The GPCR adaptor protein norbin suppresses the neutrophil-mediated immunity of mice to pneumococcal infection

Abstract

Streptococcal pneumonia is a worldwide health problem that kills ∼2 million people each year, particularly young children, the elderly, and immunosuppressed individuals. Alveolar macrophages and neutrophils provide the early innate immune response to clear pneumococcus from infected lungs. However, the level of neutrophil involvement is context dependent, both in humans and in mouse models of the disease, influenced by factors such as bacterial load, age, and coinfections. Here, we show that the G protein–coupled receptor (GPCR) adaptor protein norbin (neurochondrin, NCDN), which was hitherto known as a regulator of neuronal function, is a suppressor of neutrophil-mediated innate immunity. Myeloid norbin deficiency improved the immunity of mice to pneumococcal infection by increasing the involvement of neutrophils in clearing the bacteria, without affecting neutrophil recruitment or causing autoinflammation. It also improved immunity during Escherichia coli–induced septic peritonitis. It increased the responsiveness of neutrophils to a range of stimuli, promoting their ability to kill bacteria in a reactive oxygen species–dependent manner, enhancing degranulation, phagocytosis, and the production of reactive oxygen species and neutrophil extracellular traps, raising the cell surface levels of selected GPCRs, and increasing GPCR-dependent Rac and Erk signaling. The Rac guanine-nucleotide exchange factor Prex1, a known effector of norbin, was dispensable for most of these effects, which suggested that norbin controls additional downstream targets. We identified the Rac guanine-nucleotide exchange factor Vav as one of these effectors. In summary, our study presents the GPCR adaptor protein norbin as an immune suppressor that limits the ability of neutrophils to clear bacterial infections.

Age-related decline in the resistance of mice to bacterial infection and in LPS/TLR4 pathway-dependent neutrophil responses

“…Pathogenic E. coli O18:K1 bacteria ( 34 ) were used to induce septic peritonitis as described ( 35 ). The bacteria were grown in a biosafety level 2 facility to mid-log phase in Luria broth (LB) at 37°C, 5% CO 2 , pelleted, snap-frozen in PBS/20% glycerol and stored in aliquots at −80°C.…”

“…Mature primary neutrophils were freshly isolated each day by Percoll PLUS gradient at 4°C from mouse bone marrow using endotoxin-free reagents throughout, essentially as previously described ( 35 ). Mouse bone-marrow was flushed from femurs, tibias and pelvic bones with ice-cold Hank’s Balanced Salt Solution without Ca 2+ or Mg 2+ (HBSS – , Sigma H6648) supplemented with 15 mM HEPES, pH 7.4 (RT) (Sigma, H3784) and 0.25% fatty acid-free (FAF) BSA (Sigma, A8806) (HBSS –++ ), triturated and filtered through a 40 μm cell strainer.…”

“…The ability of isolated neutrophils to kill E. coli was measured essentially as described ( 35 ). E. coli DH5α bacteria (New England Biolabs, 2527) were grown in LB to logarithmic phase on the day of the experiment.…”

“…Phagocytosis of zymosan yeast particles was assayed essentially as described ( 35 ). Zymosan A particles (Molecular Probes, Z2894) were opsonised using IgG opsonising reagent (Molecular Probes, Z2850) according to the manufacturer’s instructions and stored in HBSS ++++ at 4°C.…”

“…NET production in response to stimulation with Staphylococcus aureus ( S. aureus, Wood 46 ) was done essentially as described ( 35 ). S. aureus were subcultured in LB to logarithmic growth at 37°C, sedimented for 2 min at 12,000 x g, washed in Dulbecco’s Modified Eagle Medium with Ca 2+ , Mg 2+ and 4.5 g/l glucose (DMEM +++ , Thermo Fisher Scientific, 31053) supplemented with 10 mM Hepes, pH 7.4 (DMEM +++ /Hepes), and opsonized with 10% mouse serum for 30 min at 37°C.…”