optical imaging research service facilities at the Institut Pasteur Paris founded from scratch in 2001. During fifteen years learning from the success and failures of introducing different fluorescence imaging technologies, methods, and technical development strategies we provide here a synopsis review of our experience to date and a synthesis of how we see the future in perspective for fluorescence imaging at the host-pathogen interface.

Arthropod-borne virus (arbovirus) infections cause several emerging and resurgent infectious diseases in humans and animals. Chikungunya-affected areas often overlap with dengue-endemic areas. Concurrent dengue virus (DENV) and chikungunya virus (CHIKV) infections have been detected in travelers returning from regions of endemicity. CHIKV and DENV co-infected Aedes albopictus have also been collected in the vicinity of co-infected human cases, emphasizing the need to study co-infections in mosquitoes. We thus aimed to study the pathogen-pathogen interaction involved in these co-infections in DENV/CHIKV co-infected Aedes aegypti mosquitoes. In mono-infections, we detected CHIKV antigens as early as 4 days post-virus exposure in both the midgut (MG) and salivary gland (SG), whereas we detected DENV serotype 2 (DENV-2) antigens from day 5 post-virus exposure in MG and day 10 post-virus exposure in SG. Identical infection rates were observed for singly and co-infected mosquitoes, and facilitation of the replication of both viruses at various times post-viral exposure. We observed a higher replication for DENV-2 in SG of co-infected mosquitoes. We showed that mixed CHIKV and DENV infection facilitated viral replication in Ae. aegypti. The outcome of these mixed infections must be further studied to increase our understanding of pathogen-pathogen interactions in host cells.

BACKGROUND:Microglial cells are tissue-resident macrophages of the central nervous system. They are extremely dynamic, sensitive to their microenvironment and present a characteristic complex and heterogeneous morphology and distribution within the brain tissue. Many experimental clues highlight a strong link between their morphology and their function in response to aggression. However, due to their complex "dendritic-like" aspect that constitutes the major pool of murine microglial cells and their dense network, precise and powerful morphological studies are not easy to realize and complicate correlation with molecular or clinical parameters.METHODS:Using the knock-in mouse model CX3CR1(GFP/+), we developed a 3D automated confocal tissue imaging system coupled with morphological modelling of many thousands of microglial cells revealing precise and quantitative assessment of major cell features: cell density, cell body area, cytoplasm area and number of primary, secondary and tertiary processes. We determined two morphological criteria that are the complexity index (CI) and the covered environment area (CEA) allowing an innovative approach lying in (i) an accurate and objective study of morphological changes in healthy or pathological condition, (ii) an in situ mapping of the microglial distribution in different neuroanatomical regions and (iii) a study of the clustering of numerous cells, allowing us to discriminate different sub-populations.RESULTS:Our results on more than 20,000 cells by condition confirm at baseline a regional heterogeneity of the microglial distribution and phenotype that persists after induction of neuroinflammation by systemic injection of lipopolysaccharide (LPS). Using clustering analysis, we highlight that, at resting state, microglial cells are distributed in four microglial sub-populations defined by their CI and CEA with a regional pattern and a specific behaviour after challenge.CONCLUSIONS:Our results counteract the classical view of a homogenous regional resting state of the microglial cells within the brain. Microglial cells are distributed in different defined sub-populations that present specific behaviour after pathological challenge, allowing postulating for a cellular and functional specialization. Moreover, this new experimental approach will provide a support not only to neuropathological diagnosis but also to study microglial function in various disease models while reducing the number of animals needed to approach the international ethical statements.

After cell entry, HIV undergoes rapid transport toward the nucleus using microtubules and microfilaments. Neither the cellular cytoplasmic components nor the viral proteins that interact to mediate transport have yet been identified. Using a yeast two-hybrid screen, we identified four cytoskeletal components as putative interaction partners for HIV-1 p24 capsid protein: MAP1A, MAP1S, CKAP1, and WIRE. Depletion of MAP1A/MAP1S in indicator cell lines and primary human macrophages led to a profound reduction in HIV-1 infectivity as a result of impaired retrograde trafficking, demonstrated by a characteristic accumulation of capsids away from the nuclear membrane, and an overall defect in nuclear import. MAP1A/MAP1S did not impact microtubule network integrity or cell morphology but contributed to microtubule stabilization, which was shown previously to facilitate infection. In addition, we found that MAP1 proteins interact with HIV-1 cores both in vitro and in infected cells and that interaction involves MAP1 light chain LC2. Depletion of MAP1 proteins reduced the association of HIV-1 capsids with both dynamic and stable microtubules, suggesting that MAP1 proteins help tether incoming viral capsids to the microtubular network, thus promoting cytoplasmic trafficking. This work shows for the first time that following entry into target cells, HIV-1 interacts with the cytoskeleton via its p24 capsid protein. Moreover, our results support a role for MAP1 proteins in promoting efficient retrograde trafficking of HIV-1 by stimulating the formation of stable microtubules and mediating the association of HIV-1 cores with microtubules.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disease caused by CAG expansion in the huntingtin gene, which adds a homopolymeric tract of polyglutamine (polyQ) to the encoded protein leading to the formation of toxic aggregates. Despite rapidly accumulating evidences supporting a role for intercellular transmission of protein aggregates, little is known about whether and how huntingtin (Htt) misfolding progresses through the brain. It has been recently reported that synthetic polyQ peptides and recombinant fragments of mutant Htt are readily internalized in cell cultures and able to seed polymerization of a reporter wild-type Htt. However, there is no direct evidence of aggregate transfer between cells and the mechanism has not been explored. By expressing recombinant fragments of mutant Htt in neuronal cells and in primary neurons, we found that aggregated fragments formed within one cell spontaneously transfer to neighbors in cell culture. We demonstrate that the intercellular spreading of the aggregates requires cell cell contact and does not occur upon aggregate secretion. Interestingly, we found that the expression of mutant, but not wild-type Htt fragments, increases the number of tunneling nanotubes, which in turn provide an efficient mechanism of transfer.

“Core facilities” have become an integral part of modern biomedical research infrastructures and today require integrated management tools to help ensure their optimization for research. Web-based software presents great opportunities, but requires innovation inasmuch as the current generation of laboratory information management systems (LIMS) is mostly comprised of automatons overseeing predictable laboratory equipment processes. By contrast, core facility processes involve not just equipment, and services, but also people: facility staff, users, administrators, PIs etc., each with their own exigencies and unpredictability. Here we present software developed during the last ten years in an academic-commercial partnership, which began as a community-driven effort. Conceived from the outset as a core facility management tool to answer the specific needs of multiple facility research infrastructures. We used an innovative ethnography approach whereby the software design extrapolates manifold use-cases using a framework of co-existing rules and policy matrices that can be constantly tuned by admin. The system can vary the outcome of multiple processes, integral to multiple users, and multiple facilities in parallel, based upon real-time context and diverse metadata that define, for example, a service, a piece of equipment, a preventative action, a training or even a metrology. Here we report on the performance and impact of the software in the singular case-history example of the Institut Pasteur Paris where the software was conceived. Our analyses of the facility evolution and the software's development during ten years reveals evangelization of the user community based on its improvements to better answer the needs of the scientific community. Our results demonstrate a key role for the software bolstering long-term downstream benefits including increased funding, higher scientific output, and quality assurances for services rendered.

ABSTRACT Mucin glycoproteins are secreted in large amounts by the intestinal epithelium and constitute an efficient component of innate immune defenses to promote homeostasis and protect against enteric pathogens. In this study, our objective was to investigate how the bacterial enteropathogen Shigella flexneri , which causes bacillary dysentery, copes with the mucin defense barrier. We report that upon in vitro infection of mucin-producing polarized human intestinal epithelial cells, virulent S. flexneri manipulates the secretion of gel-forming mucins. This phenomenon, which is triggered only by virulent strains, results in accumulation of mucins at the cell apical surface, leading to the appearance of a gel-like structure that favors access of bacteria to the cell surface and the subsequent invasion process. We identify MUC5AC, a gel-forming mucin, as a component of this structure. Formation of this gel does not depend on modifications of electrolyte concentrations, induction of trefoil factor expression, endoplasmic reticulum stress, or response to unfolded proteins. In addition, transcriptional and biochemical analyses of infected cells reveal modulations of mucin gene expression and modifications of mucin glycosylation patterns, both of which are induced by virulent bacteria in a type III secretion system-dependent manner. Thus, S. flexneri has developed a dedicated strategy to alter the mucus barrier by targeting key elements of the gel-forming capacity of mucins: gene transcription, protein glycosylation, and secretion.

forming mucins in mucus-producing cells 2 3 Brice Sperandio, Natalie Fischer, Marie Joncquel Chevalier-Curt, Yannick Rossez, 4 Pascal Roux, Catherine Robbe Masselot, and Philippe J. Sansonetti 5 6 Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, 75724 Paris cedex 15, France 7 INSERM U786, Institut Pasteur, 75724 Paris cedex 15, France 8 Université Lille Nord de France, 59000 Lille, France 9 USTL, UGSF, IFR 147, 59650 Villeneuve d’Ascq, France 10 CNRS, UMR8576, 59650 Villeneuve d’Ascq, France 11 Plateforme d’Imagerie Dynamique, Institut Pasteur, 75724 Paris cedes 15, France 12 Microbiologie et Maladies Infectieuses, Collège de France, 75231 Paris cedex 05, France 13 14 Corresponding author: Philippe J. Sansonetti, Unité de Pathogénie Microbienne Moléculaire, 15

The Gram-negative enteroinvasive bacterium Shigella flexneri is responsible for the endemic form of bacillary dysentery, an acute rectocolitis in humans. S. flexneri uses a type III secretion system to inject effector proteins into host cells, thus diverting cellular functions to its own benefit. Protective immunity to reinfection requires several rounds of infection to be elicited and is short-lasting, suggesting that S. flexneri interferes with the priming of specific immunity. Considering the key role played by T-lymphocyte trafficking in priming of adaptive immunity, we investigated the impact of S. flexneri on T-cell dynamics in vivo. By using two-photon microscopy to visualize bacterium–T-cell cross-talks in the lymph nodes, where the adaptive immunity is initiated, we provide evidence that S. flexneri , via its type III secretion system, impairs the migration pattern of CD4 + T cells independently of cognate recognition of bacterial antigens. We show that bacterial invasion of CD4 + T lymphocytes occurs in vivo, and results in cell migration arrest. In the absence of invasion, CD4 + T-cell migration parameters are also dramatically altered. Signals resulting from S. flexneri interactions with subcapsular sinus macrophages and dendritic cells, and recruitment of polymorphonuclear cells are likely to contribute to this phenomenon. These findings indicate that S. flexneri targets T lymphocytes in vivo and highlight the role of type III effector secretion in modulating host adaptive immune responses.

Fluorescence-based imaging regimes require exposure of living samples under study to high intensities of focused incident illumination. An often underestimated, overlooked, or simply ignored fact in the design of any experimental imaging protocol is that exposure of the specimen to these excitation light sources must itself always be considered a potential source of phototoxicity. This can be problematic, not just in terms of cell viability, but much more worrisome in its more subtle manifestation where phototoxicity causes anomalous behaviors that risk to be interpreted as significant, whereas they are mere artifacts. This is especially true in the case of microbial pathogenesis, where host-pathogen interactions can prove especially fragile to light exposure in a manner that can obscure the very processes we are trying to observe. For these reasons, it is important to be able to bring the parameter of phototoxicity into the equation that brings us to choose one fluorescent imaging modality, or setup, over another. Further, we need to be able to assess the risk that phototoxicity may occur during any specific imaging experiment. To achieve this, we describe here a methodological approach that allows meaningful measurement, and therefore relative comparison of phototoxicity, in most any variety of different imaging microscopes. In short, we propose a quantitative approach that uses microorganisms themselves to reveal the range over which any given fluorescent imaging microscope will yield valid results, providing a metrology of phototoxic damage, distinct from photobleaching, where a clear threshold for phototoxicity is identified. Our method is widely applicable and we show that it can be adapted to other paradigms, including mammalian cell models.

The extracellular matrix (ECM) and its role in the outcome of infectious diseases have been poorly investigated. In this study, we determined the impact of the collagen fibres architecture on the invasive process of the enteric parasite Entamoeba histolytica. The behaviour of E. histolytica wild-type and silenced for the cysteine protease A5 (CP-A5) were compared on a three-dimensional collagen matrix and within human colon fragments for fibrillar collagen cleavage and migration. The interstitial collagen fibres within the connective tissue of the human colon, visualized by multiphoton and second harmonic generation signals imaging, presented a dense scaffold at the subepithelial level and a loose meshwork within the chorion. To penetrate the tissue, E. histolytica migrated on the dense scaffold that remained intact, reached the crypt of Lieberkhün, migrated along and then disorganized the loose scaffold to escape into the mucosa. Interestingly, in vitro, CP-A5 was not required for collagenase activity and migration through the matrix but was necessary within the tissue environment for collagen meshwork remodelling and subsequent invasion. The data point out that further step of invasion relay with ECM destruction that requires human components induced or activated in the presence of CP-A5.

The nuclear pore complex (NPC) mediates nucleo-cytoplasmic transport of macromolecules and is an obligatory point of passage and functional bottleneck in the replication of some viruses. The Human Immunodeficiency Virus (HIV) has evolved the required mechanisms for active nuclear import of its genome through the NPC. However the mechanisms by which the NPC allows or even assists HIV translocation are still unknown. We investigated the involvement of four key nucleoporins in HIV-1 docking, translocation, and integration: Nup358/RanBP2, Nup214/CAN, Nup98 and Nup153. Although all induce defects in infectivity when depleted, only Nup153 actually showed any evidence of participating in HIV-1 translocation through the nuclear pore. We show that Nup358/RanBP2 mediates docking of HIV-1 cores on NPC cytoplasmic filaments by interacting with the cores and that the C-terminus of Nup358/RanBP2 comprising a cyclophilin-homology domain contributes to binding. We also show that Nup214/CAN and Nup98 play no role in HIV-1 nuclear import per se: Nup214/CAN plays an indirect role in infectivity read-outs through its effect on mRNA export, while the reduction of expression of Nup98 shows a slight reduction in proviral integration. Our work shows the involvement of nucleoporins in diverse and functionally separable steps of HIV infection and nuclear import.

BACKGROUND:Arthropod borne virus infections cause several emerging and resurgent infectious diseases. Among the diseases caused by arboviruses, dengue and chikungunya are responsible for a high rate of severe human diseases worldwide. The midgut of mosquitoes is the first barrier for pathogen transmission and is a target organ where arboviruses must replicate prior to infecting other organs. A proteomic approach was undertaken to characterize the key virus/vector interactions and host protein modifications that happen in the midgut for viral transmission to eventually take place.METHODOLOGY AND PRINCIPAL FINDINGS:Using a proteomics differential approach with two-Dimensional Differential in-Gel Electrophoresis (2D-DIGE), we defined the protein modulations in the midgut of Aedes aegypti that were triggered seven days after an oral infection (7 DPI) with dengue 2 (DENV-2) and chikungunya (CHIKV) viruses. Gel profile comparisons showed that the level of 18 proteins was modulated by DENV-2 only and 12 proteins were modulated by CHIKV only. Twenty proteins were regulated by both viruses in either similar or different ways. Both viruses caused an increase of proteins involved in the generation of reactive oxygen species, energy production, and carbohydrate and lipid metabolism. Midgut infection by DENV-2 and CHIKV triggered an antioxidant response. CHIKV infection produced an increase of proteins involved in detoxification.CONCLUSION/SIGNIFICANCE:Our study constitutes the first analysis of the protein response of Aedes aegypti's midgut infected with viruses belonging to different families. It shows that the differentially regulated proteins in response to viral infection include structural, redox, regulatory proteins, and enzymes for several metabolic pathways. Some of these proteins like antioxidant are probably involved in cell protection. On the other hand, we propose that the modulation of other proteins like transferrin, hsp60 and alpha glucosidase, may favour virus survival, replication and transmission, suggesting a subversion of the insect cell metabolism by the arboviruses.

Environmental cues are critical determinants of the fate of neural progenitors (NPs) upon transplantation into the central nervous system. In the present study, we assessed the differentiation potential of NPs implanted in a cholinergic environment of the adult mouse brain. Neurospheres containing NPs issued from fetal ganglionic eminences of transgenic mice expressing the green fluorescent protein (GFP) were transplanted either inside or outside the mouse cholinergic facial motor nucleus. In some mice, a pre-degenerated nerve releasing trophic factors was grafted into this nucleus to favor NP survival and improve axonal growth into the graft. The fate of NPs was analyzed 6 to 9 days or 2 months post-transplantation by immunofluorescence under confocal microscopy. Transplanted NPs were observed both inside and outside the facial nucleus after 6 to 9 days, but almost exclusively inside after 2 months regardless of the presence of a pre-degenerated nerve. NPs expressed markers of undifferentiated cells, astrocytes, oligodendrocytes, neurons, or cholinergic cells. The cholinergic phenotype of NPs engrafted inside the facial nucleus increased with time and the presence of a pre-degenerated nerve. Large GFP cholinergic somata and abundant long cholinergic GFP axons projecting into the nerve graft were also observed. Our results show that NPs, isolated from fetal mouse brain and transplanted into the non-neurogenic environment of the adult mouse facial nucleus, differentiate into cholinergic cells capable to project axons. This environment and the nerve graft favored NP differentiation into cholinergic neurons.

Human neurons express the innate immune response receptor, Toll-like receptor 3 (TLR3). TLR3 levels are increased in pathological conditions such as brain virus infection. Here, we further investigated the production, cellular localisation, and function of neuronal TLR3 during neuronotropic rabies virus (RABV) infection in human neuronal cells. Following RABV infection, TLR3 is not only present in endosomes, as observed in the absence of infection, but also in detergent-resistant perinuclear inclusion bodies. As well as TLR3, these inclusion bodies contain the viral genome and viral proteins (N and P, but not G). The size and composition of inclusion bodies and the absence of a surrounding membrane, as shown by electron microscopy, suggest they correspond to the previously described Negri Bodies (NBs). NBs are not formed in the absence of TLR3, and TLR3(-/-) mice -- in which brain tissue was less severely infected -- had a better survival rate than WT mice. These observations demonstrate that TLR3 is a major molecule involved in the spatial arrangement of RABV-induced NBs and viral replication. This study shows how viruses can exploit cellular proteins and compartmentalisation for their own benefit.

Pulmonary infections due to Aspergillus fumigatus result from the development of a colony of tightly associated hyphae in contact with the air, either in the alveoli (invasive aspergillosis) or in an existing cavity (aspergilloma). The fungal ball observed in vivo resembles an aerial colony obtained in agar medium in vitro more than a mycelial mass obtained in liquid shaken conditions that have been classically used to date to study A. fumigatus physiology. For this reason, we embarked on an analysis of the characteristics of A. fumigatus colonies grown in aerial static conditions. (i) Under static aerial conditions, mycelial growth is greater than in shaken, submerged conditions. (ii) The colony surface of A. fumigatus revealed the presence of an extracellular hydrophobic matrix that acts as a cohesive linkage bonding hyphae into a contiguous sheath. (iii) The extracellular matrix is composed of galactomannan, alpha 1,3 glucans, monosaccharides and polyols, melanin and proteins including major antigens and hydrophobins. (iv) A. fumigatus colonies were more resistant to polyenes than shake, submerged mycelium. This is the first analysis of the three dimensional structure of a mycelial colony. Knowledge of this multicellular organization will impact our future understanding of the pathobiology of aerial mold pathogens.