Interplay between androgen and CXCR4 chemokine signaling in myelin repair.

In men, reduced levels of testosterone are associated with the prevalence and progression of multiple sclerosis (MS), a chronic and disabling demyelinating disorder. Testosterone has been shown to promote myelin repair. Here, we demonstrate that the cooperation between testosterone and CXCR4 signaling involving astrocytes is required for myelin regeneration after focal demyelination produced in the ventral mouse spinal cord by the infusion of lysolecithin. The testosterone-dependent remyelination of axons by oligodendrocytes was accompanied by an increase in astrocytes expressing CXCR4, its ligand CXCL12 and the androgen receptor (AR) within the demyelinated area. Depriving males of their testosterone or pharmacological inhibition of CXCR4, with the selective antagonist AMD3100, prevented the appearance of astrocytes expressing CXCR4, CXCL12 and AR within the demyelinated area and the concomitant recruitment of myelin forming oligodendrocytes. Conditional genetic ablation of either CXCR4 or AR in astrocytes also completely blocked the formation of new myelin by oligodendrocytes. Interestingly, the gain of function mutation in CXCR4 causing WHIM syndrome allows remyelination to take place, even in the absence of testosterone, but its potentiating effects remained observable. After testosterone deprivation or CXCR4 inhibition, the absence of astrocytes within the demyelinated area led to the incursion of Schwann cells, most likely derived from spinal nerves, and the formation of peripheral nerve type myelin. In patients with progressive MS, astrocytes expressing CXCR4 and AR surrounded myelin lesions, and their presence opposed the incursion of Schwann cells. These results highlight a mechanism of promyelinating testosterone signaling and the importance of normalizing its levels in combined myelin repair therapies.

Optimized protocol for 3D epithelial cultures supporting human papillomavirus replication.

Human papillomaviruses (HPVs) are commensal viruses with pathogenic potential. Their life cycle requires the proliferation and differentiation of keratinocytes (KCs) to form pluristratified epithelia. Based on the original organotypic epithelial raft cultures protocol, we provide an updated workflow to optimally generate pluristratified human epithelia supporting the complete HPV replicative life cycle, here called 3D full-thickness epithelial cultures (3Deps). We describe steps for HPV genome preparation, KC transfection, and dermal equivalent preparation. We then detail procedures for 3Deps culture, harvesting, and analysis.

Reprogramming of connexin landscape fosters fast gap junction intercellular communication in human papillomavirus-infected epithelia.

Human papillomaviruses (HPVs) are highly prevalent commensal viruses that require epithelial stratification to complete their replicative cycle. While HPV infections are most often asymptomatic, certain HPV types can cause lesions, that are usually benign. In rare cases, these infections may progress to non-replicative viral cycles associated with high HPV oncogene expression promoting cell transformation, and eventually cancer when not cleared by host responses. While the consequences of HPV-induced transformation on keratinocytes have been extensively explored, the impact of viral replication on epithelial homeostasis remains largely unexplored. Gap junction intercellular communication (GJIC) is critical for stratified epithelium integrity and function. This process is ensured by a family of proteins named connexins (Cxs), including 8 isoforms that are expressed in stratified squamous epithelia. GJIC was reported to be impaired in HPV-transformed cells, which was attributed to the decreased expression of the Cx43 isoform. However, it remains unknown whether and how HPV replication might impact on the expression of Cx isoforms and GJIC in stratified squamous epithelia. To address this question, we have used 3D-epithelial cell cultures (3D-EpCs), the only model supporting the productive HPV life cycle. We report a transcriptional downregulation of most epithelial Cx isoforms except Cx45 in HPV-replicating epithelia. At the protein level, HPV replication results in a reduction of Cx43 expression while that of Cx45 increases and displays a topological shift toward the cell membrane. To quantify GJIC, we pioneered quantitative gap-fluorescence loss in photobleaching (FLIP) assay in 3D-EpCs, which allowed us to show that the reprogramming of Cx landscape in response to HPV replication translates into accelerated GJIC in living epithelia. Supporting the pathophysiological relevance of our observations, the HPV-associated Cx43 and Cx45 expression pattern was confirmed in human cervical biopsies harboring HPV. In conclusion, the reprogramming of Cx expression and distribution in HPV-replicating epithelia fosters accelerated GJIC, which may participate in epithelial homeostasis and host immunosurveillance.

[The cutaneous virome: from virology to personalized medicine]. / Le virome cutané : de la virologie à la médecine personnalisée.

The virome of the skin, defined as all viruses detected in the skin, represents a significant part of the microbiota. A much more recent discovery than the bacterial flora, the existence of the cutaneous virome has been revealed by recent metagenomic studies. The normal human skin virome is dominated by bacteriophages, Papillomaviridae, whose genomic diversity has proved extraordinary, and Polyomaviridae. Many yet unknown viral genomes within this virome await identification. The composition of the virome of the skin has been shown to be strictly individual and relatively stable over time, resulting from adaptation to everyone's genetics, lifestyle and mechanisms of immunological tolerance finely selected over the course of evolution. Yet little studied, the virome of the skin and all its interactions with other microbiota and the host are attracting growing interest. Indeed, constitutional or acquired alterations in the homeostasis between the commensal virome and the skin, ranging from sub-clinical viral dysbiosis to severe transformation of keratinocytes or adnexal cells, have been observed. These recent observations are stimulating the search for innovative solutions aimed at measuring or even modulating its pathological expression, with a view to personalized medicine.

HPV infection alters vaginal microbiome through down-regulating host mucosal innate peptides used by Lactobacilli as amino acid sources.

Despite the high prevalence of both cervico-vaginal human papillomavirus (HPV) infection and bacterial vaginosis (BV) worldwide, their causal relationship remains unclear. While BV has been presumed to be a risk factor for HPV acquisition and related carcinogenesis for a long time, here, supported by both a large retrospective follow-up study (n = 6,085) and extensive in vivo data using the K14-HPV16 transgenic mouse model, we report a novel blueprint in which the opposite association also exists. Mechanistically, by interacting with several core members (NEMO, CK1 and ß-TrCP) of both NF-κB and Wnt/ß-catenin signaling pathways, we show that HPV E7 oncoprotein greatly inhibits host defense peptide expression. Physiologically secreted by the squamous mucosa lining the lower female genital tract, we demonstrate that some of these latter are fundamental factors governing host-microbial interactions. More specifically, several innate molecules down-regulated in case of HPV infection are hydrolyzed, internalized and used by the predominant Lactobacillus species as amino acid source sustaining their growth/survival. Collectively, this study reveals a new viral immune evasion strategy which, by its persistent/negative impact on lactic acid bacteria, ultimately causes the dysbiosis of vaginal microbiota.

The Chemokine System in Oncogenic Pathways Driven by Viruses: Perspectives for Cancer Immunotherapy.

Chemokines interact with glycosaminoglycans of the extracellular matrix and activate heptahelical cellular receptors that mainly consist of G Protein-Coupled Receptors and a few atypical receptors also with decoy activity. They are well-described targets of oncogenic pathways and key players in cancer development, invasiveness, and metastasis acting both at the level of cancer cells and cells of the tumor microenvironment. Hence, they can regulate cancer cell proliferation and survival and promote immune or endothelial cell migration into the tumor microenvironment. Additionally, oncogenic viruses display the potential of jeopardizing the chemokine system by encoding mimics of chemokines and receptors as well as several products such as oncogenic proteins or microRNAs that deregulate their human host transcriptome. Conversely, the chemokine system participates in the host responses that control the virus life cycle, knowing that most oncoviruses establish asymptomatic latent infections. Therefore, the deregulated expression and function of chemokines and receptors as a consequence of acquired or inherited mutations could bias oncovirus infection toward pro-oncogenic pathways. We here review these different processes and discuss the anticancer therapeutic potential of targeting chemokine availability or receptor activation, from signaling to decoy-associated functions, in combination with immunotherapies.

Fine-tuning of MEK signaling is pivotal for limiting B and T cell activation.

MEK1 and MEK2, the only known activators of ERK, are attractive therapeutic candidates for both cancer and autoimmune diseases. However, how MEK signaling finely regulates immune cell activation is only partially understood. To address this question, we specifically delete Mek1 in hematopoietic cells in the Mek2 null background. Characterization of an allelic series of Mek mutants reveals the presence of distinct degrees of spontaneous B cell activation, which are inversely proportional to the levels of MEK proteins and ERK activation. While Mek1 and Mek2 null mutants have a normal lifespan, 1Mek1 and 1Mek2 mutants retaining only one functional Mek1 or Mek2 allele in hematopoietic cell lineages die from glomerulonephritis and lymphoproliferative disorders, respectively. This establishes that the fine-tuning of the ERK/MAPK pathway is critical to regulate B and T cell activation and function and that each MEK isoform plays distinct roles during lymphocyte activation and disease development.

Multi-Tissue Characterization of GILZ Expression in Dendritic Cell Subsets at Steady State and in Inflammatory Contexts.

Dendritic cells (DCs) are key players in the control of tolerance and immunity. Glucocorticoids (GCs) are known to regulate DC function by promoting their tolerogenic differentiation through the induction of inhibitory ligands, cytokines, and enzymes. The GC-induced effects in DCs were shown to critically depend on increased expression of the Glucocorticoid-Induced Leucine Zipper protein (GILZ). GILZ expression levels were further shown to control antigen-presenting cell function, as well as T-cell priming capacity of DCs. However, the pattern of GILZ expression in DC subsets across tissues remains poorly described, as well as the modulation of its expression levels in different pathological settings. To fill in this knowledge gap, we conducted an exhaustive analysis of GILZ relative expression levels in DC subsets from various tissues using multiparametric flow cytometry. This study was performed at steady state, in the context of acute as well as chronic skin inflammation, and in a model of cancer. Our results show the heterogeneity of GILZ expression among DC subsets as well as the complexity of its modulation, that varies in a cell subset- and context-specific manner. Considering the contribution of GILZ in the control of DC functions and its potential as an immune checkpoint in cancer settings, these results are of high relevance for optimal GILZ targeting in therapeutic strategies.

A recombinant measles virus vaccine strongly reduces SHIV viremia and virus reservoir establishment in macaques.

Replicative vectors derived from live-attenuated measles virus (MV) carrying additional non-measles vaccine antigens have long demonstrated safety and immunogenicity in humans despite pre-existing immunity to measles. Here, we report the vaccination of cynomolgus macaques with MV replicative vectors expressing simian-human immunodeficiency virus Gag, Env, and Nef antigens (MV-SHIV Wt) either wild type or mutated in the immunosuppressive (IS) domains of Nef and Env antigens (MV-SHIV Mt). We found that the inactivation of Nef and Env IS domains by targeted mutations led to the induction of significantly enhanced post-prime cellular immune responses. After repeated challenges with low doses of SHIV-SF162p3, vaccinees were protected against high viremia, resulting in a 2-Log reduction in peak viremia, accelerated viral clearance, and a decrease -even complete protection for nearly half of the monkeys- in reservoir cell infection. This study demonstrates the potential of a replicative viral vector derived from the safe and widely used measles vaccine in the development of a future human vaccine against HIV-1.

CXCR3 and CXCR5 are highly expressed in HIV-1-specific CD8 central memory T cells from infected patients.

New ways of characterizing CD8+ memory T cell responses in chronic infections are based on the measurement of chemokine receptor expression (CXCR3, CXCR5, and CX3CR1). We applied these novel phenotyping strategies to chronic HIV infection by comparing healthy donors (HDs), HIV-infected patients receiving antiretroviral therapy (ART), and spontaneous HIV controllers (HICs). In all groups, the memory cells exhibited high proportion of CXCR3+ cells. Proportions of CXCR5+ and CX3CR1+ cells were preferentially observed among central memory cells (Tcm) and effector memory cells (Tem) respectively. Chronic controlled HIV infection impacted the chemokine receptor profile of both HIV-specific and nonspecific CD8+ T cells. In total CD8+ T cells, the proportions of CXCR3- CXCR5- CX3CR1- Tcm and Tem were lower in HIV-infected patients than in HDs with subtle differences between ART and HICs. Such phenotyping strategy also revealed differences in exhaustion and senescence phenotypes, the CXCR3+ CXCR5+ CX3CR1- being more exhausted and senescent than the CXCR3+ CXCR5- CX3CR1- Tcm fraction. Among HIV-specific CD8+ T cells, the vast majority of Tcm cells were CXCR3+ and CXCR5+ cells in contrast with their nonspecific counterparts. In conclusion, the addition of migration markers contributes to better characterize Tcm/Tem compartment.

The G Protein-Coupled Receptor Kinases (GRKs) in Chemokine Receptor-Mediated Immune Cell Migration: From Molecular Cues to Physiopathology.

Although G protein-coupled receptor kinases (GRKs) have long been known to regulate G protein-coupled receptor (GPCR) desensitization, their more recently characterized functions as scaffolds and signalling adapters underscore that this small family of proteins governs a larger array of physiological functions than originally suspected. This review explores how GRKs contribute to the complex signalling networks involved in the migration of immune cells along chemokine gradients sensed by cell surface GPCRs. We outline emerging evidence indicating that the coordinated docking of several GRKs on an active chemokine receptor determines a specific receptor phosphorylation barcode that will translate into distinct signalling and migration outcomes. The guidance cues for neutrophil migration are emphasized based on several alterations affecting GRKs or GPCRs reported to be involved in pathological conditions.

CXCR4 signaling controls dendritic cell location and activation at steady state and in inflammation.

Dendritic cells (DCs) encompass several cell subsets that collaborate to initiate and regulate immune responses. Proper DC localization determines their function and requires the tightly controlled action of chemokine receptors. All DC subsets express CXCR4, but the genuine contribution of this receptor to their biology has been overlooked. We addressed this question using natural CXCR4 mutants resistant to CXCL12-induced desensitization and harboring a gain of function that cause the warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome (WS), a rare immunodeficiency associated with high susceptibility to the pathogenesis of human papillomavirus (HPV). We report a reduction in the number of circulating plasmacytoid DCs (pDCs) in WHIM patients, whereas that of conventional DCs is preserved. This pattern was reproduced in an original mouse model of WS, enabling us to show that the circulating pDC defect can be corrected upon CXCR4 blockade and that pDC differentiation and function are preserved, despite CXCR4 dysfunction. We further identified proper CXCR4 signaling as a critical checkpoint for Langerhans cell and DC migration from the skin to lymph nodes, with corollary alterations of their activation state and tissue inflammation in a model of HPV-induced dysplasia. Beyond providing new hypotheses to explain the susceptibility of WHIM patients to HPV pathogenesis, this study shows that proper CXCR4 signaling establishes a migration threshold that controls DC egress from CXCL12-containing environments and highlights the critical and subset-specific contribution of CXCR4 signal termination to DC biology.

The CXCL12/CXCR4/ACKR3 Axis in the Tumor Microenvironment: Signaling, Crosstalk, and Therapeutic Targeting.

Elevated expression of the chemokine receptors CXCR4 and ACKR3 and of their cognate ligand CXCL12 is detected in a wide range of tumors and the tumor microenvironment (TME). Yet, the molecular mechanisms by which the CXCL12/CXCR4/ACKR3 axis contributes to the pathogenesis are complex and not fully understood. To dissect the role of this axis in cancer, we discuss its ability to impinge on canonical and less conventional signaling networks in different cancer cell types; its bidirectional crosstalk, notably with receptor tyrosine kinase (RTK) and other factors present in the TME; and the infiltration of immune cells that supporttumor progression. We discuss current and emerging avenues that target the CXCL12/CXCR4/ACKR3 axis. Coordinately targeting both RTKs and CXCR4/ACKR3 and/or CXCL12 is an attractive approach to consider in multitargeted cancer therapies. In addition, inhibiting infiltrating immune cells or reactivating the immune system along with modulating the CXCL12/CXCR4/ACKR3 axis in the TME has therapeutic promise.

The atypical chemokine receptor 3 interacts with Connexin 43 inhibiting astrocytic gap junctional intercellular communication.

The atypical chemokine receptor 3 (ACKR3) plays a pivotal role in directing the migration of various cellular populations and its over-expression in tumors promotes cell proliferation and invasiveness. The intracellular signaling pathways transducing ACKR3-dependent effects remain poorly characterized, an issue we addressed by identifying the interactome of ACKR3. Here, we report that recombinant ACKR3 expressed in HEK293T cells recruits the gap junction protein Connexin 43 (Cx43). Cx43 and ACKR3 are co-expressed in mouse brain astrocytes and human glioblastoma cells and form a complex in embryonic mouse brain. Functional in vitro studies show enhanced ACKR3 interaction with Cx43 upon ACKR3 agonist stimulation. Furthermore, ACKR3 activation promotes ß-arrestin2- and dynamin-dependent Cx43 internalization to inhibit gap junctional intercellular communication in primary astrocytes. These results demonstrate a functional link between ACKR3 and gap junctions that might be of pathophysiological relevance.

Stress-glucocorticoid-TSC22D3 axis compromises therapy-induced antitumor immunity.

Psychological distress has long been suspected to influence cancer incidence and mortality. It remains largely unknown whether and how stress affects the efficacy of anticancer therapies. We observed that social defeat caused anxiety-like behaviors in mice and dampened therapeutic responses against carcinogen-induced neoplasias and transplantable tumors. Stress elevated plasma corticosterone and upregulated the expression of glucocorticoid-inducible factor Tsc22d3, which blocked type I interferon (IFN) responses in dendritic cell (DC) and IFN-γ+ T cell activation. Similarly, close correlations were discovered among plasma cortisol levels, TSC22D3 expression in circulating leukocytes and negative mood in patients with cancer. In murine models, exogenous glucocorticoid injection, or enforced expression of Tsc22d3 in DC was sufficient to abolish therapeutic control of tumors. Administration of a glucocorticoid receptor antagonist or DC-specific Tsc22d3 deletion reversed the negative impact of stress or glucocorticoid supplementation on therapeutic outcomes. Altogether, these results indicate that stress-induced glucocorticoid surge and Tsc22d3 upregulation can subvert therapy-induced anticancer immunosurveillance.

Atypical Chemokine Receptor 3 (ACKR3): A Comprehensive Overview of its Expression and Potential Roles in the Immune System.

Atypical chemokine receptor 3 (ACKR3), previously known as C-X-C chemokine receptor type 7 (CXCR7), has emerged as a key player in several biologic processes, particularly during development. Its CXCL11 and CXCL12 scavenging activity and atypical signaling properties, together with a new array of other nonchemokine ligands, have established ACKR3 as a main regulator of physiologic processes at steady state and during inflammation. Here, we present a comprehensive review of ACKR3 expression in mammalian tissues in search of a possible connection with the receptor function. Besides the reported roles of ACKR3 during development, we discuss the potential contribution of ACKR3 to the function of the immune system, focusing on the myeloid lineage.

OMIP-048 MC: Quantification of calcium sensors and channels expression in lymphocyte subsets by mass cytometry.

Calcium (Ca2+ ) signaling controls T-cell activation and functions. Ca2+ concentrations are locally detected and controlled by Ca2+ -sensors (STIM1 and 2 detecting the depletion from ER stores channels) and Ca2+ -channels (ORAI1-3 in the cell membrane and VDAC1 in the outer mitochondrial membrane). We first validated and titrated antibodies to assess the expression of these Ca2+ -sensors and -channels in human and murine cells, and further devised a 18-antibodies mass cytometry panel to characterize their expression in primary murine lymphocyte subsets.

Glucocorticoid-Induced Leucine Zipper: Fine-Tuning of Dendritic Cells Function.

Dendritic cells (DCs) are key antigen-presenting cells that control the induction of both tolerance and immunity. Understanding the molecular mechanisms regulating DCs commitment toward a regulatory- or effector-inducing profile is critical for better designing prophylactic and therapeutic approaches. Initially identified in dexamethasone-treated thymocytes, the glucocorticoid-induced leucine zipper (GILZ) protein has emerged as a critical factor mediating most, but not all, glucocorticoids effects in both non-immune and immune cells. This intracellular protein exerts pleiotropic effects through interactions with transcription factors and signaling proteins, thus modulating signal transduction and gene expression. GILZ has been reported to control the proliferation, survival, and differentiation of lymphocytes, while its expression confers anti-inflammatory phenotype to monocytes and macrophages. In the past twelve years, a growing set of data has also established that GILZ expression in DCs is a molecular switch controlling their T-cell-priming capacity. Here, after a brief presentation of GILZ isoforms and functions, we summarize current knowledge regarding GILZ expression and regulation in DCs, in both health and disease. We further present the functional consequences of GILZ expression on DCs capacity to prime effector or regulatory T-cell responses and highlight recent findings pointing to a broader role of GILZ in the fine tuning of antigen capture, processing, and presentation by DCs. Finally, we discuss future prospects regarding the possible roles for GILZ in the control of DCs function in the steady state and in the context of infections and chronic pathologies.