Immature neutrophils in cord blood exert increased expression of genes associated with antimicrobial function.

Introduction: The immune systems of both the mother and the newborn face significant challenges during birth. Proper immune regulation after birth is essential for the survival of neonates. Numerous studies have demonstrated that the neonatal immune system is relatively immature, particularly in its adaptive arm, placing the primary responsibility for immune surveillance on innate immunity. Methods: Given the significant role of neutrophils in protecting the neonate after birth, we conducted a study investigating the properties of neutrophils in newborn cord blood using various methodological approaches. Results: Our findings demonstrate the presence of immature low-density neutrophils in the cord blood, which are likely responsible for the observed elevated expression of genes coding for proteins essential to antimicrobial response, including myeloperoxidase, neutrophils elastase, and defensins. Discussion: We propose that these cells function normally and support the protection of newborns early after birth. Furthermore, our results suggest that the mode of delivery might significantly influence the programming of neutrophil function. The presented findings emphasize the importance of distinct neutrophil subpopulations in neonatal immunity and their potential impact on early postnatal health.

Interferon Lambda Signaling Restrains Experimental Autoimmune Encephalomyelitis.

IFN-λ is a type III interferon (IFN) with pleiotropic functions in modulating immune responses. To address its function in autoimmune neuroinflammation, we evaluated the development and progression of experimental autoimmune encephalitis (EAE) in IFNLR1KO (Ifnlr1-/-) and C57Bl/6 (WT) mice following immunization with MOG35-55 peptide. The results show that Ifnlr1-/-mice developed significantly more severe EAE than WT littermates with a similar day of onset, suggesting the potential of IFN-λ in reducing disease severity. We next interrogated whether IFN-λ differentially modulates EAE induced by encephalitogenic Th1 cells or Th17 cells. Encephalitogenic Th1 or Th17 generated from WT donors were transferred into WT or Ifnlr1-/-recipient mice. Whereas encephalitogenic Th1 cells induced more severe EAE in Ifnlr1-/- than WT recipients, the disease severity induced by encephalitogenic Th17 cells was similar. Additionally, in vitro experiments showed that Ifnlr1-/-macrophages promoted the expansion of myelin peptide-reactive Th17 cells but not Th1 cells. Early in the disease, the spinal cords of EAE mice displayed a significantly greater proportion of Ly6C-Ly6G+ cells with CXCR2+CD62Llo phenotype, indicating activated neutrophils. These findings suggest that IFN-λ signaling restrains activation and migration of neutrophils to the CNS, potentially attenuating neutrophil-mediated disease progression in autoimmune neuroinflammation. Recombinant IFN-λ can be used as a potential therapeutic target for treatment of patients with multiple sclerosis as it has fewer side effects due to the restricted expression of its receptor.

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.

Distinct phenotype of neutrophil, monocyte, and eosinophil populations indicates altered myelopoiesis in a subset of patients with multiple myeloma.

Hematologic malignancies, including multiple myeloma (MM), promote systemic immune dysregulation resulting in an alteration and increased plasticity of myeloid cell subsets. To determine the heterogeneity of the myeloid cell compartment in the peripheral blood of patients with MM, we performed a detailed investigation of the phenotype and function of myeloid subpopulations. We report that a subset of MM patients exhibits a specific myeloid cell phenotype indicative of altered myelopoiesis characterized by significant changes in the properties of circulating granulocytic, monocytic, and eosinophilic populations. The subset, referred to as MM2, is defined by a markedly elevated level of CD64 (FcγRI) on the surface of circulating neutrophils. Compared to healthy controls or MM1 patients displaying intermediate levels of CD64, neutrophils from MM2 patients exhibit a less differentiated phenotype, low levels of CD10 and CXC chemokine receptor 2 (CXCR2), increased capacity for the production of mitochondrial reactive oxygen species, and an expansion of CD16neg immature neutrophil subset. Classical and patrolling monocytes from MM2 patients express elevated levels of CD64 and activation markers. MM2 eosinophils display lower levels of C-C Chemokine receptor 3 (CCR3), Toll-like receptor 4 (TLR4, CD284), and tissue factor (TF, CD142). The MM2 (CD64high) phenotype is independent of age, race, sex, and treatment type. Characteristic features of the MM2 (CD64high) phenotype are associated with myeloma-defining events including elevated involved/uninvolved immunoglobulin free light chain (FLC) ratio at diagnosis. Detailed characterization of the altered myeloid phenotype in multiple myeloma will likely facilitate the identification of patients with an increased risk of disease progression and open new avenues for the rational design of novel therapeutic approaches.

C-Reactive Protein Promotes the Expansion of Myeloid Derived Cells With Suppressor Functions.

Previously we established that human C-reactive protein (CRP) exacerbates mouse acute kidney injury and that the effect was associated with heightened renal accumulation of myeloid derived cells with suppressor functions (MDSC). Herein we provide direct evidence that CRP modulates the development and suppressive actions of MDSCs in vitro. We demonstrate that CRP dose-dependently increases the generation of MDSC from wild type mouse bone marrow progenitors and enhances MDSC production of intracellular reactive oxygen species (iROS). When added to co-cultures, CRP significantly enhanced the ability of MDSCs to suppress CD3/CD28-stimulated T cell proliferation. Experiments using MDSCs from FcγRIIB deficient mice (FcγRIIB-/-) showed that CRP's ability to expand MDSCs and trigger their increased production of iROS was FcγRIIB-independent, whereas its ability to enhance the MDSC T cell suppressive action was FcγRIIB-dependent. Importantly, CRP also enabled freshly isolated primary human neutrophils to suppress proliferation of autologous T cells. These findings suggest that CRP might be an endogenous regulator of MDSC numbers and actions in vivo.

Common variable immunodeficiency patients display elevated plasma levels of granulocyte activation markers elastase and myeloperoxidase.

Common variable immunodeficiency disorders (CVIDs) represent a group of primary immunodeficiency diseases characterized by hypogammaglobulinemia and dysfunctional immune response to invading pathogens. Previous studies have indicated that CVID is associated with microbial translocation and systemic myeloid cell activation. The goal of this study was to determine whether patients with CVID display elevated systemic levels of markers of granulocyte activation and whether the levels are further influenced by intravenous immunoglobulin (IVIg) infusions. The plasma levels of granulocyte activation markers elastase and myeloperoxidase were determined using enzyme-linked immunosorbent assay (ELISA) in 46 CVID patients and 44 healthy controls. All CVID patients were in a stable state with no apparent acute infection. In addition, granulocyte activation markers' plasma levels in 24 CVID patients were determined prior to and 1 h following IVIg administration. Neutrophil elastase and myeloperoxidase plasma levels were significantly higher in CVID patients than in healthy controls. Systemic elastase levels were further increased following IVIg administration. In vitro stimulation of 13 CVID patients' whole blood using IVIg in a therapeutically relevant dose for 2 h resulted in a significant increase in plasma elastase levels compared to unstimulated blood. The data presented here indicate that CVID is associated with chronic granulocytic activation which is further exacerbated by administering IVIg. Increased myeloperoxidase and elastase levels may contribute to associated comorbidities in CVID patients.

Neutrophil and Granulocytic Myeloid-Derived Suppressor Cell-Mediated T Cell Suppression Significantly Contributes to Immune Dysregulation in Common Variable Immunodeficiency Disorders.

Common variable immunodeficiency disorders (CVID) represent a group of primary immunodeficiency diseases characterized by hypogammaglobulinemia and impaired specific Ab response, resulting in recurrent infections due to dysfunctional immune response. The specific mechanisms mediating immune deficiency in CVID remain to be determined. Previous studies indicated that immune dysregulation in CVID patients is associated with chronic microbial translocation, systemic immune activation, and altered homeostasis of lymphocytic and myeloid lineages. A detailed phenotypic, functional characterization of plasma markers and immune cell populations was performed in 46 CVID patients and 44 healthy donors. CVID patients displayed significantly elevated plasma levels of a marker of neutrophil activation neutrophil gelatinase-associated lipocalin. Neutrophils from CVID patients exhibited elevated surface levels of CD11b and PD-L1 and decreased levels of CD62L, CD16, and CD80, consistent with a phenotype of activated neutrophils with suppressive properties. Neutrophils from CVID patients actively suppressed T cell activation and release of IFN-γ via the production of reactive oxygen species. Furthermore, CVID was associated with an increased frequency of low-density neutrophils (LDNs)/granulocytic myeloid-derived suppressor cells. LDN/granulocytic myeloid-derived suppressor cell frequency in CVID patients correlated with reduced T cell responsiveness. Exogenous stimulation of whole blood with bacterial LPS emulated some but not all of the phenotypic changes observed on neutrophils from CVID patients and induced neutrophil population with LDN phenotype. The presented data demonstrate that neutrophils in the blood of CVID patients acquire an activated phenotype and exert potent T cell suppressive activity. Specific targeting of myeloid cell-derived suppressor activity represents a novel potential therapeutic strategy for CVID.

Hormonal Contraception and HIV-1 Acquisition: Biological Mechanisms.

Access to effective affordable contraception is critical for individual and public health. A wide range of hormonal contraceptives (HCs), which differ in composition, concentration of the progestin component, frequency of dosage, and method of administration, is currently available globally. However, the options are rather limited in settings with restricted economic resources that frequently overlap with areas of high HIV-1 prevalence. The predominant contraceptive used in sub-Saharan Africa is the progestin-only three-monthly injectable depot medroxyprogesterone acetate. Determination of whether HCs affect HIV-1 acquisition has been hampered by behavioral differences potentially confounding clinical observational data. Meta-analysis of these studies shows a significant association between depot medroxyprogesterone acetate use and increased risk of HIV-1 acquisition, raising important concerns. No association was found for combined oral contraceptives containing levonorgestrel, nor for the two-monthly injectable contraceptive norethisterone enanthate, although data for norethisterone enanthate are limited. Susceptibility to HIV-1 and other sexually transmitted infections may, however, be dependent on the type of progestin present in the formulation. Several underlying biological mechanisms that may mediate the effect of HCs on HIV-1 and other sexually transmitted infection acquisition have been identified in clinical, animal, and ex vivo studies. A substantial gap exists in the translation of basic research into clinical practice and public health policy. To bridge this gap, we review the current knowledge of underlying mechanisms and biological effects of commonly used progestins. The review sheds light on issues critical for an informed choice of progestins for the identification of safe, effective, acceptable, and affordable contraceptive methods.

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.

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.

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.

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.

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.

The Neonatal Fc receptor (FcRn) enhances human immunodeficiency virus type 1 (HIV-1) transcytosis across epithelial cells.

The mechanisms by which human immunodeficiency virus type 1 (HIV-1) crosses mucosal surfaces to establish infection are unknown. Acidic genital secretions of HIV-1-infected women contain HIV-1 likely coated by antibody. We found that the combination of acidic pH and Env-specific IgG, including that from cervicovaginal and seminal fluids of HIV-1-infected individuals, augmented transcytosis across epithelial cells as much as 20-fold compared with Env-specific IgG at neutral pH or non-specific IgG at either pH. Enhanced transcytosis was observed with clinical HIV-1 isolates, including transmitted/founder strains, and was eliminated in Fc neonatal receptor (FcRn)-knockdown epithelial cells. Non-neutralizing antibodies allowed similar or less transcytosis than neutralizing antibodies. However, the ratio of total:infectious virus was higher for neutralizing antibodies, indicating that they allowed transcytosis while blocking infectivity of transcytosed virus. Immunocytochemistry revealed abundant FcRn expression in columnar epithelia lining the human endocervix and penile urethra. Acidity and Env-specific IgG enhance transcytosis of virus across epithelial cells via FcRn and could facilitate translocation of virus to susceptible target cells following sexual exposure.

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.

Optimization of the transductional efficiency of lentiviral vectors: effect of sera and polycations.

Lentiviral vectors are widely used as effective gene-delivery vehicles. Optimization of the conditions for efficient lentiviral transduction is of a high importance for a variety of research applications. Presence of positively charged polycations reduces the electrostatic repulsion forces between a negatively charged cell and an approaching enveloped lentiviral particle resulting in an increase in the transduction efficiency. Although a variety of polycations are commonly used to enhance the transduction with retroviruses, the relative effect of various types of polycations on the efficiency of transduction and on the potential bias in the determination of titer of lentiviral vectors is not fully understood. Here, we present data suggesting that DEAE-dextran provides superior results in enhancing lentiviral transduction of most tested cell lines and primary cell cultures. Specific type and source of serum affects the efficiency of transduction of target cell populations. Non-specific binding of enhanced green fluorescent protein (EGFP)-containing membrane aggregates in the presence of DEAE-dextran does not significantly affect the determination of the titer of EGFP-expressing lentiviral vectors. In conclusion, various polycations and types of sera should be tested when optimizing lentiviral transduction of target cell populations.