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.

Host defense against bacterial and fungal infections diminishes with age. In humans, impaired neutrophil responses are thought to contribute to this decline. However, it remains unclear whether neutrophil responses are also impaired in old mice. Here, we investigated neutrophil function in old mice, focusing on responses primed by lipopolysaccharide (LPS), an endotoxin released by gram-negative bacteria like E. coli, which signals through toll-like receptor (TLR) 4. We show that old mice have a reduced capacity to clear pathogenic E. coli during septic peritonitis. Neutrophil recruitment was elevated during LPS-induced but not aseptic peritonitis. Neutrophils from old mice showed reduced killing of E. coli. Their reactive oxygen species (ROS) production was impaired upon priming with LPS but not with GM-CSF/TNFα. Phagocytosis and degranulation were reduced in a partially LPS-dependent manner, whereas impairment of NET release in response to S. aureus was independent of LPS. Unexpectedly, chemotaxis was normal, as were Rac1 and Rac2 GTPase activities. LPS-primed activation of Erk and p38 Mapk was defective. PIP3 production was reduced upon priming with LPS but not with GM-CSF/TNFα, whereas PIP2 levels were constitutively low. The expression of 5% of neutrophil proteins was dysregulated in old age. Granule proteins, particularly cathepsins and serpins, as well as TLR-pathway proteins and membrane receptors were upregulated, whereas chromatin and RNA regulators were downregulated. The upregulation of CD180 and downregulation of MyD88 likely contribute to the impaired LPS signaling. In summary, all major neutrophil responses except chemotaxis decline with age in mice, particularly upon LPS priming. This LPS/TLR4 pathway dependence resolves previous controversy regarding effects of age on murine neutrophils and confirms that mice are an appropriate model for the decline in human neutrophil function.

IntroductionRac-GTPases and their Rac-GEF activators play important roles in neutrophil-mediated host defence. These proteins control the adhesion molecules and cytoskeletal dynamics required for neutrophil recruitment to inflamed and infected organs, and the neutrophil effector responses that kill pathogens.MethodsHere, we used live cell TIRF-FRET imaging in neutrophils from Rac-FRET reporter mice with deficiencies in the Rac-GEFs Dock2, Tiam1 or Prex1/Vav1 to evaluate if these proteins activate spatiotemporally distinct pools of Rac, and to correlate patterns of Rac activity with the neutrophil responses they control.ResultsAll the GEFs were required for neutrophil adhesion, and Prex1/Vav1 were important during spreading and for the velocity of migration during chemotaxis. However, Dock2 emerged as the prominent regulator of neutrophil responses, as this GEF was required for neutrophil polarisation and random migration, for migration velocity during chemokinesis, for the likelihood to migrate and for the speed of migration and of turning during chemotaxis, as well as for rapid particle engulfment during phagocytosis. We identified characteristic spatiotemporal patterns of Rac activity generated by Dock2 which correlate with the importance of the Rac-GEF in these neutrophil responses. We also demonstrate a requirement for Dock2 in neutrophil recruitment during aseptic peritonitis.DiscussionCollectively, our data provide a first direct comparison of the pools of Rac activity generated by different types of Rac-GEFs, and identify Dock2 as a key regulator of polarisation, migration and phagocytosis in primary neutrophils.