Neutrophil priming occurs in a sequential manner and can be visualized in living animals by monitoring IL-1ß promoter activation.

Rapid enhancement of phagocyte functionality is a hallmark of neutrophil priming. GeneChip analyses unveiled elevated CD54, dectin-2, and IL-1ß mRNA expression by neutrophils isolated from inflammatory sites. In fact, CD54 and dectin-2 protein expression was detected on neutrophils recovered from skin, peritoneal, and lung inflammation lesions but not on those in bone marrow or peripheral blood. Neutrophils increased CD54 and dectin-2 mRNA during migration in Boyden chambers and acquired CD54 and dectin-2 surface expression after subsequent exposure to GM-CSF. Neutrophils purified from IL-1ß promoter-driven DsRed-transgenic mice acquired DsRed signals during cell migration or exposure to GM-CSF. CD54 and dectin-2 were expressed by DsRed(+) (but not DsRed(-)) neutrophils in GM-CSF-supplemented cultures, and neutrophils recovered from inflammatory sites exhibited strong DsRed signals. The dynamic process of neutrophil priming was studied in chemically induced inflammatory skin lesions by monitoring DsRed expression using confocal microscopy. A majority (>80%) of Ly6G(+) neutrophils expressed DsRed, and those DsRed(+)/Ly6G(+) cells exhibited crawling motion with a higher velocity compared with their DsRed(-)/Ly6G(+) counterparts. This report unveils motile behaviors of primed neutrophils in living animals. We propose that neutrophil priming occurs in a sequential manner with rapid enhancement of phagocyte functionality, followed by CD54 and dectin-2 mRNA and protein expression, IL-1ß promoter activation, and accelerated motility. Not only do these findings provide a new conceptual framework for our understanding of the process of neutrophil priming, they also unveil new insights into the pathophysiology of many inflammatory disorders that are characterized by neutrophil infiltration.

Race-related differences in functional antibody response to pneumococcal vaccination in HIV-infected individuals.

BACKGROUND: Both HIV positivity and African American (AA) ethnicity are associated with increased incidence of invasive pneumococcal disease (IPD). Poor immune response to pneumococcal polysaccharide-based vaccines may contribute to the race related increased frequency of IPD in African American HIV positive individuals. METHODS: Caucasian and AA HIV-infected (HIV+) individuals 40-65 years old with CD4+ T cells/µl (CD4) >200 on antiretroviral therapy (ART) received either the 13-valent pneumococcal conjugate vaccine (PCV) followed by the 23-valent pneumococcal polysaccharide vaccine (PPV) or PPV only. Serum IgG, IgM and opsonophagocytic antibody responses to serotypes 14 and 23F as well as serum IgG and opsonophagocytic antibody responses to serotype 19A were measured pre- and post-vaccination. We measured serum markers of inflammation in all participants and performed single cell gene expression profiling at the baseline by HD Biomark in Caucasians and African Americans. RESULTS: There were no significant differences in pre-immunization inflammatory markers or post-vaccination IgG and IgM concentrations between Caucasian and African American participants. However, we found significantly lower opsonophagocytic activity in response to serotypes 14 and 19A in the AA group compared to the Caucasian group. There was no association between inflammatory markers and immune response to vaccination, however we found extensive biomodal variation in gene expression levels in single IgM+ memory B cells. Differentially expressed genes may be related to differences in the immune response between ethnic groups. CONCLUSIONS: Distinct racial differences were found in the functional immune response following either PPV and/or PCV/PPV immunization in HIV-positive adults, although these differences were serotype dependent. Decreased ability to respond to vaccination may in part explain racial disparities in pneumococcal disease epidemiology. ClinicalTrials.gov ID: NCT03039491.

Inflammatory Markers and Immune Response to Pneumococcal Vaccination in HIV-Positive and -Negative Adults.

BACKGROUND: Members of the Tumor Necrosis Factor (TNF)-superfamily have speculated roles in the response against T-independent type II antigens (TI-II) including pneumococcal polysaccharides (PPS). Dysregulation in their expression is associated with an enhanced risk for pneumococcal disease in neonates but their expression in other high-risk populations including HIV-positive individuals remains to be elucidated. OBJECTIVE: To investigate signals that contribute towards PPS-response and identify potential anomalies that may account for diminished serological response in HIV-positive individuals post Pneumovax (PPV23) immunization. METHODS: Markers of inflammation, C-reactive protein (CRP), IL-6, sCD27 and sCD30, were assessed in HIV-positive and -negative individuals as potential predictors of PPV23 response. Serum levels of B cell activating factor (BAFF), transmembrane activator and calcium-modulator and cytophilin ligand interactor (TACI), B cell maturation antigen (BCMA) and B cell expression of BAFF-R, TACI, BCMA, CD40 and CD21 were assessed in total (unselected) and PPS23F (antigen)-specific B cells of PPV23 immunized HIV-positive and -negative individuals. RESULTS: CRP, sCD27, sCD30 and BAFF were significantly elevated in the serum of HIV-positive individuals but did not adversely affect PPV23 response. Assessment of PPS-specific B cells revealed enhanced TACI and reduced BAFF-R expression compared to unselected B cells in HIV-positive and -negative individuals. Surface TACI was similar but soluble TACI was significantly lower in HIV-positive compared to HIV-negative individuals. CONCLUSION: Current studies highlight a potential role for TACI in PPV23 response based on its enhanced expression on PPS-specific B cells. Although surface levels of TACI were similar, diminished soluble TACI (sTACI) in HIV-positive compared to HIV-negative individuals could potentially decrease BAFF responsiveness and Ig response. A better understanding of the role of TNF receptors could contribute to the design of improved pneumococcal vaccines. TRIAL REGISTRATION: ClinicalTrials.gov NCT02515240.

Quantitative and Functional Antibody Responses to the 13-Valent Conjugate and/or 23-Valent Purified Polysaccharide Vaccine in Aging HIV-Infected Adults.

BACKGROUND: The number of aging human immunodeficiency virus-infected (HIV+) individuals living in the United States has substantially grown over the past two decades. Advanced age and HIV infection both increase susceptibility to Streptococcus pneumoniae infection due to B cell dysfunction. The combined impact of these factors on pneumococcal vaccine responses remains unknown. METHODS: We assessed serum immunoglobulin (Ig) G and IgM levels and opsonophagocytic killing assay (OPA) titers to pneumococcal serotypes 14 and 23F in HIV+ subjects and HIV-uninfected (HIV-) controls 50-65 years old. HIV+ individuals with CD4+ T cells/µl (CD4) >200 and ≥1 year of antiretroviral therapy (ART) received either a dose of the 13-valent pneumococcal conjugate vaccine followed by the 23-valent pneumococcal polysaccharide vaccine 8 weeks later (PCV/PPV) as currently recommended (n=15) or a single dose of PPV only (n=22). HIV- controls received PCV/PPV (n=14). RESULTS: HIV+ PCV/PPV and PPV groups exhibited similar increases in IgG levels and OPA titers for both serotypes after immunization. Postvaccination IgM levels for serotype 23F, but not 14, were significantly higher in HIV+ PCV/PPV compared to PPV groups. IgG and IgM levels for serotype 14 and OPA titers to serotype 23F were significantly reduced in HIV+ compared to HIV- PCV/PPV groups. Serotype-specific IgG levels correlated with OPA titers for all groups. CONCLUSIONS: Our data suggest that the recommended PCV/PPV regimen may not significantly improve quantitative or functional antibody responses compared to PPV only in aging HIV+ subjects. Continued efforts aimed at improving vaccine responses in this high risk population are warranted.

Alterations in serotype-specific B cell responses to the 13-valent pneumococcal conjugate vaccine in aging HIV-infected adults.

BACKGROUND: Advanced age and human immunodeficiency virus (HIV) infection are associated with increased pneumococcal disease risk. The impact of these factors on cellular responses to vaccination is unknown. METHODS: HIV-infected (HIV+) individuals 50-65 years old with CD4(+) Tcells/µl (CD4) >200 on antiretroviral therapy (ART) ≥1 year received either the 13-valent pneumococcal conjugate vaccine followed by the 23-valent pneumococcal polysaccharide vaccine (PCV/PPV) or PPV only. HIV-uninfected (HIV-) controls received PCV/PPV. Phenotype distribution and surface expression of complement receptor CD21 and tumor necrosis factor superfamily receptors (TNFRs) were compared on serotype-specific B cells postvaccination. RESULTS: Postvaccination serotype-specific B cell percentages were significantly lower in HIV+ PCV/PPV compared to PPV groups, but similar between HIV+ or HIV- PCV/PPV groups. Transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI)(+) serotype-specific B cell percentages were significantly decreased in HIV+ PCV/PPV compared to PPV groups. CD21(+) serotype-specific B cells were significantly higher in HIV- compared to HIV+ PCV/PPV groups. CONCLUSIONS: An initial dose of PCV reduced the frequency, but not phenotype distribution, of serotype-specific B cells and also lowered TACI expression in aging HIV+ subjects postvaccination with PPV. These findings suggest that PCV does not enhance cellular responses to revaccination with PPV.

Age-related immune response to pneumococcal polysaccharide vaccination: lessons for the clinic.

Due to distinct immunological limitations, both infants and elderly individuals are highly susceptible to Streptococcus pneumoniae. Routine immunization of children with the conjugate vaccine over the past decade has substantially reduced incidence of vaccine-serotype related invasive pneumococcal disease in both vaccinated and unvaccinated persons of all ages. However, disease burden remains high in the elderly despite the effects of herd protection and recommended use of polysaccharide vaccine in this population for over 30 years. An increase in drug resistance and incidence of infections caused by non-vaccine serotypes emphasize the need to improve current vaccination strategies. Recent efforts to identify age-associated defects in vaccine response and the use of conjugate vaccine and potential alternatives in adults are discussed.

Response to Pneumococcal Polysaccharide Vaccination in Newly Diagnosed HIV-Positive Individuals.

BACKGROUND: Newly diagnosed HIV-positive individuals are 35 to 100-fold more susceptible to Streptococcus pneumoniae infection compared to non-infected individuals. Therefore, the 23-valent pneumococcal polysaccharide vaccine (PPV23) has previously been recommended, though efficacy and effectiveness of vaccination remains controversial. Early severe B cell dysfunction is a central feature of HIV infection. The specific nature of the immune cells involved in the production of protective antigen-specific antibodies in HIV-positive individuals remains to be elucidated. OBJECTIVES: Evaluate the antibody and antigen-specific B cell response to the 23-valent pneumococcal polysaccharide vaccine in newly diagnosed HIV-positive patients. Moreover, determine if newly diagnosed patients with CD4<200 cells/µl benefit from 6-12 months of HAART, allowing partial viral suppression and immune reconstitution, prior to immunization. METHODS: Newly diagnosed HIV-positive patients with CD4>200 cells/µl and CD4<200 cells/µl were immunized with PPV23. Patients with CD4<200 cells/µl received either immediate or delayed immunization following 6-12 months of HAART. Antibody responses, opsonophagocytic activity and phenotypic analysis of pneumococcal polysaccharide-specific B cells were studied. RESULTS: Newly diagnosed HIV-positive patients demonstrated CD4-dependent increases in antibody and opsonophagocytic titers thought to be commensurate with protection. Functional opsonophagocytic titers of patients with CD4<200 cells/µl immunized immediately compared to patients with CD4<200 cells/µl receiving HAART for 6-12 months were not significantly different. Pneumococcal polysaccharide-specific B cells were distributed evenly between IgM memory and switched memory B cells for all groups, but IgM memory B cells were significantly lower than in HIV-negative individuals. CONCLUSIONS: Despite CD4-dependent pneumococcal polysaccharide-specific deficiencies in newly diagnosed HIV-positive patients, vaccination was beneficial based on opsonophagocytic titers for all newly diagnosed HIV-positive groups. In HIV-positive patients with CD4<200 cells/µl, 6-12 months of HAART did not improve opsonophagocytic titers or antibody concentrations. Based on these findings, immunization with the 23-valent pneumococcal polysaccharide vaccine should not be delayed in newly diagnosed HIV-positive patients with CD4<200 cells/µl.

Response to Pneumococcal Polysaccharide Vaccination in HIV-Positive Individuals on Long Term Highly Active Antiretroviral Therapy.

BACKGROUND AND OBJECTIVES: Streptococcus pneumoniae continues to cause serious infections in HIV-positive individuals in the era of highly active anti-retroviral therapy. This led to the recommendation to revaccinate HIV-positive individuals with PPV23 five years after primary vaccination. The benefits of revaccination and the impact of long term highly active anti-retroviral therapy (HAART) on antigen-specific B cell reconstitution have remained unclear thus far and were investigated. DESIGN AND METHODS: We assessed antibody levels, opsonophagocytic activity and phenotype of pneumococcal polysaccharide (PPS) specific-B cells post-revaccination in long term HAART cohorts stratified according to CD4 count as group A (CD4>200) and group B (CD4<200). Anti-PPS IgG, IgM and functional antibody response against vaccine serotypes 14 and 23F were measured by ELISA and opsonophagocytic assay followed by phenotypic analysis of PPS14 and 23F-specific B cells using fluorescently labeled PPS. RESULTS: Significant increases in total and functional antibody titers were noted in groups A and B post-vaccination concomitant with significant rise in PPS-specific IgM memory B cells, a critical B cell subset required for protection against PPS although the overall response remained significantly diminished compared to HIV-negative volunteers. CONCLUSION: Comparable increases in opsonophagocytic titers between study groups A and B concomitant with a comparable rise in PPS-specific IgM memory B cells indicate revaccination to be beneficial regardless of the degree of CD4 T cell reconstitution. These findings emphasize the importance of defining effective vaccination practices amongst high-risk individuals.

Local release of properdin in the cellular microenvironment: role in pattern recognition and amplification of the alternative pathway of complement.

Properdin, the only positive regulatory protein of the complement system, acts as both a stabilizer of the alternative pathway (AP) convertases and as a selective pattern recognition molecule of certain microorganisms and host cells (i.e., apoptotic/necrotic cells) by serving as a platform for de novo C3b,Bb assembly. Properdin, a highly positively charged protein, normally exists as cyclic dimers (P(2)), trimers (P(3)), and tetramers (P(4)) of head-to-tail associations of monomeric 53 kDa subunits. While most complement proteins are produced mainly in the liver, properdin is synthesized primarily by various cell types, including neutrophils, monocytes, primary T cells, and shear-stressed endothelial cells resulting in properdin serum levels of 4-25 µg/ml. Multiple inflammatory agonists stimulate the release of properdin from stimulated leukocytes into the cellular microenvironment. Concentrated, focused increases in properdin levels may lead to stabilization and initiation of AP convertases, thus greatly amplifying the complement response to a local stimulus. This review highlights current knowledge related to these properties and discusses the implications of properdin production in a pro-inflammatory microenvironment.