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Abstract Background Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease characterized by B-cell hyper-reactivity and the production of pathogenic anti-nuclear-directed auto-antibodies (Abs). B-cell ontogeny is partly dependent on the CXCL12/CXCR4 axis for which the contribution to SLE pathogenesis remains unclear. CXCR7, the novel receptor for CXCL12, is differentially expressed among memory B-cell subsets. However, its biological role in SLE remains to be explored. Methods Relative CXCR4 and CXCR7 expression levels were compared by quantitative PCR in leukocytes from blood samples of 41 Mexican Mestizos patients with SLE and 45 ethnicity-matched healthy subjects. Intracellular and membrane expression of both receptors was analyzed by flow cytometry in naive and Ab-secreting B cells. B-cell responsiveness to CXCL12 was investigated using Transwell-based chemotaxis assays. Data were analyzed using the Kruskal-Wallis test for comparisons of values amongst healthy controls and patients with inactive or active SLE, and non-parametrically using the Mann–Whitney U-test for multiple comparisons and unpaired samples. Correlations were determined by Spearman’s ranking. Result SLE leukocytes displayed reduced levels of CXCR4 and CXCR7 transcripts. In SLE patients, a significant defect in CXCR4 expression was detected at the surface of naive and Ab-secreting B cells, associated with an abnormal intracellular localization of the receptor. CXCR7 predominantly localized in cytosolic compartments of B cells from healthy and SLE individuals. Disease activity did not impact on these expression patterns. Altered receptor compartmentalization correlated with an impaired CXCL12-promoted migration of SLE B cells. Conclusions Our data highlight a down-regulation of CXCL12 receptors on circulating B cells from SLE patients that likely influences their migratory behavior and distribution.

Abstract Background WHIM syndrome (WS), a rare congenital neutropenia due to mutations of the CXCR4 chemokine receptor, is associated with Human Papillomavirus (HPV)-induced Warts, Hypogammaglobulinemia, bacterial Infections and Myelokathexis. The long term follow up of eight patients highlights the clinical heterogeneity of this disease as well as the main therapeutic approaches and remaining challenges in the light of the recent development of new CXCR4 inhibitors. Objective This study aims to describe the natural history of WS based on a French cohort of 8 patients. Methods We have reviewed the clinical, biological and immunological features of patients with WS enrolled into the French Severe Chronic Neutropenia Registry. Results We identified four pedigrees with WS comprised of eight patients and one foetus. Estimated incidence for WS was of 0.23 per million births. Median age at the last visit was 29 years. Three pedigrees encompassing seven patients and the fetus displayed autosomal dominant heterozygous mutations of the CXCR4 gene, while one patient presented a wild-type CXCR4 gene. Two subjects exhibited congenital conotruncal heart malformations. In addition to neutropenia and myelokathexis, all patients presented deep monocytopenia and lymphopenia. Seven patients presented repeated bacterial Ears Nose Throat as well as severe bacterial infections that were curable with antibiotics. Four patients with late onset prophylaxis developed chronic obstructive pulmonary disease (COPD). Two patients reported atypical mycobacteria infections which in one case may have been responsible for one patient’s death due to liver failure at the age of 40.6 years. HPV-related disease manifested in five subjects and progressed as invasive vulvar carcinoma with a fatal course in one patient at the age of 39.5 years. In addition, two patients developed T cell lymphoma skin cancer and basal cell carcinoma at the age of 38 and 65 years. Conclusions Continuous prophylactic anti-infective measures, when started in early childhood, seem to effectively prevent further bacterial infections and the consequent development of COPD. Long-term follow up is needed to evaluate the effect of early anti-HPV targeted prophylaxis on the development of skin and genital warts.

Abstract Background CXCL12 has been widely reported to play a biologically relevant role in tumor growth and spread. In epithelial ovarian cancer (EOC), CXCL12 enhances tumor angiogenesis and contributes to the immunosuppressive network. However, its prognostic significance remains unclear. We thus compared CXCL12 status in healthy and malignant ovaries, to assess its prognostic value. Methods Immunohistochemistry was used to analyze CXCL12 expression in the reproductive tracts, including the ovaries and fallopian tubes, of healthy women, in benign and borderline epithelial tumors, and in a series of 183 tumor specimens from patients with advanced primary EOC enrolled in a multicenter prospective clinical trial of paclitaxel/carboplatin/gemcitabine-based chemotherapy (GINECO study). Univariate COX model analysis was performed to assess the prognostic value of clinical and biological variables. Kaplan-Meier methods were used to generate progression-free and overall survival curves. Results Epithelial cells from the surface of the ovary and the fallopian tubes stained positive for CXCL12, whereas the follicles within the ovary did not. Epithelial cells in benign, borderline and malignant tumors also expressed CXCL12. In EOC specimens, CXCL12 immunoreactivity was observed mostly in epithelial tumor cells. The intensity of the signal obtained ranged from strong in 86 cases (47%) to absent in 18 cases ( Conclusion Our findings highlight the previously unappreciated constitutive expression of CXCL12 on healthy epithelia of the ovary surface and fallopian tubes, indicating that EOC may originate from either of these epithelia. We reveal that CXCL12 production by malignant epithelial cells precedes tumorigenesis and we confirm in a large cohort of patients with advanced EOC that CXCL12 expression level in EOC is not a valuable prognostic factor in itself. Trial Registration ClinicalTrials.gov: NCT00052468

Abstract Background Little is known about the molecules that contribute to tumor progression of epithelial ovarian cancer (EOC), currently a leading cause of mortality from gynecological malignancies. Glucocorticoid-Induced Leucine Zipper (GILZ), an intracellular protein widely expressed in immune tissues, has been reported in epithelial tissues and controls some of key signaling pathways involved in tumorigenesis. However, there has been no report on GILZ in EOC up to now. The objectives of the current study were to examine the expression of GILZ in EOC and its effect on tumor cell proliferation. Results GILZ expression was measured by immunohistochemical staining in tissue sections from 3 normal ovaries, 7 benign EOC and 50 invasive EOC. GILZ was not detected on the surface epithelium of normal ovaries and benign tumors. In contrast, it was expressed in the cytoplasm of tumor cells in 80% EOC specimens. GILZ immunostaining scores correlated positively to the proliferation marker Ki-67 (Spearman test in univariate analysis, P < 0.00001, r = 0.56). They were also higher in tumor cells containing large amounts of phosphorylated protein kinase B (p-AKT) (unpaired t test, P < 0.0001). To assess the effect of GILZ on proliferation and AKT activation, we used the BG-1 cell line derived from ovarian tumor cells as a cellular model. GILZ expression was either enhanced by stable transfection or decreased by the use of small interfering (si) RNA targeting GILZ. We found that GILZ increased cell proliferation, phospho-AKT cellular content and AKT kinase activity. Further, GILZ upregulated cyclin D1 and phosphorylated retinoblastoma (p-Rb), downregulated cyclin-dependent kinase inhibitor p21, and promoted the entry into S phase of cell cycle. Conclusion The present study is the first to identify GILZ as a molecule produced by ovarian cancer cells that promotes cell cycle progression and proliferation. Our findings clearly indicate that GILZ activates AKT, a crucial signaling molecule in tumorigenesis. GILZ thus appears as a potential key molecule in EOC.

Chemokines present in the tumor microenvironment are essential for the control of tumor progression. We show here that several ligands of the chemokine receptor Cxcr2 were up-regulated in the PyMT (polyoma middle T oncogene) model of breast cancer. Interestingly, the knock-down of Cxcr2 in PyMT animals led to an increased growth of the primary tumor and lung metastasis. The analysis of tumor content of PyMT-Cxcr2-/- animals highlighted an increased infiltration of tumor associated neutrophils (TANs), mirrored by a decreased recruitment of tumor associated macrophages (TAMs) compared to PyMT animals. Analysis of PyMT-Cxcr2-/- TANs revealed that they lost their killing ability compared to PyMT-Cxcr2+/+ TANs. The transcriptomic analysis of PyMT-Cxcr2-/- TANs showed that they had a more pronounced pro-tumor TAN2 profile compared to PyMT TANs. In particular, PyMT-Cxcr2-/- TANs displayed an up-regulation of the pathways involved in reactive oxygen species (ROS) production and angiogenesis and factors favoring metastasis, but reduced apoptosis. In summary, our data reveal that a lack of Cxcr2 provides TANs with pro-tumor effects.

Background: WHIM (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis) syndrome is an ultra-rare, combined primary immunodeficiency and chronic neutropenic disorder characterized by a range of clinical presentations, including peripheral neutropenia, lymphopenia, and recurrent infections. WHIM syndrome is most often caused by gain-of-function mutations in the gene encoding C-X-C chemokine receptor 4 (CXCR4). As such, inhibition of CXCR4 with XOLREMDI® (mavorixafor), an orally bioavailable CXCR4 antagonist, demonstrated clinically meaningful increases in absolute neutrophil and lymphocyte counts and concomitant reduction in infections in patients with WHIM syndrome, resulting in its recent U.S. Food and Drug Administration approval. The impact of CXCR4 antagonism on other aspects of the pathobiology in WHIM syndrome, such as lymphopoiesis and leukocyte trafficking between primary and secondary lymphoid organs, is less understood. Methods: In the current study, the effects of CXCR4 antagonism on leukocyte trafficking and distribution in primary and secondary lymphoid organs were investigated in a mouse model of WHIM syndrome carrying the heterozygous Cxcr41013 mutation. Cxcr4+/1013 and Cxcr4 wild-type mice received the orally bioavailable CXCR4 antagonist X4-185. Blood, spleen and bone marrow samples were collected for numeration, flow cytometry, and functional studies. Results: Cxcr4+/1013 mice exhibited profound peripheral blood leukopenia as seen in patients with WHIM syndrome. CXCR4 antagonism corrected circulating leukopenia and mobilized functional neutrophils without disrupting granulopoiesis in the bone marrow of Cxcr4+/1013 mice. Furthermore, Cxcr4+/1013 displayed aberrant splenic T and B-cell counts and frequency. Treatment with X4-185 normalized splenic T-cell abnormalities, correcting the reduced CD8+ T-cell numbers, restoring the CD4/CD8 T-cell ratio, and ameliorating peripheral blood T-cell lymphopenia. In addition, CXCR4 antagonism was able to correct the abnormal frequencies and numbers of splenic marginal zone and follicular B cells in Cxcr4+/1013 mice, and ultimately normalize B-cell lymphopenia in the peripheral circulation. Conclusions: Our study provides comprehensive evidence that oral dosing with a CXCR4 antagonist can effectively correct WHIM-associated neutrophil and lymphocyte abnormalities in a mouse model of WHIM syndrome. These findings extend our understanding of how targeting the dysregulated CXCR4 signaling pathway can ameliorate the pathogenesis of WHIM syndrome. [ABSTRACT FROM AUTHOR]

It is well established that Ly6Chi monocytes develop from common monocyte progenitors (cMoPs) and reside in the bone marrow (BM) until they are mobilized into the circulation. In our study, we found that BM Ly6Chi monocytes are not a homogenous population, as current data would suggest. Using computational analysis approaches to interpret multidimensional datasets, we demonstrate that BM Ly6Chi monocytes consist of two distinct subpopulations (CXCR4hi and CXCR4lo subpopulations) in both mice and humans. Transcriptome studies and in vivo assays revealed functional differences between the two subpopulations. Notably, the CXCR4hi subset proliferates and is immobilized in the BM for the replenishment of functionally mature CXCR4lo monocytes. We propose that the CXCR4hi subset represents a transitional premonocyte population, and that this sequential step of maturation from cMoPs serves to maintain a stable pool of BM monocytes. Additionally, reduced CXCR4 expression on monocytes, upon their exit into the circulation, does not reflect its diminished role in monocyte biology. Specifically, CXCR4 regulates monocyte peripheral cellular activities by governing their circadian oscillations and pulmonary margination, which contributes toward lung injury and sepsis mortality. Together, our study demonstrates the multifaceted role of CXCR4 in defining BM monocyte heterogeneity and in regulating their function in peripheral tissues.

Summary: Warts, hypogammaglobulinaemia, infections and myelokathexis syndrome (WHIMS) is a rare combined primary immunodeficiency caused by the gain of function of the CXCR4 chemokine receptor. We present the prevalence of cancer in WHIMS patients based on data from the French Severe Chronic Neutropenia Registry and an exhaustive literature review. The median follow‐up of the 14 WHIMS 'patients was 28.5 years. A central review and viral evaluation of pathological samples were organized, and we conducted a thorough literature review to identify all reports of WHIMS cases. Six French patients were diagnosed with cancer at a median age of 37.6 years. The 40‐year risk of malignancy was 39% (95% confidence interval [CI]: 6%–74%). We observed two human papillomavirus (HPV)‐induced vulvar carcinomas, three lymphomas (two Epstein–Barr virus [EBV]‐related) and one basal cell carcinoma. Among the 155 WHIMS cases from the literature, 22 cancers were reported in 16 patients, with an overall cancer 40‐year risk of 23% (95% CI: 13%–39%). Malignancies included EBV‐associated lymphoproliferative disorders and HPV‐positive genital and anal cancers as in the French cohort. Worldwide, nine cases of malignancy were associated with HPV and four with EBV. Immunocompromised WHIMS patients appear to be particularly susceptible to developing early malignancy, mainly HPV‐induced carcinomas, followed by EBV‐related lymphomas. [ABSTRACT FROM AUTHOR]

Bone marrow (BM) long‐lived plasma cells (PCs) are essential for long‐term protection against infection, and their persistence within this organ relies on interactions with Cxcl12‐expressing stromal cells that are still not clearly identified. Here, using single cell RNAseq and in silico transinteractome analyses, we identified Leptin receptor positive (LepR+) mesenchymal cells as the stromal cell subset most likely to interact with PCs within the BM. Moreover, we demonstrated that depending on the isotype they express, PCs may use different sets of integrins and adhesion molecules to interact with these stromal cells. Altogether, our results constitute an unprecedented characterization of PC subset stromal niches and open new avenues for the specific targeting of BM PCs based on their isotype. [ABSTRACT FROM AUTHOR]

Neutrophils play a major role in the protection from infections but also in inflammation related to tumor microenvironment. However, cell-extrinsic and -intrinsic cues driving their function at steady state is still fragmentary. Using Cxcr2 knock-out mice, we have evaluated the function of the chemokine receptor Cxcr2 in neutrophil physiology. We show here that Cxcr2 deficiency decreases the percentage of mature neutrophils in the spleen, but not in the bone marrow (BM). There is also an increase of aged CD62Llo CXCR4hi neutrophils in the spleen of KO animals. Spleen Cxcr2-/- neutrophils display a reduced phagocytic ability, whereas BM neutrophils show an enhanced phagocytic ability compared to WT neutrophils. Spleen Cxcr2-/- neutrophils show reduced reactive oxygen species production, F-actin and a-tubulin levels. Moreover, spleen Cxcr2-/- neutrophils display an altered signaling with reduced phosphorylation of ERK1/2 and p38 MAPK, impaired PI3K-AKT, NF-kB, TGFβ and IFNγ pathways. Altogether, these results suggest that Cxcr2 is essential for neutrophil physiology. [ABSTRACT FROM AUTHOR]

Introduction WHIM syndrome is a rare combined immunodeficiency disorder characterized by disseminated human papillomavirus--induced warts, hypogammaglobulinemia, recurrent bacterial infections, and myelokathexis, a form of neutropenia associated with abnormal retention of [...], Leukocytes from individuals with warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, a rare immunodeficiency, and bearing a wild-type CXCR4 ORF ([WHIM.sup.WT]) display impaired CXCR4 internalization and desensitization upon exposure to CXCL12. The resulting enhanced CXCR4-dependent responses, including chemotaxis, probably impair leukocyte trafficking and account for the immunohematologic clinical manifestations of WHIM syndrome. We provided here evidence that GPCR kinase-3 (GRK3) specifically regulates CXCL12-promoted internalization and desensitization of CXCR4. GRK3-silenced control cells displayed altered CXCR4 attenuation and enhanced chemotaxis, as did [WHIM.sup.WT] cells. These findings identified GRK3 as a negative regulator of CXCL12-induced chemotaxis and as a candidate responsible for CXCR4 dysfunction in [WHIM.sup.WT] leukocytes. Consistent with this, we showed that GRK3 overexpression in both leukocytes and skin fibroblasts from 2 unrelated [WHIM.sup.WT] patients restored CXCL12-induced internalization and desensitization of CXCR4 and normalized chemotaxis. Moreover, we found in cells derived from one patient a profound and selective decrease in GRK3 products that probably resulted from defective mRNA synthesis. Taken together, these results have revealed a pivotal role for GRK3 in regulating CXCR4 attenuation and have provided a mechanistic link between the GRK3 pathway and the CXCR4-related [WHIM.sup.WT] disorder.