Neutrophil L-Plastin Controls Ocular Paucibacteriality and Susceptibility to Keratitis

Abstract

Why ocular mucosa is paucibacterial is unknown. Many different mechanisms have been suggested but the comprehensive experimental studies are sparse. We found that a deficiency in L-plastin (LCP1), an actin bundling protein, resulted in an ocular commensal overgrowth, characterized with increased presence of conjunctival Streptococcal spp. The commensal overgrowth correlated with susceptibility to P. aeruginosa-induced keratitis. L-plastin knockout (KO) mice displayed elevated bacterial burden in the P. aeruginosa-infected corneas, altered inflammatory responses, and compromised bactericidal activity. Mice with ablation of LPL under the LysM Cre (LysM. Cre pos LPL fl/fl) and S100A8 Cre (S100A8.Cre pos LPL fl/fl) promoters had a similar phenotype to the LPL KOs mice. In contrast, infected CD11c.Cre pos LPL fl/fl mice did not display elevated susceptibility to infection, implicating the myeloid L-plastin-sufficient cells (e.g., macrophages and neutrophils) in maintaining ocular homeostasis. Mechanistically, the elevated commensal burden and the susceptibility to infection were linked to defects in neutrophil frequencies at steady state and during infection and compromised bactericidal activities upon priming. Macrophage exposure to commensal organisms primed neutrophil responses to P. aeruginosa, augmenting PMN bactericidal capacity in an L-plastin dependent manner. Cumulatively, our data highlight the importance of neutrophils in controlling ocular paucibacteriality, reveal molecular and cellular events involved in the process, and suggest a link between commensal exposure and resistance to infection.

Conjunctival Commensal Isolation and Identification in Mice

“…The results are a measure of relative viable commensal abundance, and not actual conjunctival bacterial burden, with the aim of defining the ocular commensal community. The relative abundance and diversity have been used in our studies for comparative purposes in bacterial keratitis, as well as, to investigate the innate immune response in knock-out and wild type mice 12 . In addition, identification by MALDI-TOF MS depends on a substantial database 28 , delivering a result of "no ID" for unknown isolate spectra profiles, highlighting the importance of using a current database.…”

“…Our data shows that there is a difference between skin and conjunctival microbiota, as well as greater diversity with increased age and a sex specific difference in abundance. Furthermore, this approach has reproducibly found commensal differences in knock-out mice 12 . This protocol can be applied to describe the ocular microbiome which may vary due to caging practices, geography, or disease state, as well as the local effects of commensal metabolites and products on immune system development and response.…”

Prenylcysteine oxidase 1 like protein is required for neutrophil bactericidal activities

“…Cornea tissues were dissected from infected animals and processed as in 62 . 1–2 × 10 6 cells were resuspended in 50 μL FACS buffer containing Fc block (Biolegend, cat.…”