ABSTRACT Inflammation is a vastly underappreciated player in meibomian gland dysfunction – a common pathological condition of eyelid sebaceous glands responsible for producing tear film lipids. Pathologic blockage of the gland orifice and central duct, referred to as “obstructive” meibomian gland dysfunction, is regularly accompanied by ocular surface inflammation in patients. Moreover, this type of obstruction is ubiquitous, and even more aggressive, in patients with ocular surface immune disease. The latter is defined here as ocular surface pathology associated with systemic immune disorders, such as atopy (allergy), Stevens-Johnson syndrome/toxic epidermal necrolysis, Sjogren’s syndrome, graft versus host disease, cicatricial pemphigoid, and others. Still, the exact immune mechanisms involved in meibomian gland dysfunction remain elusive. As no pharmacological agents are indicated for meibomian gland dysfunction, there is a critical need for uncovering the pertinent immune responses of this pathobiology in patients with ocular surface immune disease. In the last funding cycle, we showed a direct role for neutrophils in meibomian gland dysfunction in our model of chronic-like allergic eye disease. Based on this work and the current literature, our central hypothesis is that neutrophils play a role in driving meibomian gland dysfunction across different forms of ocular surface immune diseases. In the current proposal, we will: i) elucidate the pathogenic neutrophil mechanisms that drive meibomian gland dysfunction in our established model of chronic-like allergic eye disease; ii) decipher the role of neutrophils in meibomian gland dysfunction in a novel and spontaneous genetic mouse model of ocular surface immune disease; and iii) map the tear immune cell landscape of meibomian gland dysfunction in patients with different ocular surface immune disease entities. In addition to mouse models, we will leverage our unpublished single cell RNA-seq dataset of immune cells from blood, conjunctiva, and tears from our allergic eye disease mouse model, paired with computational, genetic, and pharmacologic tools to pinpoint the pathogenic neutrophil mechanisms. For human samples, we established a 36-color flow cytometry panel for deep immune profiling of patient tears and have incorporated clinical expertise for the annotation of each individual with detailed clinical metadata. The rationale for this project is that identifying the critical immune cell subsets and associated pathogenic cellular programs that drive meibomian gland dysfunction will offer a strong scientific framework by which to delineate the role of inflammation in human meibomian gland dysfunction, as well as identify novel therapeutic targets and diagnostic immune markers to support efforts in the clinical space. Completion of these studies is therefore expected to have a significant impact by expanding the breadth and depth of knowledge regarding the role of inflammation, specifically neu...