Per- and polyfluoroalkyl substances (PFASs) are used in a multitude of processes and products, including nonstick coatings, food wrappers, and fire-fighting foams. These chemicals are environmentally-persistent, ubiquitous, and can be detected in the serum of 98% of Americans. Despite evidence that PFASs alter adaptive immunity, few studies have investigated their effects on innate immunity. The report here presents results of studies that investigated the impact of nine environmentally-relevant PFASs [e.g. perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid potassium salt (PFOS-K), perfluorononanoic acid (PFNA), perfluorohexanoic acid (PFHxA), perfluorohexane sulfonic acid (PFHxS), perfluorobutane sulfonic acid (PFBS), ammonium perfluoro(2-methyl-3-oxahexanoate) (GenX), 7H-perfluoro-4-methyl-3,6-dioxa-octane sulfonic acid (Nafion byproduct 2), and perfluoromethoxyacetic acid sodium salt (PFMOAA-Na)] on one component of the innate immune response, the neutrophil respiratory burst. The respiratory burst is a key innate immune process by which microbicidal reactive oxygen species (ROS) are rapidly induced by neutrophils in response to pathogens; defects in the respiratory burst can increase susceptibility to infection. The study here utilized larval zebrafish, a human neutrophil-like cell line, and primary human neutrophils to ascertain whether PFAS exposure inhibits ROS production in the respiratory burst. It was observed that exposure to PFHxA and GenX suppresses the respiratory burst in zebrafish larvae and a human neutrophil-like cell line. GenX also suppressed the respiratory burst in primary human neutrophils. This report is the first to demonstrate that these PFASs suppress neutrophil function and support the utility of employing zebrafish larvae and a human cell line as screening tools to identify chemicals that may suppress human immune function.

Neutrophils are important innate immune cells that respond during inflammation and infection. These migratory cells utilize β₂-integrin cell surface receptors to move out of the vasculature into inflamed tissues and to perform various anti-inflammatory responses. Although critical for fighting off infection, neutrophil responses can also become dysregulated and contribute to disease pathophysiology. In order to limit neutrophil-mediated damage, investigators have focused on β₂-integrins as potential therapeutic targets, but so far these strategies have failed in clinical trials. As the field continues to move forward, a better understanding of β₂-integrin function and signaling will aid the design of future therapeutics. Here, we provide a detailed review of resources, tools, experimental methods, and in vivo models that have been and will continue to be utilized to investigate the vitally important cell surface receptors, neutrophil β₂-integrins.

Analyses of human clinical HIV-1 vaccine trials and preclinical vaccine studies performed in rhesus macaque (RM) models have identified associations between non-neutralizing Fc Receptor (FcR)-dependent antibody effector functions and reduced risk of infection. Specifically, antibody-dependent phagocytosis (ADP) has emerged as a common correlate of reduced infection risk in multiple RM studies and the human HVTN505 trial. This recurrent finding suggests that antibody responses with the capability to mediate ADP are most likely a desirable component of vaccine responses aimed at protecting against HIV-1 acquisition. As use of RM models is essential for development of the next generation of candidate HIV-1 vaccines, there is a need to determine how effectively ADP activity observed in RMs translates to activity in humans. In this study we compared ADP activity of human and RM monocytes and polymorphonuclear leukocytes (PMN) to bridge this gap in knowledge. We observed considerable variability in the magnitude of monocyte and PMN ADP activity across individual humans and RM that was not dependent on FcR alleles, and only modestly impacted by cell-surface levels of FcRs. Importantly, we found that for both human and RM phagocytes, ADP activity of antibodies targeting the CD4 binding site was greatest when mediated by human IgG3, followed by RM and human IgG1. These results demonstrate that there is functional homology between antibody and FcRs from these two species for ADP. We also used novel RM IgG1 monoclonal antibodies engineered with elongated hinge regions to show that hinge elongation augments RM ADP activity. The RM IgGs with engineered hinge regions can achieve ADP activity comparable to that observed with human IgG3. These novel modified antibodies will have utility in passive immunization studies aimed at defining the role of IgG3 and ADP in protection from virus challenge or control of disease in RM models. Our results contribute to a better translation of human and macaque antibody and FcR biology, and may help to improve testing accuracy and evaluations of future active and passive prevention strategies.

MARCKS is an actin and PIP2-binding protein that plays an essential role in neutrophil migration and adhesion; however, the molecular details regarding MARCKS function in these processes remains unclear. Neutrophil adhesion and migration also require the cell surface receptors & beta;(2)-integrins. We hypothesized that MARCKS inhibition would alter neutrophil & beta;(2)-integrin activation and signaling. We utilized a MARCKS-targeting peptide to inhibit MARCKS in inside-out and outside-in & beta;(2)-integrin activation in neutrophils. MANS-mediated MARCKS inhibition had no significant effect on inside-out & beta;(2)-integrin activation. MANS treatment significantly attenuated ICAM-1/Mn2+-stimulated static adhesion, cell spreading and & beta;(2)-integrin clustering, suggesting a role for MARCKS function in outside-in & beta;(2)-integrin activation. Additional work is needed to better understand the molecular mechanisms of MARCKS role in outside-in & beta;(2)-integrin activation and signaling.

Neutrophils are innate immune cells that respond quickly to sites of bacterial infection and play an essential role in host defense. Interestingly, some bacterial pathogens benefit from exuberant neutrophil inflammation. Salmonella is one such pathogen that can utilize the toxic mediators released by neutrophils to colonize the intestine and cause enterocolitis. Because neutrophils can aid gut colonization during Salmonella infection, neutrophils represent a potential host-directed therapeutic target. Myristoylated alanine-rich C-kinase substrate (MARCKS) is an actin-binding protein that plays an essential role in many neutrophil effector responses. We hypothesized that inhibition of MARCKS protein would alter bovine neutrophil responses to Salmonella Typhimurium (STm) ex vivo. We used a MARCKS inhibitor peptide to investigate the role of MARCKS in neutrophil responses to STm. This study demonstrates that MARCKS inhibition attenuated STm-induced neutrophil adhesion and chemotaxis. Interestingly, MARCKS inhibition also enhanced neutrophil phagocytosis and respiratory burst in response to STm. This is the first report describing the role of MARCKS protein in neutrophil antibacterial responses.

Background: The HVTN108 trial evaluated the safety and immunogenicity of a DNA prime, adjuvanted protein boost HIV vaccine in the United States and South Africa. The underlying factors influencing individual variation in vaccine responsiveness are unknown. In this study, we defined the IgG Fc and Fc receptor (FcR) genotypes in the HVTN108 cohort to test our hypothesis that IgG and FcR genetic variation can affect vaccine-elicited functional antibody responses. Methods: IgG Fc and FcR alleles were determined by targeted PCR amplification and next-generation sequencing. Vaccine-elicited functional antibody responses, including binding antibody multiplex assay (BAMA), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP) activity, were measured using standardized and qualified methods. Relationships between alleles and antibody responses were identified by linear regression controlling for treatment group and region. Results: The distribution of many polymorphisms significantly differed between the United States and South Africa. Within the subset of the cohort tested for functional antibody responses (IgG, n = 41; FcR, n = 55), IgG genotypes such as IGHG1*12 ( P = 0.012), IGHG3*11 ( P = 0.033), IGHG2*02 ( P = 0.038), IGHG4*07 ( P = 0.076), and others were associated with ADCC antibody responses when corrected for vaccine group and regional effects. In the same way, we identified that the FCER1A rs2427827 mutation had a significant association with lower peak ADCC activity and the FCER2 rs2228137 mutation was associated with lower antibody binding to Con6 gp120 protein. Conclusions: Genetic variation in both antibodies and FcRs associated with levels of HIV—vaccine-elicited functional antibodies. Significant regional differences in distribution of this variation support the need for vaccine testing in diverse populations.