Dysregulation of Systemic and Mucosal Humoral Responses to Microbial and Food Antigens as a Factor Contributing to Microbial Translocation and Chronic Inflammation in HIV-1 Infection.

HIV-1 infection is associated with an early and profound depletion of mucosal memory CD4+ T cells, a population that plays an indispensable role in the regulation of isotype switching and transepithelial transport of antibodies. In this study, we addressed whether the depletion of CD4+ T cell in HIV-1-infected individuals results in altered humoral responses specific to antigens encountered at mucosal surfaces. Comprehensive protein microarray of systemic humoral responses to intestinal microbiota demonstrated reduced IgG responses to antigens derived from Proteobacteria and Firmicutes but not Bacteroidetes. Importantly, intestinal secretions of antiretroviral therapy-treated HIV-1-infected individuals exhibited a significant elevation of IgM levels and decreased IgA/IgM and IgG/IgM ratios of antibodies specific to a variety of microbial and food antigens. The presented findings indicate reduced competence of mucosal B cells for class switch recombination from IgM to other isotypes limiting their capacity to react to changing antigenic variety in the gut lumen. Decreased availability of microbiota-specific IgA and IgG may be an important factor contributing to the translocation of microbial antigens across the intestinal mucosal barrier and their systemic dissemination that drives chronic inflammation in HIV-1-infected individuals.

Immune suppression by neutrophils in HIV-1 infection: role of PD-L1/PD-1 pathway.

HIV-1 infection is associated with a progressive loss of T cell functional capacity and reduced responsiveness to antigenic stimuli. The mechanisms underlying T cell dysfunction in HIV-1/AIDS are not completely understood. Multiple studies have shown that binding of program death ligand 1 (PD-L1) on the surface of monocytes and dendritic cells to PD-1 on T cells negatively regulates T cell function. Here we show that neutrophils in the blood of HIV-1-infected individuals express high levels of PD-L1. PD-L1 is induced by HIV-1 virions, TLR-7/8 ligand, bacterial lipopolysaccharide (LPS), and IFNα. Neutrophil PD-L1 levels correlate with the expression of PD-1 and CD57 on CD4+ and CD8+ T cells, elevated levels of neutrophil degranulation markers in plasma, and increased frequency of low density neutrophils (LDNs) expressing the phenotype of granulocytic myeloid-derived suppressor cells (G-MDSCs). Neutrophils purified from the blood of HIV-1-infected patients suppress T cell function via several mechanisms including PD-L1/PD-1 interaction and production of reactive oxygen species (ROS). Collectively, the accumulated data suggest that chronic HIV-1 infection results in an induction of immunosuppressive activity of neutrophils characterized by high expression of PD-L1 and an inhibitory effect on T cell function.

Altered serum cytokine signature in common variable immunodeficiency.

PURPOSE: Common variable immunodeficiency (CVID) is the most frequent form of primary symptomatic hypogammaglobulinemia. CVID patients display a number of abnormalities in lymphocyte subpopulations including chronic T-cell activation and decreased numbers of circulating CD4(+) T cells and NK cells. We and others have recently shown that CVID is associated with increased concentration of soluble CD14 (sCD14) and other factors indicating limited microbial translocation. METHODS: To address the mechanisms of chronic immune activation in CVID, we performed a detailed analysis of cytokine serum levels in 36 patients with CVID, 52 patients with selective IgA deficiency (IgAD), and 56 healthy volunteers. RESULTS: We show that CVID is associated with elevated serum levels of CXCL-10/IP-10, IL-1R antagonist, TNF-α, IL-10, IL-12 (p40), CCL-2/MCP-1, G-CSF, and CCL-11/eotaxin. The detected cytokine signature is consistent with an ongoing activation of cells of myeloid lineage. In contrast, the levels of cytokines typically produced by CD4(+) T helper cells of Th1 (IFN-γ, IL-2), Th2 (IL-9, IL-13), and Th17 (IL-17) subtypes were suppressed in CVID patients compared to healthy donors. CONCLUSIONS: Presented data suggest that the altered cytokine profile observed in patients with CVID may be attributed to the activation of monocyte-macrophage and granulocyte lineages, possibly driven by the translocation of bacterial components across the gastrointestinal or respiratory tracts mucosal barrier.

Transgenic mice overexpressing neuregulin-1 model neurofibroma-malignant peripheral nerve sheath tumor progression and implicate specific chromosomal copy number variations in tumorigenesis.

Patients with neurofibromatosis type 1 (NF1) develop benign plexiform neurofibromas that frequently progress to become malignant peripheral nerve sheath tumors (MPNSTs). A genetically engineered mouse model that accurately models plexiform neurofibroma-MPNST progression in humans would facilitate identification of somatic mutations driving this process. We previously reported that transgenic mice overexpressing the growth factor neuregulin-1 in Schwann cells (P(0)-GGFß3 mice) develop MPNSTs. To determine whether P(0)-GGFß3 mice accurately model human neurofibroma-MPNST progression, cohorts of these animals were monitored through death and were necropsied; 94% developed multiple neurofibromas, with 70% carrying smaller numbers of MPNSTs. Nascent MPNSTs were identified within neurofibromas, suggesting that these sarcomas arise from neurofibromas. Although neurofibromin expression was maintained, P(0)-GGFß3 MPNSTs exhibited Ras hyperactivation, as in human NF1-associated MPNSTs. P(0)-GGFß3 MPNSTs also exhibited abnormalities in the p16(INK4A)-cyclin D/CDK4-Rb and p19(ARF)-Mdm-p53 pathways, analogous to their human counterparts. Array comparative genomic hybridization (CGH) demonstrated reproducible chromosomal alterations in P(0)-GGFß3 MPNST cells (including universal chromosome 11 gains) and focal gains and losses affecting 39 neoplasia-associated genes (including Pten, Tpd52, Myc, Gli1, Xiap, and Bbc3/PUMA). Array comparative genomic hybridization also identified recurrent focal copy number variations affecting genes not previously linked to neurofibroma or MPNST pathogenesis. We conclude that P(0)-GGFß3 mice represent a robust model of neurofibroma-MPNST progression useful for identifying novel genes driving neurofibroma and MPNST pathogenesis.

Zebrafish class 1 phosphatidylinositol transfer proteins: PITPbeta and double cone cell outer segment integrity in retina.

Phosphatidylinositol transfer proteins (PITPs) in yeast co-ordinate lipid metabolism with the activities of specific membrane trafficking pathways. The structurally unrelated metazoan PITPs (mPITPs), on the other hand, are an under-investigated class of proteins. It remains unclear what biological activities mPITPs discharge, and the mechanisms by which these proteins function are also not understood. The soluble class 1 mPITPs include the PITPalpha and PITPbeta isoforms. Of these, the beta-isoforms are particularly poorly characterized. Herein, we report the use of zebrafish as a model vertebrate for the study of class 1 mPITP biological function. Zebrafish express PITPalpha and PITPbeta-isoforms (Pitpna and Pitpnb, respectively) and a novel PITPbeta-like isoform (Pitpng). Pitpnb expression is particularly robust in double cone cells of the zebrafish retina. Morpholino-mediated protein knockdown experiments demonstrate Pitpnb activity is primarily required for biogenesis/maintenance of the double cone photoreceptor cell outer segments in the developing retina. By contrast, Pitpna activity is essential for successful navigation of early developmental programs. This study reports the initial description of the zebrafish class 1 mPITP family, and the first analysis of PITPbeta function in a vertebrate.

Functional analysis of an Orc6 mutant in Drosophila.

The origin recognition complex (ORC) is a 6-subunit complex required for the initiation of DNA replication in eukaryotic organisms. ORC is also involved in other cell functions. The smallest Drosophila ORC subunit, Orc6, is important for both DNA replication and cytokinesis. To study the role of Orc6 in vivo, the orc6 gene was deleted by imprecise excision of P element. Lethal alleles of orc6 are defective in DNA replication and also show abnormal chromosome condensation and segregation. The analysis of cells containing the orc6 deletion revealed that they arrest in both the G(1) and mitotic stages of the cell cycle. Orc6 deletion can be rescued to viability by a full-length Orc6 transgene. The expression of mutant transgenes of Orc6 with deleted or mutated C-terminal domain results in a release of mutant cells from G(1) arrest and restoration of DNA replication, indicating that the DNA replication function of Orc6 is associated with its N-terminal domain. However, these mutant cells accumulate at mitosis, suggesting that the C-terminal domain of Orc6 is important for the passage through the M phase. In a cross-species complementation experiment, the expression of human Orc6 in Drosophila Orc6 mutant cells rescued DNA replication, suggesting that this function of the protein is conserved among metazoans.

Optimization of methods for the accurate characterization of whole blood neutrophils.

Neutrophils are the most abundant circulating leukocyte population with critical roles in immune defense, regulation of innate and adaptive immune systems, and disease pathogenesis. Our progress in understanding precise mechanisms of neutrophil activation, recruitment, and function has been hampered by the lack of optimized and standardized methods for the characterization and phenotyping of this readily activated population. By comparing eight methods of neutrophil characterization, we demonstrate that the level of neutrophil activation and degranulation is associated with specific experimental conditions and the number and type of manipulation steps employed. Staining whole blood at 4 °C and removal of remaining unbound antibodies prior to one-step fixation and red blood cell lysis minimizes neutrophil activation, decreases phenotypic alterations during processing, and prevents nonspecific antibody binding. The effects of anticoagulants used for collection, processing delays, and time and temperature during sample analysis on neutrophil phenotype are addressed. The presented data provide a foundation for higher quality standards of neutrophil characterization improving consistency and reproducibility among studies.

Role of the Orc6 protein in origin recognition complex-dependent DNA binding and replication in Drosophila melanogaster.

The six-subunit origin recognition complex (ORC) is a DNA replication initiator protein in eukaryotes that defines the localization of the origins of replication. We report here that the smallest Drosophila ORC subunit, Orc6, is a DNA binding protein that is necessary for the DNA binding and DNA replication functions of ORC. Orc6 binds DNA fragments containing Drosophila origins of DNA replication and prefers poly(dA) sequences. We have defined the core replication domain of the Orc6 protein which does not include the C-terminal domain. Further analysis of the core replication domain identified amino acids that are important for DNA binding by Orc6. Alterations of these amino acids render reconstituted Drosophila ORC inactive in DNA binding and DNA replication. We show that mutant Orc6 proteins do not associate with chromosomes in vivo and have dominant negative effects in Drosophila tissue culture cells. Our studies provide a molecular analysis for the functional requirement of Orc6 in replicative functions of ORC in Drosophila and suggest that Orc6 may contribute to the sequence preferences of ORC in targeting to the origins.

Specific and nonspecific membrane-binding determinants cooperate in targeting phosphatidylinositol transfer protein beta-isoform to the mammalian trans-Golgi network.

Phosphatidylinositol transfer proteins (PITPs) regulate the interface between lipid metabolism and specific steps in membrane trafficking through the secretory pathway in eukaryotes. Herein, we describe the cis-acting information that controls PITPbeta localization in mammalian cells. We demonstrate PITPbeta localizes predominantly to the trans-Golgi network (TGN) and that this localization is independent of the phospholipid-bound state of PITPbeta. Domain mapping analyses show the targeting information within PITPbeta consists of three short C-terminal specificity elements and a nonspecific membrane-binding element defined by a small motif consisting of adjacent tryptophan residues (the W(202)W(203) motif). Combination of the specificity elements with the W(202)W(203) motif is necessary and sufficient to generate an efficient TGN-targeting module. Finally, we demonstrate that PITPbeta association with the TGN is tolerant to a range of missense mutations at residue serine 262, we describe the TGN localization of a novel PITPbeta isoform with a naturally occurring S262Q polymorphism, and we find no other genetic or pharmacological evidence to support the concept that PITPbeta localization to the TGN is obligately regulated by conventional protein kinase C (PKC) or the Golgi-localized PKC isoforms delta or epsilon. These latter findings are at odds with a previous report that conventional PKC-mediated phosphorylation of residue Ser262 is required for PITPbeta targeting to Golgi membranes.

Hypertrophic neuropathies and malignant peripheral nerve sheath tumors in transgenic mice overexpressing glial growth factor beta3 in myelinating Schwann cells.

The neuregulin-1 (NRG-1) family of growth and differentiation factors exerts a variety of effects on Schwann cells and their precursors during nervous system development; however, NRG-1 effects on adult Schwann cells are poorly defined. Several lines of evidence suggest that NRG-1 actions on adult Schwann cells are distinct from those observed during development. To test this hypothesis, we generated transgenic mice overexpressing the NRG-1 isoform glial growth factor beta3 (GGFbeta3) in myelinating Schwann cells [protein zero (P0)GGFbeta3 mice]. P0-GGFbeta3 mice develop resting tremors, gait abnormalities, decreased hindlimb strength, and paralysis by approximately 7 months of age. Sciatic nerves from these animals show a hypertrophic neuropathy characterized by demyelination, remyelination, and "onion bulb" formation. Development of this hypertrophic neuropathy is preceded by Schwann cell hyperplasia that is prominent in 1-month-old mice and present but decreased in 2- and 4-month-old animals. P0-GGFbeta3 mice also develop peripheral ganglion-associated malignant peripheral nerve sheath tumors. Motor, sensory, and sympathetic ganglia from 1-, 2-, and 4-month-old P0-GGFbeta3 mice uniformly contain intraganglionic, likely preneoplastic, Schwann cell proliferations. Examination of bromodeoxyuridine incorporation and caspase-3 activation in sciatic nerves and trigeminal ganglia indicates that Schwann cell hyperplasia in P0-GGFbeta3 mice reflects increased proliferation rather than decreased apoptosis. These observations are consistent with the hypothesis that GGFbeta3 induces proliferation of adult Schwann cells and demyelination of peripheral nerve axons. Furthermore, overexpression of this NRG-1 isoform frequently induces neoplastic Schwann cell proliferation within PNS ganglia, suggesting that NRG-1 may contribute to human Schwann cell neoplasia.

Functional insight into the role of Orc6 in septin complex filament formation in Drosophila.

Septins belong to a family of polymerizing GTP-binding proteins that are important for cytokinesis and other processes that involve spatial organization of the cell cortex. We reconstituted a recombinant Drosophila septin complex and compared activities of the wild-type and several mutant septin complex variants both in vitro and in vivo. We show that Drosophila septin complex functions depend on the intact GTP-binding and/or hydrolysis domains of Pnut, Sep1, and Sep2. The presence of the functional C-terminal domain of septins is required for the integrity of the complex. Drosophila Orc6 protein, the smallest subunit of the origin recognition complex (ORC), directly binds to septin complex and facilitates septin filament formation. Orc6 forms dimers through the interactions of its N-terminal, TFIIB-like domains. This ability of the protein suggests a direct bridging role for Orc6 in stimulating septin polymerization in Drosophila. Studies reported here provide a functional dissection of a Drosophila septin complex and highlight the basic conserved and divergent features among metazoan septin complexes.

Effect of hormonal contraception on the function of plasmacytoid dendritic cells and distribution of immune cell populations in the female reproductive tract.

OBJECTIVE: Epidemiological evidence suggests an association between the use of hormonal contraception and an increased risk of acquiring sexually transmitted diseases including HIV-1. We sought to elucidate the biological mechanisms underlying the effect of hormonal contraception on the immune system. DESIGN: Cross-sectional study. METHODS: To delineate the biological mechanisms underlying the effect of hormonal contraceptives on the immune system, we analyzed the functional capacity of circulating plasmacytoid dendritic cells (pDCs), the distribution of vaginal immune cell populations, and the systemic and genital levels of immune mediators in women using depot medroxyprogesterone acetate (DMPA), NuvaRing, or combined oral contraceptives (COC). RESULTS: The use of DMPA or NuvaRing was associated with reduced capacity of circulating pDCs to produce interferon (IFN)-α and tumor necrosis (TNF-α) in response to TLR-9 stimulation. Systemic levels of IFN-α and cervicovaginal fluid levels of IFN-α, CXCL10, monocyte chemotactic protein-1, and granulocyte-colony stimulating factor were significantly lower in DMPA users compared to control volunteers not using hormonal contraception. The density of CD207 Langerhans cells in the vaginal epithelium was reduced in NuvaRing and combined oral contraceptive users but not in DMPA users. CONCLUSIONS: The presented evidence suggests that the use of some types of hormonal contraception is associated with reduced functional capacity of circulating pDCs and altered immune environment in the female reproductive tract.

Effect of progestins on immunity: medroxyprogesterone but not norethisterone or levonorgestrel suppresses the function of T cells and pDCs.

OBJECTIVES: The potential effect of hormonal contraception on HIV-1 acquisition and transmission represents an important public health issue. Several observational studies have suggested an association between the use of hormonal contraception, in particular injectable depot medroxyprogesterone acetate (DMPA), and an increased risk of HIV-1 acquisition and transmission. We and others have previously demonstrated that DMPA acts as a potent inhibitor of innate and adaptive immune mechanisms. The study presented here addresses the immunomodulatory properties of several common progestins with a potential to replace DMPA. STUDY DESIGN: To identify safe alternatives to DMPA, we tested the effect of commonly used progestins on the function of human primary T cells and plasmacytoid dendritic cells (pDCs) obtained from the blood of healthy premenopausal women. RESULTS: Medroxyprogesterone acetate (MPA) inhibited the activation of T cells and pDCs in response to T cell receptor- and Toll-like receptor-mediated activation at physiological concentrations. Etonogestrel exerted a partial suppressive activity at high concentrations. In sharp contrast, norethisterone (NET) and levonorgestrel (LNG) did not exhibit detectable immunosuppressive activity. CONCLUSION: Evidence indicating the immunosuppressive properties of DMPA strongly suggests that DMPA should be discontinued and replaced with other forms of long-term contraception. Since NET and LNG do not exert immunosuppressive properties at physiological concentrations, these progestins should be considered as alternative contraceptives for women at high risk for HIV-1 infection. IMPLICATIONS: The presented data suggest that, at physiological levels, the progestins NET and LNG do not suppress cytokine production by immune cells and should be considered as alternatives to DMPA; however, more in vivo testing is needed to confirm this data.

Hormonal contraception and HIV-1 infection: medroxyprogesterone acetate suppresses innate and adaptive immune mechanisms.

Recent observational studies indicate an association between the use of hormonal contraceptives and acquisition and transmission of HIV-1. The biological and immunological mechanisms underlying the observed association are unknown. Depot medroxyprogesterone acetate (DMPA) is a progestin-only injectable contraceptive that is commonly used in regions with high HIV-1 prevalence. Here we show that medroxyprogesterone acetate (MPA) suppresses the production of key regulators of cellular and humoral immunity involved in orchestrating the immune response to invading pathogens. MPA inhibited the production of interferon (IFN)-γ, IL-2, IL-4, IL-6, IL-12, TNFα, macrophage inflammatory protein-1α (MIP-1α), and other cytokines and chemokines by peripheral blood cells and activated T cells and reduced the production of IFNα and TNFα by plasmacytoid dendritic cells in response to Toll-like receptor-7, -8, and -9 ligands. Women using DMPA displayed lower levels of IFNα in plasma and genital secretions compared with controls with no hormonal contraception. In addition, MPA prevented the down-regulation of HIV-1 coreceptors CXCR4 and CCR5 on the surface of T cells after activation and increased HIV-1 replication in activated peripheral blood mononuclear cell cultures. The presented results suggest that MPA suppresses both innate and adaptive arms of the immune system resulting in a reduction of host resistance to invading pathogens.

Drosophila Orc6 facilitates GTPase activity and filament formation of the septin complex.

The origin recognition complex or ORC is a six-subunit protein important for DNA replication and other cell functions. Orc6, the smallest subunit of ORC, is essential for both replication and cytokinesis in Drosophila, and interacts with the septin protein Pnut, which is part of the Drosophila septin complex. In this study, we describe the analysis of the interaction of Orc6 with Pnut and whole Drosophila septin complex. Septin complex was purified from Drosophila embryos and also reconstituted from recombinant proteins. The interaction of Orc6 with the septin complex is dependent on the coiled-coil domain of Pnut. Furthermore, the binding of Orc6 to Pnut increases the intrinsic GTPase activity of the Drosophila septin complex, whereas in the absence of GTP it enhances septin complex filament formation. These results suggest an active role for Orc6 in septin complex function. Orc6 might be a part of a control mechanism directing the cytokinesis machinery during the final steps of mitosis.