Enhanced IgG1 -mediated antibody response towards thymus-dependent immunization in CXCR1-deficient mice.

BACKGROUND: Chemokine receptors and their corresponding ligands are key players of immunity by regulation of immune cell differentiation and migration. CXCR1 is a high-affinity receptor for CXCL8. Differential expression of CXCR1 is associated with a variety of human pathologies including cancer and inflammatory diseases. While various studies have highlighted the importance of CXCR1-mediated CXCL8-sensing for neutrophil trafficking and function, its role in B-cell responses remains unsolved. Therefore, our aim was to investigate innate and adaptive antibody responses in CXCR1-deficient mice. METHODS: Cell populations of the spleen and the peritoneal cavity were identified and quantified via flow cytometry. To investigate thymus-independent (TI) and thymus-dependent (TD) antibody responses, mice were immunized intraperitoneally with TNP-Ficoll, Pneumovax23, and TNP-Chicken Gamma Globulin. Mice were bled before as well as 7 and 14 days after vaccination to collect serum. Serum antibody levels overtime were analyzed according to their specificity by enzyme-linked immunosorbent assay. B-1 cell functionality was examined by IL-5/IL-5Rα-dependent stimulation of peritoneal and splenic cells in vitro. To analyze CXCR1/2-expression, CD19+ splenocytes were enriched by magnetic-activated cell sorting before isolation of total RNA contents, followed by reverse transcription and real-time polymerase chain reaction. RESULTS: The distribution of natural B-1 cell populations was disturbed in the absence of CXCR1, while their responsiveness towards TI antigens and in vitro stimulation remained functional. Besides, CXCR1-deficiency was accompanied by increased frequencies of follicular B-2 cells in the spleen. Interestingly, these mice produced elevated levels of antigen-specific IgG1 upon TD immunization and harbored a significantly enlarged proportion of CXCR5-expressing T helper (H) cells. CXCR1-expression was detectable in CD19+ splenocytes derived from wild-type, but not CXCR1-deficient mice. CONCLUSION: Our data demonstrate a previously unknown relevance of CXCR1 for the production of specific IgG1 in response to vaccination. These findings identify CXCR1 as a promising candidate for future studies on the regulation of adaptive antibody responses.

SLy2-deficiency promotes B-1 cell immunity and triggers enhanced production of IgM and IgG2 antibodies against pneumococcal vaccine.

BACKGROUND: Despite the benefits of existing vaccines, Streptococcus pneumoniae is still responsible for the greatest proportion of respiratory tract infections around the globe, thereby substantially contributing to morbidity and mortality in humans. B-1 cells are key players of bacterial clearance during pneumococcal infection and even provide long-lasting immunity towards S. pneumoniae. Previous reports strongly suggest an essential role of the immunoinhibitory adapter Src homology domain 3 lymphocyte protein 2 (SLy2) for B-1 cell-mediated antibody production. The objective of this study is to evaluate S. pneumoniae-directed B cell responses in the context of SLy2 deficiency. METHODS: B-1 cell populations were analyzed via flow cytometry before and after pneumococcal immunization of SLy2-deficient and wild-type control mice. Global and vaccine-specific immunoglobulin M (IgM) and IgG antibody titers were assessed by enzyme-linked immunosorbent assay. To investigate survival rates during acute pneumococcal lung infection, mice were intranasally challenged with S. pneumoniae (serotype 3). Complementary isolated splenic B cells were stimulated in vitro and their proliferative response was assessed by fluorescent staining. In vitro antibody secretion was quantified by LEGENDplex. RESULTS: We demonstrate increased frequencies of B-1 cells and elevated titers of preantigenic IgM in SLy2-deficient mice. In addition, these mice produce significantly more amounts of IgM and IgG2 upon pneumococcal vaccination. Knocking out SLy2 did not induce survival advantages in our murine model of acute pneumonia, indicating the presence of compensatory mechanisms. CONCLUSION: Our results reveal reinforced specific antibody responses towards pneumococcal polysaccharides and enhanced IgG2 secretion as a consequence of SLy2 deficiency, which could be relevant to the development of more efficient vaccines.

Deficiency of PI3-Kinase catalytic isoforms p110γ and p110δ in mice enhances the IL-17/G-CSF axis and induces neutrophilia.

BACKGROUND: Phosphoinositide 3-kinase γ (PI3Kγ) and PI3Kδ are second messenger-generating enzymes with key roles in proliferation, differentiation, survival, and function of leukocytes. Deficiency of the catalytic subunits p110γ and p110δ of PI3Kγ and PI3Kδ in p110γ/δ-/- mice leads to defective B- and T-cell homeostasis. Here we examined the role of p110γ and p110δ in the homeostasis of neutrophils by analyzing p110γ-/-, p110δ-/- and p110γ/δ-/- mice. METHODS: Neutrophils and T cells in leukocyte suspensions from the bone marrow (BM), blood, spleen and lung were analyzed by flow cytometry. Serum concentrations of IL-17, of the neutrophilic growth factor G-CSF, and of the neutrophil mobilizing CXC chemokines CXCL1/KC and CXCL2/MIP-2 were measured by Bio-Plex assay. Production of G-CSF and CXCL1/KC by IL-17-stimulated primary lung tissue cells were determined by ELISA, whereas IL-17-dependent signaling in lung tissue cells was analyzed by measuring Akt phosphorylation using immunoblot. RESULTS: We found that in contrast to single knock-out mice, p110γ/δ-/- mice exhibited significantly elevated neutrophil counts in blood, spleen, and lung. Increased granulocytic differentiation stages in the bone marrow of p110γ/δ-/- mice were paralleled by increased serum concentrations of G-CSF, CXCL1/KC, and CXCL2/MIP-2. As IL-17 induces neutrophilia via the induction of G-CSF and CXC chemokines, we measured IL-17 and IL-17-producing T cells. IL-17 serum concentrations and frequencies of IL-17+ splenic T cells were significantly increased in p110γ/δ-/- mice. Moreover, IFN-γ+, IL-4+, and IL-5+ T cell subsets were drastically increased in p110γ/δ-/- mice, suggesting that IL-17+ T cells were up-regulated in the context of a general percentage increase of other cytokine producing T cell subsets. CONCLUSIONS: We found that p110γ/δ deficiency in mice induces complex immunological changes, which might in concert contribute to neutrophilia. These findings emphasize a crucial but indirect role of both p110γ and p110δ in the regulation of neutrophil homeostasis.

SLy1 regulates T-cell proliferation during Listeria monocytogenes infection in a Foxo1-dependent manner.

Infection of mice with Listeria monocytogenes results in a strong T-cell response that is critical for an efficient defense. Here, we demonstrate that the adapter protein SLy1 (SH3-domain protein expressed in Lymphocytes 1) is essential for the generation of a fully functional T-cell response. The lack of SLy1 leads to reduced survival rates of infected mice. The increased susceptibility of SLy1 knock-out (KO) mice was caused by reduced proliferation of differentiated T cells. Ex vivo analyses of isolated SLy1 KO T cells displayed a dysregulation of Forkhead box protein O1 shuttling after TCR signaling, which resulted in an increased expression of cell cycle inhibiting genes, and therefore, reduced expansion of the T-cell population. Forkhead box protein O1 shuttles to the cytoplasm after phosphorylation in a protein complex including 14-3-3 proteins. Interestingly, we observed a similar regulation for the adapter protein SLy1, where TCR stimulation results in SLy1 phosphorylation and SLy1 export to the cytoplasm. Moreover, immunoprecipitation analyses revealed a binding of SLy1 to 14-3-3 proteins. Altogether, this study describes SLy1 as an immunoregulatory protein, which is involved in the generation of adaptive immune responses during L. monocytogenes infection, and provides a model of how SLy1 regulates T-cell proliferation.

Fluorescent Ly6G antibodies determine macrophage phagocytosis of neutrophils and alter the retrieval of neutrophils in mice.

Fluorescently labeled Ly6G antibodies enable the tracking of neutrophils in mice, whereas purified anti-Ly6G rapidly depletes neutrophils from the circulation. The mechanisms underlying neutrophil depletion are still under debate. Here, we examined how identical Ly6G antibodies coupled to different fluorochromes affect neutrophil fate in vivo. BM cells stained with Ly6G antibodies were injected into mice. The number of retrieved anti-Ly6G-FITC(+) cells was reduced significantly in comparison with anti-Ly6G-APC(+) or anti-Ly6G-PE(+) cells. Flow cytometry and multispectral imaging flow cytometry analyses revealed that anti-Ly6G-FITC(+) neutrophils were preferentially phagocytosed by BMMs in vitro and by splenic, hepatic, and BM macrophages in vivo. Direct antibody injection of anti-Ly6G-FITC but not anti-Ly6G-PE depleted neutrophils to the same degree as purified anti-Ly6G, indicating that the FITC-coupled antibody eliminates neutrophils by a similar mechanism as the uncoupled antibody. With the use of a protein G-binding assay, we demonstrated that APC and PE but not FITC coupling inhibited access to interaction sites on the anti-Ly6G antibody. We conclude the following: 1) that neutrophil phagocytosis by macrophages is a central mechanism in anti-Ly6G-induced neutrophil depletion and 2) that fluorochrome-coupling can affect functional properties of anti-Ly6G antibodies, thereby modifying macrophage uptake of Ly6G-labeled neutrophils and neutrophil retrieval following adoptive cell transfer or injection of fluorescent anti-Ly6G.

SLy2 controls the antibody response to pneumococcal vaccine through an IL-5Rα-dependent mechanism in B-1 cells.

The adaptor protein SLy2 (Src homology domain 3 lymphocyte protein 2) is located on human chromosome 21 and was reported to be among a group of genes amplified in Down's syndrome (DS) patients. DS patients characteristically show an impaired immunity to pneumococcal infections. However, molecular mechanisms linking gene amplifications with specific DS phenotypes remain elusive. To investigate the effect of SLy2 gene amplification on the mammalian immune system, we studied SLy2 overexpressing transgenic-SLy2 (TG) mice. We found that baseline immunoglobulin M (IgM) levels as well as IgM responses following Pneumovax immunizations were reduced in TG mice. Moreover, B-1 cells, the major natural IgM-producing population in mice, were reduced in the peritoneal cavity of TG mice, while other immune cell compartments were unaltered. Mechanistically, SLy2 overexpression attenuated the expression of the IL-5 receptor α chain on B-1 cells, resulting in decreased B-1 cell numbers and decreased differentiation into Ab-secreting cells. Since B-1 cells essentially contribute to immunity against Streptococcus pneumoniae, the present study provides a novel molecular link between SLy2 expression and pneumococcal-specific IgM responses in vivo. These studies suggest that the adaptor protein SLy2 is a potential future target for immunomodulatory strategies for pneumococcal infections.

Eph receptor B4 is a regulator of estrogen receptor alpha in breast cancer cells.

BACKGROUND: Estrogen receptor alpha (ER-α) plays an important role in breast cancer initiation and progression and represents a major target in cancer therapy. The expression and activity of ER-α is regulated by multiple mechanisms at the transcriptional and post-translational level. Interaction of tyrosine kinase receptor-activated signaling pathways with ER-α function has been reported. We previously performed a kinome-wide small interfering RNA high-throughput screen to identify novel protein kinases involved in the regulation of ER-α transcriptional activity in human breast cancer cells. Our screening analysis identified the Eph receptor tyrosine kinases (Eph) as potential positive regulators of ER-α. RESULTS: In this study, we demonstrate Eph receptor B4 (EphB4), a member of Eph kinase family, a positive regulator of ER-α in human breast cancer cell lines (MCF-7, T-47D and BT-474). Down-regulation of EphB4 by RNA interference technology impairs estrogen-dependent ER-α transcriptional activity in breast cancer cells. Decreased activity of ER-α after EphB4 knockdown is the consequence of diminished ER-α messenger RNA and protein expression. Furthermore, phosphorylation of Akt, a downstream mediator of EphB4, is reduced following EphB4 silencing. CONCLUSIONS: Our data suggests EphB4 as an upstream regulator of ER-α in human breast cancer cells by modulating ER-α transcription. The results also suggest Akt as a relevant downstream signaling molecule in this novel EphB4-ER-α pathway.

ß-Catenin Is a Positive Regulator of Estrogen Receptor-α Function in Breast Cancer Cells.

Estrogen receptor-alpha (ERα) is a key factor in the development of breast cancer in humans. The expression and activity of ERα is regulated by a multitude of intracellular and extracellular signals. Here we show a cross-talk between ß-catenin and ERα in human breast cancer cells. Knockdown of ß-catenin by RNAi resulted in significant reduction of ERα mRNA and/or protein levels in MCF-7, T-47D, and BT-474 breast cancer cells and in significant reduction of estradiol-induced expression of the ERα target genes pS2 and GREB1. In addition ß-catenin silencing resulted in significant decrease of growth of MCF-7 cells both in the absence and presence of estradiol. ß-catenin and ERα could not be co-immunoprecipitated by ERα antibodies from lysates of E2-treated or untreated cells suggesting lack of direct physical interaction. It is concluded that ß-catenin is a positive regulator of ERα mRNA and protein expression.