Gérard Eberl

RORt, a multitask nuclear receptor at mucosal surfaces

RORt is a nuclear hormone receptor that has followed an exponential success carrier.

Its modest origins as an orphan receptor cloned from human pancreas blossomed within 15 years into a critical regulator of anti-microbial immunity and a major target in the fight against inflammatory pathologies. Here, I review its role as a transcription factor required for the generation of type 3 lymphoid cells, which induce the development of lymphoid tissues, provide resistance of epithelial stem cells to injury, maintain homeostasis with the symbiotic microbiota,orchestrate defense against extracellular microbes and regulate allergic responses. RORt is also an intriguing molecule that is regulated by the circadian rhythm and includes cholesterol metabolites as ligands. RORttherefore links anti-microbial immunity with circadian rhythms and steroids, the logic of which remains to be understood.

A brief history of RORt

Two independent lines of research lead to the discovery ofROR and its shorter isoform RORt. In the first line of research, PCR primers were designed to amplify cDNAs coding for conserved regions of the DNA binding domain of nuclear hormone receptors. This strategy aimed for the cloning of presumably all nuclear receptors expressed in a given tissue or cell line. Using this approach, novel orphan receptors named retinoid Z receptor (RZR) and RZRwere then identified in umbilical vain endothelial cells [START_REF] Becker-Andre | Identification of nuclear receptor mRNAs by RT-PCR amplification of conserved zinc-finger motif sequences[END_REF] and rat brain [START_REF] Carlberg | a new family of retinoid-related orphan receptors that function as both monomers and homodimers[END_REF] . In addition, using hybridization cloning with the cDNA of the retinoic acid receptor (RAR), retinoic acid related orphan receptor (ROR) was identified in rat brain and shown to be identical to RZR [START_REF] Giguere | Isoform-specific amino-terminal domains dictate DNAbinding properties of ROR alpha, a novel family of orphan hormone nuclear receptors[END_REF] . This lead to the identification of the third member of the RZR/ROR family, ROR, in a cDNA library from human pancreas,and later from mouse muscle, using the degenerate primer approach [START_REF] Hirose | the third member of ROR/RZR orphan receptor subfamily that is highly expressed in skeletal muscle[END_REF][START_REF] Medvedev | Cloning of a cDNA encoding the murine orphan receptor RZR/ROR and characterization of its response element[END_REF] . Interestingly, melatonin was reported to activate the transcription factor activity of ROR and RORin cultured cells [START_REF] Becker-Andre | Pineal gland hormone melatonin binds and activates an orphan of the nuclear receptor superfamily[END_REF][START_REF] Wiesenberg | Transcriptional activation of the nuclear receptor RZR  by the pineal gland hormone melatonin and identification of CGP 52608 as a synthetic ligand[END_REF][START_REF] Carlberg | The orphan receptor family RZR/ROR, melatonin and 5-lipoxygenase: an unexpected relationship[END_REF] , suggesting early on a role forthe ROR family of receptors in circadian rhythms.

In the second line of research, in a screen for proteins that confer resistance to TCR-mediated apoptosis,a shorter isoform of ROR was identified by expression cloning of a thymocyte cDNA library in a T cell hybridoma [START_REF] He | a novel isoform of an orphan receptor, negatively regulates Fas ligand expression and IL-2 production in T cells[END_REF] . While ROR is expressed in a variety of organs, this new isoform was found to be primarily expressed in the thymus and was therefore named RORt. Mice that lacked the expression of both ROR and RORt showed increased thymocyte apoptosis [START_REF] He | a novel isoform of an orphan receptor, negatively regulates Fas ligand expression and IL-2 production in T cells[END_REF][START_REF] Sun | Requirement for ROR in thymocyte survival and lymphoid organ development[END_REF][START_REF] Kurebayashi | Retinoid-related orphan receptor  (ROR) is essential for lymphoid organogenesis and controls apoptosis during thymopoiesis[END_REF] . Such mice also had theremarkable phenotype of lacking lymph nodes and Peyer's patches.

In knock-in mice in which the Rorc(t) locus coded for GFP instead of RORt, but still expressed ROR, it was shown that RORt is expressed by and is required for the development of fetal lymphoid tissue inducer (LTi) cells [START_REF] Eberl | An essential function for the nuclear receptor RORγt in the generation of fetal lymphoid tissue inducer cells[END_REF] , a then enigmatic cell type that colonizes developing lymphoid tissues in the fetus before any other hematopoietic cell type [START_REF] Mebius | A developmental switch in lymphocyte homing receptor and endothelial vascular addressin expression regulates lymphocyte homing and permits CD4 + CD3cells to colonize lymph nodes[END_REF][START_REF] Mebius | Developing lymph nodes collect CD4 + CD3 -LT + cells that can differentiate to APC, NK cells, and follicular cells but not T or B cells[END_REF][START_REF] Adachi | Three distinctive steps in Peyer's patch formation of murine embryo[END_REF] .

It was nevertheless the association of RORt with Th17 cells that shot RORt to universal fame. In a landmark paper by Dan Cua and colleagues, it was demonstrated that IL-23, rather than IL-12,is the critical cytokine in experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis [START_REF] Cua | Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain[END_REF] . In a second paper, Cua reported that IL-23 promotes the generation of Th17 cells, the effector cells that driveEAE [START_REF] Langrish | IL-23 drives a pathogenic T cell population that induces autoimmune inflammation[END_REF] . On the basis of transcriptome profiles, Th17 cells were found to express high levels of RORt as compared to Th1 cells, an observation that lead to the demonstration that Th17, like LTi cells, expressed RORt and required RORt for their generation [START_REF] Ivanov | The orphan nuclear receptor RORt directs the differentiation program of proinflammatory IL-17 + T helper cells[END_REF] .

Two years later, the world of RORt + cells expanded again. A population of LTi-like cells was identified that express markers of NK cells, such as NKp46, as well as the signature cytokines IL-17 and IL-22, but do not cluster and induce the development of lymphoid tissues [START_REF] Satoh-Takayama | Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense[END_REF][START_REF] Luci | Influence of the transcription factor RORgammat on the development of NKp46+ cell populations in gut and skin[END_REF][START_REF] Sanos | RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp46+ cells[END_REF][START_REF] Cella | A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity[END_REF] . To keep controlof the expanding universe of LTi-likecells, the term "innate lymphoid cells" (ILCs) was coined [START_REF] Spits | Innate lymphoid cells--a proposal for uniform nomenclature[END_REF][START_REF] Spits | The expanding family of innate lymphoid cells: regulators and effectors of immunity and tissue remodeling[END_REF] . It was progressively realized that the ILC universe includes ILC1s, ILC2s and (RORt + ) ILC3s, which mirror Th1, Th2 and Th17 cells in the expression of signature transcription factors, surface markers and effector cytokines [START_REF] Eberl | Innate lymphoid cells: a new paradigm in immunology[END_REF] .

A recent observation brings RORt back full circle to its original description as a cousin of the melatonin receptorsROR and ROR [START_REF] Becker-Andre | Pineal gland hormone melatonin binds and activates an orphan of the nuclear receptor superfamily[END_REF][START_REF] Wiesenberg | Transcriptional activation of the nuclear receptor RZR  by the pineal gland hormone melatonin and identification of CGP 52608 as a synthetic ligand[END_REF][START_REF] Carlberg | The orphan receptor family RZR/ROR, melatonin and 5-lipoxygenase: an unexpected relationship[END_REF] . RORis a component of the transcriptional network of peripheral circadian clocks [START_REF] Preitner | The orphan nuclear receptor REV-ERB controls circadian transcription within the positive limb of the mammalian circadian oscillator[END_REF][START_REF] Ueda | A transcription factor response element for gene expression during circadian night[END_REF] , which regulates the transcription factor Nfil3, which in turn represses the expression of RORt [START_REF] Yu | TH17 cell differentiation is regulated by the circadian clock[END_REF] .

Deregulation of the circadian clock thus leads to a deregulation of Th17 cells and increased susceptibility to inflammatory pathology. Finally, and no less intriguing, cholesterol metabolites are natural ligands of RORt [START_REF] Santori | Identification of natural RORgamma ligands that regulate the development of lymphoid cells[END_REF][START_REF] Cyster | 25-Hydroxycholesterols in innate and adaptive immunity[END_REF] , possibly linking type 3 immunity to the endocrine system and metabolism, even though the biology of these ligands remains to be understood.

RORt and lymphoid tissue development

LTi cells were first describedas CD3 -CD4 + or CD3 -IL-7R + cells in the developing lymph nodes (LNs) [START_REF] Mebius | A developmental switch in lymphocyte homing receptor and endothelial vascular addressin expression regulates lymphocyte homing and permits CD4 + CD3cells to colonize lymph nodes[END_REF][START_REF] Mebius | Developing lymph nodes collect CD4 + CD3 -LT + cells that can differentiate to APC, NK cells, and follicular cells but not T or B cells[END_REF] and Peyer's patches (PPs) [START_REF] Adachi | Three distinctive steps in Peyer's patch formation of murine embryo[END_REF] (Figure 1). Mebius' and Nishikawa's labs characterizedLTi cells and their interactions with stroma cellswithin the lymphoid tissue anlagen [START_REF] Yoshida | IL-7 receptor  + CD3cells in the embryonic intestine induces the organizing center of Peyer's patches[END_REF][START_REF] Honda | Molecular basis for hematopoietic/mesenchymal interaction during initiation of Peyer's patch organogenesis[END_REF] , but it is the discovery of RORt that allowed to demonstrate formally that LTi cells are required for the development of lymphoid tissues [START_REF] Eberl | An essential function for the nuclear receptor RORγt in the generation of fetal lymphoid tissue inducer cells[END_REF] . In the fetus, LTi cells are the only cells expressing RORt. In RORtdeficient mice, LTi cells are absent and LNs and PPs fail to develop. These cells express several members of the TNF superfamily, such as soluble lymphotoxin (LT)  3 and its membrane-bound variant LT 1  2 , TRANCE and TRANCE-L [START_REF] Mebius | Developing lymph nodes collect CD4 + CD3 -LT + cells that can differentiate to APC, NK cells, and follicular cells but not T or B cells[END_REF][START_REF] Yoshida | IL-7 receptor  + CD3cells in the embryonic intestine induces the organizing center of Peyer's patches[END_REF][START_REF] Kim | Regulation of peripheral lymph node genesis by the tumor necrosis factor family member TRANCE[END_REF] . LT 1  2 is essential for the development of lymphoid tissues [START_REF] De Togni | Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin[END_REF][START_REF] Rennert | Surface lymphotoxin / complex is required for the development of peripheral lymphoid organs[END_REF][START_REF] Alimzhanov | Abnormal development of secondary lymphoid tissues in lymphotoxin -deficient mice[END_REF] , as it engages LTR on specialized stroma cells, which in turninduces the expression of adhesion molecules and chemokines that recruit lymphocytes and myeloid cells [START_REF] Dejardin | The lymphotoxin- receptor induces different patterns of gene expression via two NF-kappaB pathways[END_REF] .

After birth, LTi cells cluster in hundreds of so-called cryptopatches (CPs)located between crypts of the intestinal lamina propria [START_REF] Kanamori | Identification of novel lymphoid tissues in murine intestinal mucosa where clusters of c-kit + IL-7R + Thy1 + lympho-hemopoietic progenitors develop[END_REF][START_REF] Eberl | Thymic origin of intestinal  T cells revealed by fate mapping of RORt + cells[END_REF] . CPs collect B cells todevelop into isolated lymphoid follicles (ILFs), which generate mostly T cellindependent IgA + B cells [START_REF] Hamada | Identification of multiple isolated lymphoid follicles on the antimesenteric wall of the mouse small intestine[END_REF][START_REF] Lorenz | Isolated lymphoid follicle formation is inducible and dependent upon lymphotoxin-sufficient B lymphocytes, lymphotoxin  receptor, and TNF receptor I function[END_REF][START_REF] Tsuji | Requirement for lymphoid tissue-inducer cells in isolated follicle formation and T cell-independent immunoglobulin a generation in the gut[END_REF] . Surprisingly, the formation of ILFs from CPs requires bacterial microbiota [START_REF] Lorenz | Isolated lymphoid follicle formation is inducible and dependent upon lymphotoxin-sufficient B lymphocytes, lymphotoxin  receptor, and TNF receptor I function[END_REF][START_REF] Bouskra | Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis[END_REF] . Proliferating bacteria release peptidoglycans from their cell wall, which is recognized by the innate receptor NOD-1 in epithelial cells [START_REF] Girardin | Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan[END_REF] . This unleashes an activation cascade through the release of CCL-20, the activation of the CCR6 + (the receptor for CCL-20) LTi cells and stroma cells in CPs and the recruitment of B cells [START_REF] Bouskra | Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis[END_REF][START_REF] Mcdonald | CC chemokine receptor 6 expression by B lymphocytes is essential for the development of isolated lymphoid follicles[END_REF] . In RORt-deficient mice, ILFs-like structures still develop [START_REF] Lochner | Microbiota-induced tertiary lymphoid tissues aggravate inflammatory disease in the absence of RORγt and LTi cells[END_REF] . However, these structures are termed tertiary lymphoid tissues (tLTs), which are induced by chronic inflammation in most organs independently of LTi cells, through the expression of LT 1  2 by subsets of B cells, T cells or NK cells [START_REF] Aloisi | Lymphoid neogenesis in chronic inflammatory diseases[END_REF] .

Intriguingly, LTi cells are retained in mature (adult) LNs and Peyer's patches, and locate in the cortex between B cell follicles [START_REF] Mackley | CCR7-dependent trafficking of RORgamma(+) ILCs creates a unique microenvironment within mucosal draining lymph nodes[END_REF] . Their functions in this region remains enigmatic, mainly because it has been so far impossible to investigate the consequence of a absence of LTi cells in adult lymphoid tissues, as RORt-deficient mice lack both.

ILC3s

A population of intestinal lymphoid cells expressing the NK marker NKp46 in mouse and NKp44 in human, were found to co-express RORt and IL-22 [START_REF] Satoh-Takayama | Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense[END_REF][START_REF] Luci | Influence of the transcription factor RORgammat on the development of NKp46+ cell populations in gut and skin[END_REF][START_REF] Sanos | RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp46+ cells[END_REF][START_REF] Cella | A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity[END_REF] . These cells were originally named NK22 cells, but their requirement for RORt rather than Eomesin development, as well as their cytokine profile, makes them more similar to LTi cells than to NK cells. However, in contrast to LTi cells, NK22 do not cluster in CPs, and therefore, are not involved in the development of lymph nodes, Peyer's patches and ILFs [START_REF] Sawa | Lineage relationship analysis of RORγt+ innate lymphoid cells[END_REF][START_REF] Reynders | Identity, regulation and in vivo function of gut NKp46+RORgammat+ and NKp46+RORgammat-lymphoid cells[END_REF] . Rather, NK22 are viewed as more "regular" effector cells that patrol the tissue and produce effector cytokines where it matters in terms of defense and injury [START_REF] Satoh-Takayama | The Chemokine Receptor CXCR6 Controls the Functional Topography of Interleukin-22 Producing Intestinal Innate Lymphoid Cells[END_REF] .

Given their common dependence on RORt and their similar cytokine profiles, LTi cells and NK22 cells were grouped together as ILC3s, while lymphoid cells that produce the type 2 cytokines IL-4, IL-5 and IL-13 were grouped as ILC2s, and those that produce IFN as ILC1s [START_REF] Spits | Innate lymphoid cells--a proposal for uniform nomenclature[END_REF][START_REF] Spits | The expanding family of innate lymphoid cells: regulators and effectors of immunity and tissue remodeling[END_REF] .A difficulty arose when it was found that ILC3s can downregulate RORt and upregulate Tbet to produce IFNin a context of intense inflammation [START_REF] Klose | A T-bet gradient controls the fate and function of CCR6-RORgammat+ innate lymphoid cells[END_REF] . These cells were termed ex-ILC3s.Another form of ILC3 plasticity was found in vitrousing human ILC lines. When stimulated through TLR2, such lines expressed IL-5 and IL-13 in addition to IL-22 [START_REF] Crellin | Regulation of cytokine secretion in human CD127(+) LTilike innate lymphoid cells by Toll-like receptor 2[END_REF] . It remains to be assessed to what extent such plasticity is operational in vivo, underlying for example the rapid response of ILCs to diverse types of tissue perturbations.

The prompt responsiveness of ILCsto infection and injuryplaces them upstream in the immune response. ILC3s are activated by the inducer cytokines IL-1 and IL-23 produced by macrophages and DCs, typically in response to infection by extracellular bacteria and fungi. Activated ILC3s produce the effectors cytokines IL-17, IL-22, GM-CSF, LT 3 and LT 1  2 , which in turn induce the production of antimicrobial peptides (AMPs) by epithelial cells, of neutrophil-recruiting chemokines by stromal cells and epithelial cells [START_REF] Eberl | Innate lymphoid cells: a new paradigm in immunology[END_REF][START_REF] Wang | Lymphotoxin beta receptor signaling in intestinal epithelial cells orchestrates innate immune responses against mucosal bacterial infection[END_REF] , and the activation of B cells [START_REF] Kruglov | Nonredundant function of soluble LTalpha3 produced by innate lymphoid cells in intestinal homeostasis[END_REF][START_REF] Magri | Innate lymphoid cells integrate stromal and immunological signals to enhance antibody production by splenic marginal zone B cells[END_REF] . This antigenindependent mode of activation of ILCs is similar to that of memory and "innate" T cells, at least in terms of kinetics [START_REF] Fan | Hallmarks of Tissue-Resident Lymphocytes[END_REF] . It does not require selection and expansion of antigen-specific clones, but rather relies on the expansion of populations. It remains to be understood whether sub-subsets of ILCs exist and specifically expand in reaction to particular infections and injuries, thereby providing a form of immunological memory to challenges, as described for NK cells [START_REF] Sun | Adaptive immune features of natural killer cells[END_REF] .

ILC3s play a critical role early in the protection to enteropathogens, such as the proteobacteria Citrobacter rodentium, a homologue of the human E. coliintestinal pathogens [START_REF] Satoh-Takayama | Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense[END_REF] , to Salmonella enterica [START_REF] Klose | A T-bet gradient controls the fate and function of CCR6-RORgammat+ innate lymphoid cells[END_REF] and to rotaviruses [START_REF] Hernandez | Interferon-lambda and interleukin 22 act synergistically for the induction of interferon-stimulated genes and control of rotavirus infection[END_REF] , through their production of IL-22. They also mediate containment of bacterial symbionts that live within Peyer's patches [START_REF] Sonnenberg | Innate lymphoid cells promote anatomical containment of lymphoid-resident commensal bacteria[END_REF] , and more generally protect from dextran sodium sulfate (DSS)-induced inflammation through the containment of symbiotic bacteria [START_REF] Lochner | Microbiota-induced tertiary lymphoid tissues aggravate inflammatory disease in the absence of RORγt and LTi cells[END_REF][START_REF] Sawa | RORgammat(+) innate lymphoid cells regulate intestinal homeostasis by integrating negative signals from the symbiotic microbiota[END_REF] . The secretion by ILC3s of IL-22 also plays a role in the protection of epithelial cells from apoptosisinduced by chemotherapy or irradiationin the intestine and the thymus [START_REF] Aparicio-Domingo | Type 3 innate lymphoid cells maintain intestinal epithelial stem cells after tissue damage[END_REF][START_REF] Lindemans | Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration[END_REF][START_REF] Dudakov | Interleukin-22 drives endogenous thymic regeneration in mice[END_REF] , as well as in the protection of hepatocytes from inflammation [START_REF] Zenewicz | Interleukin-22 but not interleukin-17 provides protection to hepatocytes during acute liver inflammation[END_REF] . Intriguingly, ILC3s are relatively resistant to irradiation and chemotherapy, possibly as a consequence of their low turnover [START_REF] Eberl | An essential function for the nuclear receptor RORγt in the generation of fetal lymphoid tissue inducer cells[END_REF][START_REF] Sawa | Lineage relationship analysis of RORγt+ innate lymphoid cells[END_REF][START_REF] Gasteiger | Tissue residency of innate lymphoid cells in lymphoid and nonlymphoid organs[END_REF] .

On the other hand, ILC3s and IL-22 are involved in the progression of colon cancer [START_REF] Kirchberger | Innate lymphoid cells sustain colon cancer through production of interleukin-22 in a mouse model[END_REF] , presumablybecause they protect epithelial cells from apoptosis through the activation of the transcription factor STAT-3 [START_REF] Aparicio-Domingo | Type 3 innate lymphoid cells maintain intestinal epithelial stem cells after tissue damage[END_REF][START_REF] Lindemans | Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration[END_REF][START_REF] Dudakov | Interleukin-22 drives endogenous thymic regeneration in mice[END_REF] . In addition, ILC3s contribute to inflammatory pathology through their capacity to co-expressIFNduring Salmonella infection in mouse [START_REF] Klose | A T-bet gradient controls the fate and function of CCR6-RORgammat+ innate lymphoid cells[END_REF] and in animals and patients suffering from inflammatory bowel disease (IBD) [START_REF] Buonocore | Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology[END_REF][START_REF] Geremia | IL-23-responsive innate lymphoid cells are increased in inflammatory bowel disease[END_REF] . The expression of IL-17 by ILC3s is involved in obesityassociated asthma induced by high fat diet [START_REF] Kim | Interleukin-17-producing innate lymphoid cells and the NLRP3 inflammasome facilitate obesity-associated airway hyperreactivity[END_REF] , possibly as a consequence of a loss of containment of the intestinal microbiota and an induction of ILC3s in adipose tissue by incoming bacteria and bacterial compounds [START_REF] Burcelin | Immuno-microbiota cross and talk: the new paradigm of metabolic diseases[END_REF] .

Another surprising feature of ILC3s is their expression of MHC class II [START_REF] Eberl | An essential function for the nuclear receptor RORγt in the generation of fetal lymphoid tissue inducer cells[END_REF][START_REF] Mebius | Developing lymph nodes collect CD4 + CD3 -LT + cells that can differentiate to APC, NK cells, and follicular cells but not T or B cells[END_REF] , as well as of several components of the class II antigen-processing pathway. This feature allows the ILC3s to present antigens and repress the activity of specific T cells in the intestine during homeostasis [START_REF] Hepworth | Innate lymphoid cells regulate CD4+ T-cell responses to intestinal commensal bacteria[END_REF] , and to induce T cells activation in the spleen [START_REF] Von Burg | Activated group 3 innate lymphoid cells promote T-cellmediated immune responses[END_REF] .

The relative role of ILC3s, DCs and macrophages in the MHC class II-restricted activation and regulation of T cells [START_REF] Mortha | Microbiota-dependent crosstalk between macrophages and ILC3 promotes intestinal homeostasis[END_REF] , at least in the intestine and the lymphoid tissues, remains to be measured. So, ILC3s are pivotal to many processes in mucosal immunity, from the development of lymphoid tissues and the containment of the microbiota, the early immunity to pathogens and the protection of epithelial cells, to the exacerbation of inflammatory pathology and the progression of cancer. Even though ILC3s depend on RORt for their development, it remains to be determined whether RORt is also required for their maintenance, an important consideration when targeting RORt with agonists or antagonists to regulate type 3 immune responses [START_REF] Solt | Suppression of TH17 differentiation and autoimmunity by a synthetic ROR ligand[END_REF][START_REF] Huh | Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORgammat activity[END_REF] . A recent report shows that a RORt antagonist leads to the loss of Th17 cells but not of ILC3s [START_REF] Withers | Transient inhibition of ROR-gammat therapeutically limits intestinal inflammation by reducing TH17 cells and preserving group 3 innate lymphoid cells[END_REF] .

Thymocytes and Th17 cells

RORt is required for the survival of immature CD4 + CD8 + thymocytes by regulating the level of the anti-apoptotic factor Bcl-x L [START_REF] Sun | Requirement for ROR in thymocyte survival and lymphoid organ development[END_REF] . In the absence of RORt, immature thymocytes can spend less time at the CD4 + CD8 + stage and therefore, recombination of the genes coding for the TCR chain is biased towards proximal V to J rearrangements [START_REF] Guo | Regulation of the TCR repertoire by the survival window of CD4 + CD8 + thymocytes[END_REF] . The thymus of a RORt-deficient mouse contains half the number of cells found in the thymus of a wild-type animal [START_REF] Sun | Requirement for ROR in thymocyte survival and lymphoid organ development[END_REF] . In the periphery, control of antiapoptotic genesby RORt has not been reported.

Expression of RORt by T cells remained confined to immature thymocytes, until Th17 cells develop that require RORt [START_REF] Ivanov | The orphan nuclear receptor RORt directs the differentiation program of proinflammatory IL-17 + T helper cells[END_REF] . Naive CD4 + T cells are induced into the Th17 pathway by IL-1 [START_REF] Sutton | A crucial role for interleukin (IL)-1 in the induction of IL-17-producing T cells that mediate autoimmune encephalomyelitis[END_REF] , IL-23 [START_REF] Langrish | IL-23 drives a pathogenic T cell population that induces autoimmune inflammation[END_REF][START_REF] Harrington | Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages[END_REF] , or the combination of IL-6 and TGF [START_REF] Mangan | Transforming growth factor-beta induces development of the T(H)17 lineage[END_REF][START_REF] Bettelli | Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells[END_REF][START_REF] Zhou | TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function[END_REF] .

IL-23 and IL-6 induce the phosphorylation of STAT-3, which in turn induces the expression of RORt [START_REF] Harris | Cutting edge: An in vivo requirement for STAT3 signaling in TH17 development and TH17-dependent autoimmunity[END_REF] . The characterization of Th17 cells was initially reported in the context of EAE as the cells that are induced by IL-23 to express IL-17 and provoke autoimmunity [START_REF] Langrish | IL-23 drives a pathogenic T cell population that induces autoimmune inflammation[END_REF] , as well as in arthritis [START_REF] Sato | Th17 functions as an osteoclastogenic helper T cell subset that links T cell activation and bone destruction[END_REF][START_REF] Hirota | T cell self-reactivity forms a cytokine milieu for spontaneous development of IL-17+ Th cells that cause autoimmune arthritis[END_REF] and IBD [START_REF] Elson | Monoclonal anti-interleukin 23 reverses active colitis in a T cell-mediated model in mice[END_REF][START_REF] Kastelein | Discovery and biology of IL-23 and IL-27: related but functionally distinct regulators of inflammation[END_REF] . Therefore, RORtwas firstperceived as a public enemy that must be targeted to block the progression of autoimmune inflammation. Another line of research nevertheless showed the importance of Th17 cells in mucosal immunity to pathogens and their role in the containment of the microbiota through both the production of IL-17 and IL-22 [START_REF] Aujla | IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia[END_REF][START_REF] Khader | Th17 cells at the crossroads of innate and adaptive immunity against infectious diseases at the mucosa[END_REF] .

Caught in the middle of these confusing perceptions, IL-17 and IL-22 have been sometimes described as both pro-and anti-inflammatory, which only reflects the homeostatic or pathologic context in which these cytokines were studied. In contrast, ILC3s, also acting both during intestinal homeostasis and pathology, were first characterized in the context of intestinal homeostasis and thus as primarily "beneficial" cells [START_REF] Eberl | Thymic origin of intestinal  T cells revealed by fate mapping of RORt + cells[END_REF][START_REF] Bouskra | Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis[END_REF] . Nevertheless, the distinctive role of Th17 cells and ILC3s in homeostasis, defense and pathology has been difficult to pull apart, as models to ablate Th17 cells or ILC3s, individually, have to be improved [START_REF] Satoh-Takayama | Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense[END_REF][START_REF] Sonnenberg | Innate lymphoid cells promote anatomical containment of lymphoid-resident commensal bacteria[END_REF] . Based on their innate versus adaptive nature, early responses against pathogens have generally been "assigned"to ILC3s, while chronic responses, such as autoimmune inflammation, have been assignedto Th17 cells.

The mechanisms by which microbes induce Th17 cells, and more generally type 3 immune responses,remain a hard nut to crack. Whereas it is clear that IL-1 and IL-23 play a central role in the cascade of events that lead to the generation of Th17 cells [START_REF] Langrish | IL-23 drives a pathogenic T cell population that induces autoimmune inflammation[END_REF][START_REF] Sutton | A crucial role for interleukin (IL)-1 in the induction of IL-17-producing T cells that mediate autoimmune encephalomyelitis[END_REF][START_REF] Harrington | Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages[END_REF][START_REF] Sano | An IL-23R/IL-22 Circuit Regulates Epithelial Serum Amyloid A to Promote Local Effector Th17 Responses[END_REF] , the signals that induce IL-23 are not known. It has been shown

that ATP, produced by bacteria, activates a subset of intestinal lamina propria DCs through the P2X and P2Y receptors to produce IL-23 [START_REF] Atarashi | ATP drives lamina propria T(H)17 cell differentiation[END_REF] , but this mechanism remains to be validated in the context ofbacteria that adhere to epithelial cells, such as segmented filamentous bacteria (SFB) [START_REF] Ivanov | Induction of Intestinal Th17 Cells by Segmented Filamentous Bacteria[END_REF][START_REF] Gaboriau-Routhiau | The key role of segmented filamentous bacteria in the coordinated maturation of gut helper T cell responses[END_REF] and pathogenic strains of E. coli, which efficiently induce the generation of Th17 cells [START_REF] Atarashi | Th17 Cell Induction by Adhesion of Microbes to Intestinal Epithelial Cells[END_REF] . The pathway by which these adherent bacteria induce Th17 cells remains unknown.

Type 3 Tregs

FoxP3 is the signature transcription factor for regulatory T (Treg) cells. Therefore, it came as a surprise that a significant proportion of intestinal FoxP3 + T cells also express RORt [START_REF] Lochner | In vivo equilibrium of proinflammatory IL-17+ and regulatory IL-10+ Foxp3+ RORγt+ T cells[END_REF] . It was suggested that RORt + FoxP3 + cells are not Tregs, but rather precursor T cells that express both transcription factors until expression of one is promoted over the other to generate Tregs or Th17 cells [START_REF] Zhou | TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function[END_REF] . This hypothesis was derived form the observation that 25% of intestinal Th17 cells had expressed FoxP3 at some stage of their development, as determined by genetic fate mapping of FoxP3 + cells. It was also suggested that Tregs acquire characteristics of effectors cells, induced by the inflammatory state of the tissue [START_REF] Wohlfert | Plasticity of T reg at infected sites[END_REF] . For example, colitis induces the expression of IL-23 by macrophages and DCs, which in turn induces the expression of RORt in developing effector T cells as well as in Tregs. This leads to the expression of the chemokine receptor CCR6 on both mature Th17 and RORt +

Tregs [START_REF] Lochner | In vivo equilibrium of proinflammatory IL-17+ and regulatory IL-10+ Foxp3+ RORγt+ T cells[END_REF] , and thus co-localization of both the effector and the regulator cells to augment the efficacy of immune regulation [START_REF] Yang | Foxp3(+) T cells expressing RORgammat represent a stable regulatory T-cell effector lineage with enhanced suppressive capacity during intestinal inflammation[END_REF] . Furthermore, a small proportion of RORt + Tregs was found to be "perverted"into genuine effector cells, induced by chronic inflammationinto the expression of IL-17 [START_REF] Blatner | Expression of RORgammat Marks a Pathogenic Regulatory T Cell Subset in Human Colon Cancer[END_REF] .

Nevertheless, the majority of RORt + Tregs produces IL-10 at levels that exceed the levels produced by other subsets of Tregs, and express high levels of other attributes of Tregs, such as ICOS, CTLA-4, CD39 and CD73 100 , and exert regulatory functions in vitro and in vivo [START_REF] Lochner | In vivo equilibrium of proinflammatory IL-17+ and regulatory IL-10+ Foxp3+ RORγt+ T cells[END_REF][START_REF] Yang | Foxp3(+) T cells expressing RORgammat represent a stable regulatory T-cell effector lineage with enhanced suppressive capacity during intestinal inflammation[END_REF] . Moreover, IL-10-producing RORt + Tregs dramatically expand during intestinal and lung inflammation, presumably to avoid exponential growth of the inflammation [START_REF] Lochner | In vivo equilibrium of proinflammatory IL-17+ and regulatory IL-10+ Foxp3+ RORγt+ T cells[END_REF] . Nevertheless, it is possible that the phenomenon of Treg "perversion" expandsduring chronic long-term inflammation, conditions found in the intestine of IBD and colon cancer patients [START_REF] Blatner | Expression of RORgammat Marks a Pathogenic Regulatory T Cell Subset in Human Colon Cancer[END_REF] -but rarely obtained in mouse models.

Recently, it was demonstrated that microbiota-and antigen-induced Tregs express RORt, whereas microbiota-independent Tregs include asubset expressing

Other cells expressing RORt

RORt is expressed exclusively by lymphoid cells. Or so we thought. Populations of bone marrow and human blood neutrophils express RORt, as well as IL-17 upon stimulation with IL-6 and IL-23 105 . A similar subset of RORt + neutrophils was identified in mice that expresses IL-17 upon Aspergillus fumigatus infection of the lungs 106 . These observations are reminiscent of older data showing that LPS induces lung neutrophilia, the recruitment of which is induced by CXC chemokines produced by stromal and epithelial cells. These chemokines are in turn induced by IL-17

produced by neutrophils and T cells 107 .

RORt as a molecule

The natural ligand for RORt has remained elusive for long (Figure 2). The related ROR was co-crystallized with cholesterol and cholesterol sulfate 108 . Recently, it was found that cholesterol biosynthetic intermediates, such as oxysterols, are involved in the function of RORt during the development of lymph nodes and the differentiation of Th17 cells and IL-17 + Tcells [START_REF] Santori | Identification of natural RORgamma ligands that regulate the development of lymphoid cells[END_REF]109 . These sterols are likely to be endogenous metabolites, rather than compounds derived from the microbiota, as lymph nodes and ILC3s develop in germ free animals. Interestingly, 7,27-hydroxycholesterol, is both a ligand for RORt and EBI2, a G protein-coupled receptor involved in the positioning of activated B cells in lymph nodes. The functional link between RORt and EBI2 remains unexplored [START_REF] Cyster | 25-Hydroxycholesterols in innate and adaptive immunity[END_REF] .

Synthetic inverse agonists of RORt have been identified that derive from LXR agonists [START_REF] Solt | Suppression of TH17 differentiation and autoimmunity by a synthetic ROR ligand[END_REF] or sterols such as digoxin [START_REF] Huh | Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORgammat activity[END_REF]

RORt and ROR

ROR directly regulates the expression of clock genes through binding to the promoter of Cry1, Bmal1, Rev-Erb and also Nfil3 120 . Thus, ROR regulates clock genes, and one of those clock genes, Nfil3, regulates RORt 28 . However, ROR and RORt are not usually co-expressed in the same type of cells. Whereas RORt is expressed in lymphoid cells (and neutrophils) exclusively, ROR is not expressed in hematopoietic cells, but in many other types of parenchymal cells, such as hepatocytes and muscle cells [START_REF] He | a novel isoform of an orphan receptor, negatively regulates Fas ligand expression and IL-2 production in T cells[END_REF] . Therefore, it is possible that ROR and RORt have similarbiology in different cells, both connected to circadian clocks and oxysterol ligands [START_REF] Santori | Identification of natural RORgamma ligands that regulate the development of lymphoid cells[END_REF]109 , but have different functions through the regulation of distinct set of tissue-specific genes.

Interestingly, the other members of the ROR family, ROR and ROR, also play important roles in the circadian rhythms, as both ROR-deficient mice and ROR-deficient mice show alterations in circadian oscillations 121 .

Concluding remarks

RORt has turned out to be an extraordinary nuclear receptor and transcription factor, which controls type 3 immunity, a critical branch of immune responses that contains the symbiotic microbiota at mucosal surfaces and fights bacterial and fungal pathogens, but also leads to autoimmunity and cancer. The links between RORt, oxysterols and clock genes makes RORt a potential node connecting immunity with metabolism and circadian rhythms. The biology of RORt blossoms at an exciting time as immunology expands into a more transversal field of research connecting different physiological systems.

  Gata3[100][101][102][103] . The generation of RORt + Tregs is dependent on antigen presentation by DCs and macrophages, as well as on the activation of STAT3 100 . Surprisingly, the "pro-Th17" cytokines IL-6 and IL-23, rather than IL-10, activate STAT3 for the generation of RORt + Tregs. The eventual lineage choice to generate RORt + Tregs instead of Th17 cells is dependent on the metabolism of vitamin A into retinoic acid (RA), as an absence of vitamin A, or the inhibition of the RA receptor, tips the balance in favor of Th17 cells 100 . This suggests that the normal metabolism of vitamin A by DCs, stromal cells and neurons, rather than microbes, determines the balance between RORt + Tregs and Th17, or in other words, between anti-and proinflammatory (type 3) responses. I therefore propose that RA is a "normo-signal", required in many physiological processes, which is used by the immune system to monitor the health state of a tissue. In the context of tissue damage,upon infection or injury, the production of RA is decreased and the balance between RORt + Tregs and Th17 cells is shifted in favor of Th17 cells. During homeostasis in the intestine, this balance is regulated to the level of type 3 immunity required to contain the symbiotic microbiota.In one study, the absence of RORt + Tregs lead to the increase in Th1 and Th17 cells, and the exacerbation of colitis 101 . However, in another study, RORt + Tregs were not found to control Th1 and Th17 cells, but rather Th2 cells 100 . It was proposed that bacteria induce type 3 responses, mediated by Th17 cells and RORt + Tregs, which collectively repress competing type 1 or type 2 responses, a phenomenon described by the equilibrium model of immunity 104 . As a consequence, in mice that lack RORt + Tregs or Th17 cells, anti-helminth responses are increasedand allergic inflammation is exacerbated.Thus microbiota regulates allergy through the induction of type 3 responses 100 . The exacerbated Th17 responses observed in the absence of RORt + Tregs, reported in the first study 101 , may be the consequence of an intestinal microbiota that is more potently inducing type 3 responses than in the second study 100 .The developmental origin of RORt + Tregs remains debated and is difficult to address. Do they derive from Th17 cells, RORt -FoxP3 + Tregs, or naïve T cells? Genetic fate mapping, using Foxp3 or Rorc(t), cannot be conclusive, as FoxP3 is continuously expressed in the Treg lineage, whereas RORt is already expressed by immature CD4 + CD8 + T cells in the thymus[START_REF] Eberl | Thymic origin of intestinal  T cells revealed by fate mapping of RORt + cells[END_REF] . Resolution of the differentiation pathway of RORt + Tregs requires the transfer of single T cells, or individual T cell barcoding.

  and ursolic acid110, 111 . Such compounds trigger intense interests from the pharmaceutic industry as drugs to block type 3 responses during autoimmunity and IBD. Evidently, given the multiple cells and pathways that depend on RORt, including the containment of the intestinal microbiota and the regulation of competing immune responses such as allergic responses, such a strategy may cause important collateral damage, or be inefficient in the intestine[START_REF] Lochner | Microbiota-induced tertiary lymphoid tissues aggravate inflammatory disease in the absence of RORγt and LTi cells[END_REF] 100 . It is possible that for similar reasons, anti-IL-23 treatment is inefficient or even deleterious in the context of IBD, even though it is very efficient in the context of skin disease 112 . Interestingly, it appears that Th17 cells are more sensitive to transient RORt inhibition than ILC3s[START_REF] Withers | Transient inhibition of ROR-gammat therapeutically limits intestinal inflammation by reducing TH17 cells and preserving group 3 innate lymphoid cells[END_REF] , indicating that partial blockage of type 3 responses can be achieved with RORt inhibitors that may preserve the activity of ILC3s and some level of microbial containment.Regulation of RORt also occurs at the protein-protein level. Itch is an E3 ubiquitin ligase that binds RORt and targets it for ubiquitination and degradation 113 .In the absence of Itch, mice develop IL-17-dependent intestinal inflammation and tumorigenesis. Nitric oxide (NO) also regulates the activity of RORt through nitration of tyrosine residues 114 . As a consequence, iNOS-deficient mice develop enhanced Th17 differentiation. At the transcription factor level, FoxP3 binds directly to RORtand appears to impose its anti-inflammatory programin RORt + Tregs over the pro-inflammatory program induced by RORt[START_REF] Zhou | TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function[END_REF][START_REF] Lochner | In vivo equilibrium of proinflammatory IL-17+ and regulatory IL-10+ Foxp3+ RORγt+ T cells[END_REF] . Gata3, the signature transcription factor of type 2 responses, directly binds to and inhibits the Rorc promoter 103 . Finally, T-bet, the signature transcription factor of type 1 responses, prevents Runx1 to mediate transactivation of Rorc 115 . An interesting twist in the regulation of RORt expression involves Nfil3. This basic leucine zipper transcription factor is required for the development of NK cells 116 , as well as more generally for the development of ILCs 117-119 . Nfil3 is negatively regulated by the clock protein Rev-Erb, and in turn, negatively regulates expression of RORt by directly binding to the Rorc promoter 28 . As a consequence, the expression of RORt, and the amplitude of type 3 responses, is regulated by the circadian clock, and disruption of the circadian rhythm leads to increased susceptibility to type 3 inflammatory pathology.

Figure 1 .

 1 Figure 1. The RORt + cells. RORt + cells include type 3 innate lymphoid cells (ILC3s) and several subsets of T cells (Th17 cells, invariant NKT cells, T cells). Not mentioned is a subset of IL-17producing neutrophils that has been documented in mouse and man (see text). All RORt + cells can express the effector cytokines IL-17 and IL-22 to varying degrees, except most RORt + regulatory T cells (microbiota-induced or iTregs) that express IL-10. A subset of ILC3s, named lymphoid tissue inducer (LTi) cells, expresses soluble (LT 3 ) and membrane-bound (LT 1  2 ) lymphotoxin, as well as TRANCE and TRANCE ligand, which are involved in the development of lymphoid tissues in the fetus and after birth in the intestinal lamina propria and the activation of B cells. IL-17, as well as LT 1  2 , induces the recruitment of neutrophils, IL-22 prevents apoptosis of epithelial stem cells, and both IL-17 and IL-22 induce the production of anti-microbial peptides (AMPs) by epithelial cells. RORt + Tregs are induced by the symbiotic microbiota and regulate competing type 1 and type 2 responses.

Figure 2 .

 2 Figure 2. The RORt molecule. The type 3 inducer cytokines IL-23 and IL-6 induce the phosphorylation of the transcription factor STAT3, which then activates Rorc(t), the gene coding for RORt that is essential for the generation of ILC3s and RORt + T cells. RORt then induces the expression of the type 3 effector cytokines IL-17 and IL-22. FoxP3, the signature transcription factor of Treg cells, binds RORt and generally imposes a regulatory phenotype to FoxP3 + RORt + (Treg) cells. Similarly, Gata3 and T-bet, the signature transcription factor of type 2 and type 1 lymphoid cells, respectively, block the expression of RORt. The expression of RORt is also under control of the circadian rhythm, as Nfil3, regulated by clock genes, represses Rorc(t). ROR is involved in the regulation of circadian clocks, but is not normally expressed by hematopoietic cells. Natural ligands of RORt have recently been identified as oxysterols, the biology of which remains to be fully understood.

Acknowledgements

This work has been supported by the Institut Pasteur.


Noelia López-Montero

Jost Enninga

Diverted recycling-Shigella subversion of Rabs

Keywords: Vesicular traffic, Rab subversion, Rab11, intracellular bacteria, bacterial containing 30 vacuole, vacuolar rupture, Shigella flexneri, macropinocytosis. 31 32

Intracellular bacteria have evolved molecular weapons to subvert host cells for their 78 own benefit. They manipulate eukaryotic pathways for the promotion of efficient cellular 79 invasion within a bacterial containing vacuole (BCV), intracellular survival, evasion of 80 immune responses, and for their own propagation. [START_REF] Bhavsar | Manipulation of host-cell pathways by bacterial 359 pathogens[END_REF] The bacterial weaponry that achieves 81 such complex control is often constituted of an "effector cocktail", secreted bacterial 82 proteins into the host cytosol through specialized injection devices, like the type-3 83 secretion system (T3SS). These effectors interplay with host factors involved in the 84 pathogenicity pathways. It has been recognized that the subversion of the vesicular 85 trafficking machinery represents a key strategy by the bacteria in order to physically shape 86 their local intracellular environment. [START_REF] Cossart | Manipulation of host membrane machinery by bacterial 361 pathogens[END_REF][START_REF] Sherwood | A Rab-centric perspective of bacterial pathogen-occupied 363 vacuoles[END_REF][START_REF] Stein | Bacterial pathogens commandeer Rab 365 GTPases to establish intracellular niches[END_REF] 87

Rab GTPases in vesicular trafficking 88

Cell membrane trafficking is regulated by the Rab family of proteins, which is part of the 89 Ras superfamily of small GTPases. In humans, almost 70 different Rab proteins have 90 been identified. They coordinate sequential trafficking steps by switching between their 91 active GTP-bound form and the inactive GDP-bound form, participating at different levels 92 for example during vesicle formation, motility, tethering or docking and fusion. [START_REF] Zhen | Cellular functions of Rab GTPases at a glance[END_REF]

93

Vesicles carrying internalized cargo from different entry pathways, such as clathrin 94 and caveolae-mediated endocytosis, macropinocytosis and phagocytosis traffic to 95 lysosomes for degradation. The pair Rab5-Rab7 is considered as the master regulator of 96 endosomal trafficking and maturation. Rab5 determines the identity of early endosomal 97 compartments. [START_REF] Zeigerer | Rab5 is necessary for the biogenesis of the endolysosomal 371 system in vivo[END_REF] One of its effectors, the phosphatidylinositol 3-kinase catalytic subunit 98 type 3 (PI3KC3), produces phosphatidylinositol 3-phosphate (PI3P) on endosome 99 membranes, which in turn exerts a positive feedback loop resulting in increased Rab5 100 recruitment. 7 Rabex5, a GDP/GTP exchange factor (GEF) for Rab5, contributes to the 101 positive loop by stabilizing Rab5 on the membranes. [START_REF] Lippe | Functional synergy between 377 Rab5 effector Rabaptin-5 and exchange factor Rabex-5 when physically associated in a 378 complex[END_REF] More than 60 downstream effectors 102 of Rab5 have been identified, [START_REF] Mottola | The complexity of Rab5 to Rab7 transition guarantees specificity of pathogen 380 subversion mechanisms[END_REF] but their functions remain most often poorly understood. 103

The progressive replacement of Rab5 by Rab7 is essential for proper maturation of early 104 endosomes into lysosomes. [START_REF] Rink | Rab conversion as a mechanism of 382 progression from early to late endosomes[END_REF][START_REF] Poteryaev | Caenorhabditis elegans SAND-1 is 384 erssential for RAB-7 function in endosomal traffic[END_REF][START_REF] Huotari | Endosome maturation[END_REF] Upon Rab7 recruitment and activation, Rab5 is released 105 allowing the maturation of early endosomes into late endosomes. [START_REF] Rink | Rab conversion as a mechanism of 382 progression from early to late endosomes[END_REF] Retrograde transport of 106 late endosomes along microtubules is necessary for efficient fusion with lysosomes. It is 107 mediated by the binding of active Rab7 to Rab-interacting lysosomal protein (RILP), which 108 induces the subsequent interaction with dynein-dynactin motor complexes. [START_REF] Harrison | Phagosomes fuse with late 387 endosomes and/or lysosomes by extension of membrane protrusions along microtubules: 388 role of Rab7 and RILP[END_REF] In addition, 109 the protein Hook has been shown to be involved in endosome maturation; its N-terminus 110 interacts with microtubules and its C-terminus with membranes. [START_REF] Luiro | 390 Interconnections of CLN3, Hook1 and Rab proteins link Batten disease to defects in the 391 endocytic pathway[END_REF]

111

Alternatively to catabolic degradation, and as a requirement for cell surface 112 homeostasis, a part of the endosomal membrane and cargo are recycled back to the 113 plasma membrane. [START_REF] Grant | Pathways and mechanisms of endocytic recycling[END_REF] The membrane recycling system is highly dynamic; multiple 114 pathways for entry and exit from the endocytic recycling compartment have been 115

described with Rab11 present in most of them. Rab11 attracts effectors responsible for the 116 trafficking of recycling endosomes. [START_REF] Welz | Orchestration of cell surface proteins by 395 Rab11[END_REF] It mediates vesicular transport along microfilaments 117 by binding the globular tail domain of myosin V (MyoV). 17 Rab11 also interacts with the C-118 terminal of MyoV forming a ternary complex through Rab11 family-interacting protein 2 119 (FIP2). [START_REF] Hales | Rab11 family interacting protein 2 associates 400 with Myosin Vb and regulates plasma membrane recycling[END_REF] In addition, Rab11 employs kinesins (KIFs) for anterograde transport along 120 microtubules. FIP5 drives the interaction of Rab11 with KIF3. [START_REF] Schonteich | The Rip11/Rab11-FIP5 and kinesin II complex regulates endocytic protein 404 recycling[END_REF] Rab11 can bind directly 121 KIF13 promoting the formation of tubules. [START_REF] Delevoye | Recycling endosome tubule 407 morphogenesis from sorting endosomes requires the kinesin motor KIF13A[END_REF] The effector protrudin directs the interactions 122 of Rab11 with KIF5. Intriguingly, protrudin interacts selectively with GDP-Rab11 [START_REF] Shirane | Protrudin induces neurite formation by directional membrane 410 trafficking[END_REF] . This 123 changes the concept of Rab11 being switched off in its GDP form, and could explain the 124 large promiscuity of Rab11 functions. FIP3 regulates the retrograde transport of Rab11 125 endosomes along microtubules mediated by dynein. This has been proposed for the 126 sorting of peripheral endosomes to the perinuclear recycling compartment. [START_REF] Horgan | Rab11-FIP3 links the 412 Rab11 GTPase and cytoplasmic dynein to mediate transport to the endosomal-recycling 413 compartment[END_REF] In addition, 127

Rab11 is involved in the tethering and fusion of recycling endosomes with the plasma 128 membrane [START_REF] Takahashi | Rab11 415 regulates exocytosis of recycling vesicles at the plasma membrane[END_REF][START_REF] Guichard | Rab11-mediated trafficking in host-pathogen interactions[END_REF] . 129 Although Rab11 is the hallmark of the slow recycling pathway, its highly versatile 130 nature and its complex localization profile in different cell types have led to numerous 131 hypotheses about its roles. Importantly, only one Rab11 GEF, Crag, has been recently 132 identified in Drosophila. [START_REF] Xiong | Crag is a GEF for Rab11 required 421 for rhodopsin trafficking and maintenance of adult photoreceptor cells[END_REF] In mitosis, Rab11 has a role in organizing the mitotic spindle and 133 spindle poles, and centriole distribution. [START_REF] Das | A new role for Rab GTPases during early mitotic stages[END_REF] It is involved in ciliogenesis, cytokinesis, 134 neuritogenesis, and oogenesis. [START_REF] Welz | Orchestration of cell surface proteins by 395 Rab11[END_REF] It controls the maintenance of microvilli and 135 apical/basolateral specialization in epithelial cells, [START_REF] Knowles | Rab11a regulates syntaxin 3 localization and microvillus 427 assembly in enterocytes[END_REF] and it is a regulator of autophagosome 136 maturation. [START_REF] Szatmári | Rab11 facilitates cross-talk between autophagy and endosomal pathway through 430 regulation of Hook localization[END_REF] 137

Besides the three Rabs, Rab5, Rab7 and Rab11, described in some detail above, 138 other Rabs have been analyzed in the context of several local trafficking pathways. 139

Exemplarily, Rab1 and Rab2 mediate endoplasmic reticulum (ER)-Golgi trafficking, Rab3 140 controls exocytic events, Rab32 is involved in the biogenesis of lysosome-related 141 organelles (LROs) and mitochondrial fission, Rab6 regulates intra-Golgi trafficking, Rab9 142 and Rab7L1 mediate retrograde trafficking from late endosomes and lysosomes to the 143 trans-Golgi network (TGN), Rab4 is involved in the fast recycling pathway, as has been 144

reviewed in detail by Mizuno-Yamasaki and cols. [START_REF] Mizuno-Yamasaki | GTPase networks in membrane 432 traffic[END_REF] In addition, many of the Rabs have 145 been described to be frequently involved in more than one single pathway. 146

Manipulation of Rabs by intracellular bacteria 147

Intracellular bacterial pathogens use different mechanisms to hijack Rab proteins 148 (see table 1 for an overview), especially those that reside within BCVs. They can mimic 149

Rab GEFs and GTP-hydrolysis activating proteins (GAPs) through their effector proteins, 150 either favoring recruitment to or displacement of certain Rabs from their vacuoles. For 151 instance, the phosphatase activity of the Salmonella enterica effector SopB is required for 152 Rab5 recruitment to the BCV. SopB produces PI3P at the BCV membrane, which prolongs 153 the association of Rab5 allowing BCV maturation. In addition, active Rab5 associates with 154 the PI3KC3, which is responsible for augmented PI3P formation on the BCV membrane. [START_REF] Mallo | SopB promotes phosphatidylinositol 3-phosphate formation on 435 Salmonella vacuoles by recruiting Rab5 and Vps34[END_REF] 155 SopE, another Salmonella effector, has been found to act as a GEF for Rab5, recruiting 156 non-prenylated Rab5 on the BCV. [START_REF] Mukherjee | SopE acts as an Rab5-437 specific nucleotide exchange factor and recruits non-prenylated Rab5 on Salmonella-438 containing phagosomes to promote fusion with early endosomes[END_REF] Mycobacterium tuberculosis also interferes with Rab5. 157

The glycolipid lipoarabinomannan (LAM), released from the bacterial membrane into the 158 phagosomal membrane, inactivates the PI3KC3 impairing Rab5 recruitment. [START_REF] Fratti | Role of phosphatidylinositol 441 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome 442 maturation arrest[END_REF] 159 Concomitantly, the Mycobacterium phosphatase SapM depletes PI3P contributing to the 160 arrest of BCV maturation. [START_REF] Vergne | Mechanism of 444 phagolysosome biogenesis block by viable Mycobacterium tuberculosis[END_REF] 161 Alternatively, some bacterial effectors bind Rabs and thus prevent subsequent 162 interactions with their cognate host effectors. For instance, the Legionella effector VipD 163 forms a complex with Rab5 and Rab22 preventing the interaction with their effectors, such 164 as Rabaptin-5 and early endosome antigen 1 (EEA1). [START_REF] Ku | VipD of Legionella pneumophila targets activated Rab5 and Rab22 to interfere 448 with endosomal trafficking in macrophages[END_REF] This leads to a generalized 165 impairment in endosomal maturation and, consequently, in pathogen degradation. [START_REF] Gaspar | VipD is a Rab5-activated phospholipase A1 that protects 450 Legionella pneumophila from endosomal fusion[END_REF]

166

Another example is the complex formed by the Salmonella effector SifA and host protein 167 pleckstrin homology domain-containing family M member 2 (SKIP). The SifA-SIKP 168 complex induces the formation of Salmonella induced filaments (SIFs). [START_REF] Brumell | SifA, a type III secreted effector of Salmonella 453 typhimurium, directs Salmonella-induced filament (Sif) formation along microtubules[END_REF] It also binds and 169 recruits Rab9 to the BCV and SIFs. Sequestration of Rab9 by SifA-SKIP avoids the 170 recycling of mannose phosphate receptors (MPRs), which prevents the delivery of 171 lysosomal hydrolytic enzymes to the BCV and SIFs. [START_REF] Mcgourty | Salmonella 456 inhibits retrograde trafficking of mannose-6-phosphate receptors and lysosome function[END_REF] In addition, the C-terminus of SifA, 172 which shows similar structure of other bacterial GEFs, binds RhoA although it does not 173 change its GTPase status. [START_REF] Zhao | The 459 Salmonella effector protein SifA plays a dual role in virulence[END_REF] SIF formation is indirectly supported through another 8 Salmonella effector, SopD2, which binds directly to Rab7. This effector inhibits the 175 nucleotide exchange of this Rab. It impairs the recruitment of the Rab effectors to the BCV, 176 and subsequenly avoids degradation within lysosomes, thus allowing BCV maturation. [START_REF] Costa | Salmonella Disrupts Host Endocytic Trafficking by SopD2-Mediated 462 Inhibition of Rab7[END_REF] 177

Other bacterial effectors are able to covalently modify Rab proteins and 178 therefore completely change their properties. It has been described that the Legionella 179 effector DrrA, which is a GEF for Rab1, [START_REF] Murata | The 464 Legionella pneumophila effector protein DrrA is a Rab1 guanine nucleotide-exchange 465 factor[END_REF] covalently modifies Rab1 by AMPylation of its 180 Switch II region. Then, the AMPylated Rab1 restricts the access of GAPs, becoming 181 constitutively active at the BCV. [START_REF] Müller | The Legionella 467 effector protein DrrA AMPylates the membrane traffic regulator Rab1b[END_REF] With opposed function, SidD reverses Rab1 182 AMPylation. [START_REF] Tan | Legionella pneumophila SidD is a deAMPylase that modifies Rab1[END_REF] The effector LidA has also an auxiliary role on Rab1 recruitment thought the 183 action of DrrA, [START_REF] Machner | A bifunctional bacterial protein links GDI displacement to 472 Rab1 activation[END_REF] and the effector LepB functions as a Rab1 GAP. [START_REF] Gazdag | 474 Mechanism of Rab1b deactivation by the Legionella pneumophila GAP LepB[END_REF] Another Legionella 184 effector named AnkX also covalently modifies Rab1. It transfers a phosphocoline group 185 from CDP-choline to a serine also in the Swich II region, leading to a strong inhibition of its 186 interaction with GEFs and Rab GDP dissociation inhibitors (GDIs). [START_REF] Tan | Legionella pneumophila regulates the small GTPase Rab1 477 activity by reversible phosphorylcholination[END_REF] Therefore, as 187 phosphocolinated Rab1 cannot be solubilized by the GDI, it remains membrane bound 188 even in the GDP form. The Listeria monocytogenes enzyme glyceraldehyde-3-phosphate 189 dehydrogenase (GAPDH) ADP-ribosylates Rab5. This covalent modification renders Rab5 190 unresponsive for activation by GEFs, and in turn it blocks further maturation into Rab7. [START_REF] Alvarez-Dominguez | Characterization of a Listeria monocytogenes protein interfering with 482 Rab5a[END_REF] 191 Some effectors can even degrade Rabs by proteolytic cleavage. This is the case of 192 the Salmonella effector GtgE, which is expressed in the broad-host bacterium Salmonella 193 enterica serovar Typhimurium (S. Typhimurium) and helps to overcome the host restriction 194 barrier. This bacterial effector is specific for Rab32 and Rab7L1. In mouse macrophages, 195 GtgE avoids Rab32 recruitment to the BCV preventing delivery of antimicrobial molecules 196 to the BCV, and allowing bacterial survival and replication. Conversely, the human specific 197 serovars S. Typhi and S. Parathypi do not express GtgE. Rab32 is therefore recruited to 198 the BCVs, bringing antimicrobial capacity to the BCVs and killing intravacuolar bacteria. [START_REF] Spanò | A Rab32-dependent pathway contributes to Salmonella typhi host 484 restriction[END_REF] 199

The Salmonella GAP for Rab32, SopD2, contributes to Rab32 removal from the BCV in S. 200 Typhimurium. [START_REF] Spanò | A Bacterial Pathogen 486 Targets a Host Rab-Family GTPase Defense Pathway with a GAP[END_REF] However, in S. Typhi SopD2 is a pseudogene. [START_REF] Parkhill | Complete genome sequence of a multiple drug resistant 494 Salmonella enterica serovar Typhi CT18[END_REF] In addition, the absence 201 of GtgE in the human-adapted S. Typhi and S. Parathypi allows Rab7L1 recruitment to the 202 BCV, which is required for the export of typhoid toxin, a unique virulence factor for human-203 adapted serovars. [START_REF] Spanò | Proteolytic targeting of Rab29 by an effector protein 496 distinguishes the intracellular compartments of human-adapted and broad-host 497 Salmonella[END_REF]

204

The different examples of manipulation of Rab GTPases by pathogens described 205 above have been characterized in some detail at the molecular level. However, this has 206 not yet been achieved for many other cases. For example, the Brucella abortus effector 207

RicA tethers Rab2 to its BCV by an unknown mechanism. In vitro, RicA binds preferentially 208 GDP-Rab2 but does not possess GEF activity. [START_REF] De Barsy | 500 Identification of a Brucella spp. secreted effector specifically interacting with human small 501 GTPase Rab2[END_REF] Chlamydia trachomatis recruits Rab6, 209

Rab11 and Rab14 to the BCV in order to scavenge sphingomyelin from the Golgi. [START_REF] Lipinski | Rab6 and Rab11 regulate Chlamydia trachomatis development and golgin-84-504 dependent Golgi fragmentation[END_REF][START_REF] Capmany | Chlamydia trachomatis intercepts Golgi-derived 506 sphingolipids through a Rab14-mediated transport required for bacterial development and 507 replication[END_REF] It 210 also recruits Rab39, which is involved in multi-vesicular bodies (MVBs) trafficking, for 211 inclusion growth and bacterial development. [START_REF] Tudela | The late endocytic Rab39a GTPase regulates the interaction 510 between multivesicular bodies and chlamydial inclusions[END_REF] The specific bacterial effectors responsible 212 of such subversions are still unknown. Finally, Yersinia pestis promotes the recruitment of 213 Rab1 to the BCVs. [START_REF] Connor | Yersinia pestis 512 Requires Host Rab1b for Survival in Macrophages[END_REF] It is thought that this may be a mechanism to control vacuolar pH but, 214 again, neither bacterial effectors responsible nor precise molecular events have been 215 identified so far. 216

Subversion of Rabs by Shigella: Role in vacuolar rupture 217

For the causative agent of bacillary dysentery in humans, Shigella flexneri, Rab 218 subversion has only been studied very recently. It has been shown that the Shigella 219 effector VirA functions as a GAP for Rab1, mediating the disruption of ER-Golgi trafficking 220 and suppressing host autophagy, which contributes to intracellular bacterial persistence. [START_REF] Dong | Structurally distinct bacterial TBC-like 514 GAPs link Arf GTPase to Rab1 inactivation to counteract host defenses[END_REF]

221

Even though not directly acting on Rabs, IpaJ cleaves the Arf1 and Arf2 N-myristoylated 222 host GTPases, which regulate Golgi trafficking, inhibiting thus the host secretory 223 pathway. 57

224

The scarce information about the usage of host Rabs by Shigella may be due to the 225 intricate lifestyle of this pathogen. Shigella is a cytosolic bacterium that ruptures its BCV 226 very rapidly after invasion, [START_REF] Ray | Tracking the dynamic interplay between bacterial and host factors 521 during pathogen-induced vacuole rupture in real time[END_REF] replicates in the cytosol, and propagates into the neighboring 227 cells. [START_REF] Ray | Life on the inside: the intracellular lifestyle 523 of cytosolic bacteria[END_REF] The events occurring at the initial steps of infection are highly transient, making 228 them difficult to be studied in detail. [START_REF] Mellouk | Cytosolic Access of Intracellular Bacterial Pathogens: The 525 Shigella Paradigm[END_REF] In fact, in both reports mentioned above, the effects 229 of the Shigella effectors were studied at late infection times (around 3 hours post-230 infection). At that stage bacteria are cytosolic and spread inter-cellularly. Therefore, the 231 possible implication of VirA and IpaJ on Rab subversion at the early stages, such as 232 bacterial entry and vacuolar escape, remains unclear. 233

As mentioned before, the step of vacuolar escape is a key event for Shigella 234 virulence, but the implication of vesicular trafficking and Rab subversion in the process 235 was never evaluated. Membrane damage, and thus vacuolar rupture, has been thought to 236 occur mainly through the insertion of the T3SS injectisome on the BCV membrane. 61,62 237 Contrary to the established ideas, our group has demonstrated that BCV rupture requires 238 host factors involved in vesicular trafficking, including Rab GTPases, to take place in an 239 efficient manner. [START_REF] Mellouk | Shigella subverts the host recycling compartment to rupture its vacuole[END_REF][START_REF] Weiner | 535 Macropinosomes are key players in early Shigella invasion and vacuolar escape in 536 epithelial cells[END_REF] In particular, Shigella hijacks Rab11 to newly formed 240 macropinosomes leading to efficient BCV rupture. This relation was initially discovered 241 characterizing data from a high-content siRNA library screen of host membrane trafficking 242 factors involved in Shigella BCV rupture. BCV escape was monitored by automated 243 microscopy using the CCF4 FRET probe as a reporter of vacuolar rupture. [START_REF] Nothelfer | Monitoring Shigella 538 flexneri vacuolar escape by flow cytometry[END_REF] This screen 244 provided further Rab proteins to be potentially involved in vacuolar escape, such as Rab1, 245

Rab3, Rab4, Rab5, Rab7L1 along with Rab11. [START_REF] Mellouk | Shigella subverts the host recycling compartment to rupture its vacuole[END_REF]

246

Our live imaging revealed that Rab5, Rab7 and Rab11 were recruited to the 247 Shigella entry sites upon bacterial invasion. While Rab5 was transiently recruited, Rab11 248 was massively and permanently accumulated. [START_REF] Mellouk | Shigella subverts the host recycling compartment to rupture its vacuole[END_REF] Then, in order to evaluate the specific role 249 of Rab11 in vacuolar rupture, we used fluorescently tagged galectin-3 as live cell marker 250 for loss of endomembrane integrity. [START_REF] Keller | Single 540 cell measurements of vacuolar rupture caused by intracellular pathogens[END_REF] In Rab11 knockdown cells, Shigella vacuolar escape 251 is dramatically delayed, but it does not affect bacterial entry. Importantly, the GTPase 252 activity of Rab11 is necessary for efficient BCV rupture, since expression of GDP-locked [START_REF] Weiner | 535 Macropinosomes are key players in early Shigella invasion and vacuolar escape in 536 epithelial cells[END_REF]

253

and GTP-locked Rab11 (non published results) led to a strong delay in vacuolar rupture. 254

We found that Rab11 recruitment to the entry foci was entirely controlled by the bacterial 255 effector IpgD, [START_REF] Mellouk | Shigella subverts the host recycling compartment to rupture its vacuole[END_REF] an inositol phosphate phosphatase that converts phosphatidylinositol 4,5-256 bisphosphate (PI(4,5)P2) to phosphatidylinositol 5-phosphate (PI5P). [START_REF] Niebuhr | Conversion of PtdIns(4,5)P(2) into PtdIns(5)P by the S.flexneri 544 effector IpgD reorganizes host cell morphology[END_REF] In agreement with 257 its implication in Rab11 recruitment, the Shigella effector IpgD exhibited a strong delay in 258 vacuolar rupture. In addition, we demonstrated that IpgD was also involved in the 259 regulation of macropinosome formation during bacterial invasion. The IpgD mutant, which 260 entered without any delay into epithelial cells, was dramatically impaired for 261 macropinosome formation during the time of invasion. [START_REF] Mellouk | Shigella subverts the host recycling compartment to rupture its vacuole[END_REF] Previously, IpgD, through the 262 action of PI5P production was reported to be involved in the shaping of the entry foci. [START_REF] Niebuhr | IpgD, a protein 546 secreted by the type III secretion machinery of Shigella flexneri, is chaperoned by IpgE 547 and implicated in entry focus formation[END_REF] 263 Canonical macropinocytosis has been described as clathrin-independent non-264 selective endocytosis (see figure 1 for a comparison of Shigella induced 265 macropinosomes). By macropinocytosis, cells are able to internalize considerable volumes 266 of extracellular fluid for the uptake of soluble molecules as well as particles such as 267 viruses, bacteria and apoptotic cell fragments. Antigen presenting cells are capable of 268 constitutive macropinocytosis, while in macrophages, lymphocytes, fibroblast and epithelial 269 cells macropinosomes appear upon applying a variety of external stimuli [START_REF] Lim | Macropinocytosis: an endocytic pathway for internalising large 549 gulps[END_REF] . 270

Macropinosomes are directed towards the lysosomal pathway though the Rab5/Rab7 271 cascade, Lamp1 acquisition and acidification. [START_REF] Racoosin | Macropinosome maturation and fusion with tubular 551 lysosomes in macrophages[END_REF] The recycling of them has only been 272

suggested by a few studies, furthermore the molecular mechanism of this and its 273 regulation remains unclear. Intriguingly, in some non-professional phagocytic cells 12 macropinosomes have been reported to recycle to the plasma membrane, with little or no 275 interaction with endosomal vesicles. [START_REF] Hamasaki | Association of early endosomal autoantigen 1 with 553 macropinocytosis in EGF-stimulated A431 cells[END_REF] Our recent study on Shigella induced 276 macropinosomes has been the first that proposed a direct implication of Rab11. 277

Classically, it has been considered that Shigella takes advantage of ruffling for its 278 own internalization within macropinosome-like vesicles. [START_REF] Cossart | Bacterial invasion: the paradigms of enteroinvasive 556 pathogens[END_REF] In contrast to this paradigm, our 279 results from multidimensional live cell confocal microscopy showed that the recruited 280 Rab5, Rab7 and Rab11 were located exclusively at the membranes of the surrounding 281 macropinosomes (figure 1), but never at the forming BCV. [START_REF] Weiner | 535 Macropinosomes are key players in early Shigella invasion and vacuolar escape in 536 epithelial cells[END_REF] At the ultrastructural level, the 282 different identities of both compartments was confirmed by focused ion beam scanning 283 electron microscopy (FIB/SEM), an emerging technique for tomography of large volumes, [START_REF] Murphy | Correlative 3D imaging of whole mammalian cells with light and electron 559 microscopy[END_REF] 284 using a correlative approach. This showed clearly that Shigella enters in a tight vacuole 285 distinct from the surrounding macropinosomes. In addition, FIB/SEM revealed the 286 presence of contact sites between the BCV and macropinosomes, with the appearance of 287 smaller intramacropinosomal vesicles right at their interface during vacuolar rupture. [START_REF] Weiner | 535 Macropinosomes are key players in early Shigella invasion and vacuolar escape in 536 epithelial cells[END_REF]

288

Although Rab5 and Rab7 are recruited to Shigella induced macropinosomes, they do not 289 recruit Lamp1 nor do they show acidification (non published results). Instead, they are 290 blocked for further maturation and hijack Rab11 for virulence purposes. These 291 observations suggest that BCV rupture may be mediated by direct physical contacts 292 between Rab11-positive macropinosomes and the BCV. Such contacts may be an 293 outcome of "misdirected" trafficking towards the BCV instead of their recycling to the 294 plasma membrane, which could be considered as an "internal recycling" pathway. 295

In the scenario of "internal recycling" the exocyst (a protein complex made of eight 296 distinct proteins) may play a role. Rab11-positive recycling endosomes are delivered to the 297 apical cell surface along radial actin filaments by MyoV. [START_REF] Pylypenko | Structural basis of myosin V Rab GTPase-dependent 398 cargo recognition[END_REF] Then, they are tethered to the 298 plasma membrane by binding of MyoV and Rab11 to the exocyst component Sec15 to 299 form a tripartite docking complex. Afterwards, Sec15 interacts with SNARE complexes to 300 mediate vesicle fusion with the plasma membrane. [START_REF] Takahashi | Rab11 415 regulates exocytosis of recycling vesicles at the plasma membrane[END_REF][START_REF] Guichard | Rab11-mediated trafficking in host-pathogen interactions[END_REF] Knocking down constituents of the 301 exocyst inhibited the process of Shigella internalization (our own unpublished 302 experiments), but the precise step of inhibition needs to be elucidated. 303

Since the GDP/GTP status of Rab11 is important for BCV destabilization, another 304 possible role for Rab11 in BCV rupture could be as a shuttle, providing the nascent 305 macropinosomes with downstream molecules involved in contact formation. It has been 306

described in Drosophila that under fed conditions the protein Hook has a negative 307 regulatory role in autophagosome maturation. Hook anchors late endosomes to 308 microtubules and thus impairs their fusion with amphisomes, delaying their maturation. In 309 this context Rab11 is prominently located at recycling endosomes. In starving conditions, 310

Rab11 regulates the relocalization of Hook protein in a GTP-dependent manner binding 311 and recruiting Hook to autophagic structures. Late endosomes are then not anchored 312 anymore to microtubules, and their fusion with amphisomes is favored, allowing a faster 313 maturation and catabolic degradation. [START_REF] Szatmári | Rab11 facilitates cross-talk between autophagy and endosomal pathway through 430 regulation of Hook localization[END_REF] 314 Interestingly, Rab 11 has been also reported to be involved in expulsion of pore 315 forming toxins from the surface of challenged host cells. [START_REF] Los | RAB-5-and 561 RAB-11-dependent vesicle-trafficking pathways are required for plasma membrane repair 562 after attack by bacterial pore-forming toxin[END_REF] A similar function has been 316 described for the ESCRT machinery, which is involved in local membrane deformation and 317 scission, and in repairing plasma membrane wounds. [START_REF] Jimenez | ESCRT 564 machinery is required for plasma membrane repair[END_REF] It could be considered that Rab11 318 is recruited together with the ESCRT complex to the BCV via macropinosomes to repair 319 the damage caused by the insertion of the T3SS translocon complex, pinching off small 320 intraluminal vesicles at the contact interphase, and inducing in turn vacuolar rupture. 321

Finally, the notion that endosomal compartments function as multifunctional 322 platforms on which unique sets of molecular machines are assembled have emerged 323 recently. [START_REF] Gould | New roles for endosomes: from vesicular carriers 566 to multi-purpose platforms[END_REF] Therefore, Shigella induced macropinosomes may represent a platform for 324 localized production of specific signalling lipids and docking of certain protein complexes 325 promoting vacuolar scape. 326

Conclusion and perspective 327

In summary, as a result of Rab subversion, bacterial effectors induce or avoid 328 multiple interactions between the BCV and other host cell compartments modifying the 329 BCV or its membrane composition. Therefore, bacteria establish an intracellular replicative 330 niche through the generation of a sort of hybrid organelle. [START_REF] Sherwood | A Rab-centric perspective of bacterial pathogen-occupied 363 vacuoles[END_REF][START_REF] Stein | Bacterial pathogens commandeer Rab 365 GTPases to establish intracellular niches[END_REF] 331 Most of the currently described Rab modifications by pathogens lead to an 332 impairment of BCV degradation for bacteria survival. This is achieved either by blocking 333 phagosome maturation, by avoiding fusion with lysosomes or by redirecting BCV to 334 alternative trafficking pathways such as the ER. In addition, intracellular bacteria also 335 subvert vesicular trafficking in order to gain access to nutrients or scavenge building 336 blocks for intracellular replication. 337

From our findings about the cytosolic bacterium Shigella we propose that the 338 concept of Rab subversion is not an exclusive strategy for BCV-contained bacteria, but 339 also for cytosolic ones. We have discovered a new role for recycling endosomes, via 340 Rab11, during early stages of Shigella infection. This provides a different concept about 341 bacterial vacuolar rupture. Intriguingly, none of the different Rabs recruited to the entry foci 342 (Rab5, Rab7 and Rab11) were located on BCV membranes, but only on macropinosomes. 343 Further research on Rab manipulation by cytosolic bacteria will help to fully understand the 344 process of vacuolar rupture, a key event for their virulence.

1 Title: 1 2 3 4 6 7

 1136 Diverted recycling-Shigella subversion of Rabs Noelia Lopez-Montero and Jost Enninga Institut Pasteur, Research unit "Dynamics of host-pathogen interactions", 25 rue du Dr. 5 Roux, 75724 Paris, FranceCorrespondence: Jost Enninga (jost.enninga@pasteur.fr) 8Noelia Lopez-Montero (noelia.lopez-montero@pasteur.fr)

Figure 1 :

 1 Figure 1: Schematic representation of macropinosome-like vesicles induced by 569