These findings identify extracellular β2m as a regulator of neutrophil-mediated phagocytosis and efferocytosis and demonstrate that proteolytic processing differentially influences these activities.
Abstract
Polymorphonuclear leukocytes (PMNs), predominantly neutrophil granulocytes, are key components of the innate immune system that eliminate invading pathogens through phagocytosis and clear apoptotic cells through efferocytosis. Beta-2-microglobulin (β2m) is best known as the light chain of major histocompatibility complex class I (MHC I), where it is required for antigen presentation to CD8⁺ T cells. However, emerging evidence suggests that extracellular β2m may also regulate innate immune responses. Here, we show that extracellular β2m enhances neutrophil phagocytosis and efferocytosis. Addition of soluble β2m (50 µg/ml) increased phagocytosis of latex beads by PMNs from 23% to 31%, whereas the proteolytically cleaved variant desLys58-β2m (dK58β2m) had no effect. In contrast, both β2m and dK58β2m enhanced phagocytosis of the Gram-positive bacterium Streptococcus pyogenes and the Gram-negative bacterium Acinetobacter baumannii by more than 3.6-fold. Furthermore, both β2m variants promoted efferocytosis of apoptotic Jurkat cells in a dose-dependent manner, resulting in up to a two-fold increase that was comparable to the effect of GM-CSF. Cytochalasin D abolished β2m-mediated uptake of apoptotic cells. Pre-incubation of latex beads with β2m followed by washing did not enhance phagocytosis, and pre-incubation of PMNs with β2m followed by washing did not enhance subsequent efferocytosis of apoptotic cells. These findings indicate that β2m does not act by coating phagocytic targets or by inducing sustained neutrophil priming. Collectively, these findings identify extracellular β2m as a regulator of neutrophil-mediated phagocytosis and efferocytosis and demonstrate that proteolytic processing differentially influences these activities.
Platelet Factor 4 (PF4), a cationic antimicrobial peptide, serves as a ligand for the myeloid-specific phagocytic receptor CR3 (Mac-1, CD11b/CD18). We previously demonstrated that recombinant dimeric PF4 (rdPF4) functions as a bacterial opsonin, enhancing phagocytosis of Gram-positive Staphylococcus aureus and facilitating clearance of both antibiotic-susceptible and methicillin-resistant S. aureus in a mouse model of infectious peritonitis. In this study, we examined whether rdPF4 is pathogen-agnostic by assessing its effect on phagocytosis of Gram-negative encapsulated Klebsiella pneumoniae, a WHO Bacterial Priority Pathogen. We demonstrate that rdPF4 enhances CR3-mediated phagocytosis of both live and heat-inactivated high-virulence K2 and low-virulence K3 strains of K. pneumoniae by various mouse and human macrophage cell lines, as well as primary neutrophils and macrophages. It also increased phagocytosis of carbapenem-resistant K. pneumoniae. rdPF4 did not directly kill bacteria but acted as an opsonin binding to the negatively charged bacterial capsule and creating recognition sites for CR3 on leukocytes. In a mouse sepsis model, a single dose of rdPF4 significantly enhanced bacterial clearance from the lungs, liver, and peritoneum and reduced bacteremia. Histological analyses showed that rdPF4 provided substantial protection to lung and liver tissues against K. pneumoniae-induced damage. Consistent with these findings, rdPF4 treatment increased the survival rates of infected mice. These results show that rdPF4 effectively targets the capsule, a key virulence factor of K. pneumoniae, thereby reducing the bacterium’s ability to evade the host immune response. Overall, the data suggest a common mechanism in which cationic rdPF4, by binding to the negatively charged surfaces of both Gram-negative and Gram-positive bacteria, diminishes their antiphagocytic properties.
N. Podolnikova, Iryna Klymenko, James Alagna et al.· bioRxiv· 0 citations
A novel function of Ecb is revealed, the activation of basophils to produce IL-4 in an IgE-independent manner, and a dual Ecb contribution to immune evasion-by interfering with complement activation and skewing Th2 immunity-and a role in the development of allergic inflammation-by inducing IL-4 production is suggested.
Haruka Sakakibara, Shion Kamada, Rikuto Iwata et al.· Microbiology and immunology· 0 citations
Human seminal plasma (HSP) plays an important role in shaping the reproductive immune environment, but its effects on modulating neutrophil antimicrobial functions and pathogen clearance remain unclear. In this study, we investigated the immunomodulatory effects of HSP on key neutrophil effector responses, including reactive oxygen species (ROS) production, neutrophil extracellular trap (NET) formation, phagocytosis, and bacterial killing. Human neutrophils were stimulated with calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or uropathogenic Escherichia coli (UPEC) in the presence or absence of HSP. In addition, neutrophils from NOX2- and PAD4-knockout mice were used to elucidate the molecular pathways underlying HSP-mediated regulation of NETosis. HSP significantly suppressed ROS production and NET formation induced by A23187, PMA, and UPEC, while also reducing neutrophil phagocytic capacity and impairing bacterial killing. Mechanistically, HSP-mediated inhibition of NETosis was found to be PAD4-dependent but NOX2-independent. Furthermore, the inhibitory effect of HSP on PMA-stimulated human neutrophils was diminished when HSP was obtained from donors pretreated with acetylsalicylic acid, which significantly reduced prostaglandin E2 levels in HSP. Consistent with this observation, pharmacological blockade of prostaglandin signaling restored ROS production in HSP-treated neutrophils. Overall, these findings identify HSP as a physiological inhibitor of neutrophil effector functions and support a role for HSP in maintaining immune homeostasis and tolerance within the reproductive tract through the suppression of neutrophil oxidative burst, NET formation, and antimicrobial activity.
Gabriel Mayoral-Andrade, Gabriela Vasquez-Martinez, Israel Cotzomi-Ortega et al.· Frontiers in Immunology· 0 citations
Understanding the complex network of molecular crosstalk between ESKAPE pathogen metabolites and NK cells is fundamental for developing novel immunotherapy strategies for drug-resistant infections, designing adjuvant drugs based on bacterial components as well as correcting immune disorders in critical conditions.
E. Denisova, E. Tyshchuk, L. Kraeva et al.· Russian Journal of Infection...· 0 citations
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