The role of monocytes and neutrophils in fungal keratitis

Project summary/abstract Fusarium, Aspergillus and Candida are important causes of corneal blindness and vision loss in the USA and worldwide. Preliminary data using a murine models of Aspergillus keratitis show that monocyte depletion leads to exacerbated corneal disease and impaired fungal killing without affecting neutrophil recruitment to the corneal stroma. Aim 1 will examine neutrophil activation by Ca++ influx using a novel reporter mouse developed at UCI and will determine the effect of monocyte depletion on neutrophil pro-inflammatory and fungal killing activity in infected corneas by multi-photon, intravital microscopy. Preliminary data also identified distinct neutrophil clusters by single cell RNA sequencing of total CD45+ myeloid cells from infected corneas that included ICAM-1 expressing neutrophils that were localized at the site of infection in the cornea. FACS isolated ICAM-1+ neutrophils were more transcriptionally active and produced more cytokines than either ICAM-1- from WT mice or from ICAM-1-/- mice. Aim 2 will therefore examine the role of ICAM-1+ neutrophils in Fusarium, Aspergillus and Candida keratitis, and examine the role of the Hv1 proton channel that is elevated in ICAM-1+ neutrophils using Hvcn1-/- mice and novel, highly specific Hv1 inhibitors in fungal keratitis. Aim 3 will examine the processing and the role of the pore forming proteins Gasdermin D (GSDMD) and GSDME in IL-1β secretion by neutrophils and in fungal keratitis. We will also examine the effect of small molecule inhibitors of NLRP3, GSDMD and GSDME. Combined results from these proposed studies will greatly increase our understanding of the pathogenesis of fungal keratitis and identify potential targets for immune intervention.