Regulation of IL-1α and Its Role in Microbial Keratitis

Microbial infection of the cornea is one of the leading causes of preventable blindness world-wide. The infection results in corneal opacity, inflammation, and intense pain that could lead to permanent blindness if left untreated. Treatment involves antimicrobial agents followed by corticosteroids to suppress inflammation. However, there is a need for a more targeted treatment to regulate inflammation as steroids are non-specific. In both human patients and our murine model of bacterial and fungal keratitis, neutrophils are the predominant infiltrating cell-type in the cornea comprising up to 90% of recruited cells. Neutrophils are essential for bacterial killing and acts as an important source of proinflammatory cytokines during microbial keratitis. Depletion of neutrophils leads to tremendously impaired bacterial clearance and decreased cytokine levels in the cornea. We previously reported that the proinflammatory cytokines IL-1α and IL-1β are highly upregulated in both human and mice during microbial keratitis. IL-1β is necessary for neutrophil recruitment to the corneas and subsequently protection from the infection. However, whether there is a role for IL-1α and what role that may be is unclear. This thesis explores the production and regulation of IL-1α, and what role it is playing in the cornea during infection. We found that while neutrophils are the predominant source of IL-1β in the cornea, both neutrophils and monocytes contribute to IL-1α production which peaked at 24hpi. To examine whether there is a role for IL-1α in the response to bacterial infection of the cornea, we compared WT, Il1a-/-, Il1b-/-, and Il1a-/-Il1b-/- mice infected with P. aeruginosa. In accordance with our previous studies, Il1b-/- and DKO mice were unable to control the infection due to delayed neutrophil and monocyte recruitment to the cornea. On the contrary, Il1a-/- mice had the opposite effect—showing enhanced bacteria killing compared to WT mice despite no differences in neutrophil recruitment. We found no differences in neutrophil effector functions between WT and Il1a-/- neutrophils. However, RNA sequencing revealed Il1a-/- neutrophils had a more proinflammatory transcriptomic profile than WT neutrophils with elevations in C1q expression. These findings reveal a novel role for IL-1α, that is distinct from IL-1β, in regulating the response to P. aeruginosa infection in the cornea. Further, we examined the regulation of IL-1α secretion compared to IL-1β in macrophages and neutrophils. The mechanism for IL-1β secretion is well characterized in macrophages where it is tightly regulated in a two-step process mediated by inflammasomes and GSDMD. We found that IL-1α secretion by macrophages is also mediated by GSDMD, but not NLRP3 inflammasome. However, IL-1α secretion by neutrophils is independent of both NLRP3 and GSDMD in response to stimulation with β-glucan. Secretion of IL-1α by neutrophils is also independent of cell death. Instead, we found that IL-1α secretion is mediated by extracellular vesicle release. In conclusion, we described a novel role for IL-1α that is distinct from IL-1β during bacterial infection of the corneas—where IL-1α is detrimental and IL-1β is protective. We also found that IL-1α is important for monocyte recruitment to the cornea whereas IL-1β is essential for neutrophil recruitment. Our finding adds to the increasing evidence showing non-redundant roles for the two cytokines despite sharing the same receptor, IL-1R1. Further, we found differential regulation for IL-1α and IL-1β secretion by neutrophils. While IL-1β is dependent on NLRP3 and GSDMD for secretion, IL-1α release is mediated by extracellular vesicles. Together, these studies improve our understanding of the inflammatory response during microbial keratitis and identify potential targets for therapeutics

Pathogenic Aspergillus and Fusarium as important causes of blinding corneal infections - the role of neutrophils in fungal killing, tissue damage and cytokine production.

Filamentous fungi Aspergillus and Fusarium species are major causes of visual impairment and blindness in immune competent individuals. Once conidia penetrate the corneal epithelium and enter the stroma, they undergo germination, and exposure of cell wall components induces a pronounced neutrophil-rich cellular infiltrate. In this review, we discuss Aspergillus and novel Fusarium virulence factors that are required for corneal infection, and describe the multiple functions of neutrophils in limiting hyphal growth in the cornea. This review will also discuss the role of neutrophils as an important source of cytokines in fungal keratitis, and highlight recent studies identifying unique characteristics of neutrophil secretion of IL-1α and IL-1β

β-Glucan-stimulated neutrophil secretion of IL-1α is independent of GSDMD and mediated through extracellular vesicles.

Neutrophils are an important source of interleukin (IL)-1β and other cytokines because they are recruited to sites of infection and inflammation in high numbers. Although secretion of processed, bioactive IL-1β by neutrophils is dependent on NLRP3 and Gasdermin D (GSDMD), IL-1α secretion by neutrophils has not been reported. In this study, we demonstrate that neutrophils produce IL-1α following injection of Aspergillus fumigatus spores that express cell-surface β-glucan. Although IL-1α secretion by lipopolysaccharide (LPS)/ATP-activated macrophages and dendritic cells is GSDMD dependent, IL-1α secretion by β-glucan-stimulated neutrophils occurs independently of GSDMD. Instead, we found that bioactive IL-1α is in exosomes that were isolated from cell-free media of β-glucan-stimulated neutrophils. Further, the exosome inhibitor GW4869 significantly reduces IL-1α in extracellular vesicles (EVs) and total cell-free supernatant. Together, these findings identify neutrophils as a source of IL-1α and demonstrate a role for EVs, specifically exosomes, in neutrophil secretion of bioactive IL-1α

Differential Roles for IL-1α and IL-1β in Pseudomonas aeruginosa Corneal Infection.

Pseudomonas aeruginosa is an important cause of dermal, pulmonary, and ocular disease. Our studies have focused on P. aeruginosa infections of the cornea (keratitis) as a major cause of blinding microbial infections. The infection leads to an influx of innate immune cells, with neutrophils making up to 90% of recruited cells during early stages. We previously reported that the proinflammatory cytokines IL-1α and IL-1β were elevated during infection. Compared with wild-type (WT), infected Il1b-/- mice developed more severe corneal disease that is associated with impaired bacterial killing as a result of defective neutrophil recruitment. We also reported that neutrophils are an important source of IL-1α and IL-1β, which peaked at 24 h postinfection. To examine the role of IL-1α compared with IL-1β in P. aeruginosa keratitis, we inoculated corneas of C57BL/6 (WT), Il1a-/-, Il1b-/-, and Il1a-/-Il1b-/- (double-knockout) mice with 5 × 104 ExoS-expressing P. aeruginosa. Il1b-/- and double-knockout mice have significantly higher bacterial burden that was consistent with delayed neutrophil and monocyte recruitment to the corneas. Surprisingly, Il1a-/- mice had the opposite phenotype with enhanced bacteria clearance compared with WT mice. Although there were no significant differences in neutrophil recruitment, Il1a-/- neutrophils displayed a more proinflammatory transcriptomic profile compared to WT with elevations in C1q expression that likely caused the phenotypic differences observed. To our knowledge, our findings identify a novel, non-redundant role for IL-1α in impairing bacterial clearance

Immunity to pathogenic fungi in the eye.

Fusarium, Aspergillus and Candida are important fungal pathogens that cause visual impairment and blindness in the USA and worldwide. This review will summarize the epidemiology and clinical features of corneal infections and discuss the immune and inflammatory responses that play an important role in clinical disease. In addition, we describe fungal virulence factors that are required for survival in infected corneas, and the activities of neutrophils in fungal killing, tissue damage and cytokine production