Chemokine expression profile of an innate granuloma.

Granulomas are defined by the presence of organized layers of immune cells that include macrophages. Granulomas are often characterized as a way for the immune system to contain an infection and prevent its dissemination. We recently established a mouse infection model where Chromobacterium violaceum induces the innate immune system to form granulomas in the liver. This response successfully eradicates the bacteria and returns the liver to homeostasis. Here, we sought to characterize the chemokines involved in directing immune cells to form the distinct layers of a granuloma. We use spatial transcriptomics to investigate the spatial and temporal expression of all CC and CXC chemokines and their receptors within this granuloma response. The expression profiles change dynamically over space and time as the granuloma matures and then resolves. To investigate the importance of monocyte-derived macrophages in this immune response, we studied the role of CCR2 during C. violaceum infection. Ccr2-/- mice had negligible numbers of macrophages, but large numbers of neutrophils, in the C. violaceum-infected lesions. In addition, lesions had abnormal architecture resulting in loss of bacterial containment. Without CCR2, bacteria disseminated and the mice succumbed to the infection. This indicates that macrophages are critical to form a successful innate granuloma in response to C. violaceum.

The Yin and Yang of non-immune and immune responses in meibomian gland dysfunction.

Meibomian gland dysfunction (MGD) is a leading cause of dry eye disease and one of the most common ophthalmic conditions encountered in eye clinics worldwide. These holocrine glands are situated in the eyelid, where they produce specialized lipids, or meibum, needed to lubricate the eye surface and slow tear film evaporation - functions which are critical to preserving high-resolution vision. MGD results in tear instability, rapid tear evaporation, changes in local microflora, and dry eye disease, amongst other pathological entities. While studies identifying the mechanisms of MGD have generally focused on gland obstruction, we now know that age is a major risk factor for MGD that is associated with abnormal cell differentiation and renewal. It is also now appreciated that immune-inflammatory disorders, such as certain autoimmune diseases and atopy, may trigger MGD, as demonstrated through a T cell-driven neutrophil response. Here, we independently discuss the underlying roles of gland and immune related factors in MGD, as well as the integration of these two distinct mechanisms into a unified perspective that may aid future studies. From this unique standpoint, we propose a revised model in which glandular dysfunction and immunopathogenic pathways are not primary versus secondary contributors in MGD, but are fluid, interactive, and dynamic, which we likened to the Yin and Yang of MGD.

Microglia at sites of atrophy restrict the progression of retinal degeneration via galectin-3 and Trem2.

Outer retinal degenerations, including age-related macular degeneration (AMD), are characterized by photoreceptor and retinal pigment epithelium (RPE) atrophy. In these blinding diseases, macrophages accumulate at atrophic sites, but their ontogeny and niche specialization remain poorly understood, especially in humans. We uncovered a unique profile of microglia, marked by galectin-3 upregulation, at atrophic sites in mouse models of retinal degeneration and human AMD. In disease models, conditional deletion of galectin-3 in microglia led to phagocytosis defects and consequent augmented photoreceptor death, RPE damage, and vision loss, indicating protective roles. Mechanistically, Trem2 signaling orchestrated microglial migration to atrophic sites and induced galectin-3 expression. Moreover, pharmacologic Trem2 agonization led to heightened protection but in a galectin-3-dependent manner. In elderly human subjects, we identified this highly conserved microglial population that expressed galectin-3 and Trem2. This population was significantly enriched in the macular RPE-choroid of AMD subjects. Collectively, our findings reveal a neuroprotective population of microglia and a potential therapeutic target for mitigating retinal degeneration.

An innate granuloma eradicates an environmental pathogen using Gsdmd and Nos2.

Granulomas often form around pathogens that cause chronic infections. Here, we discover an innate granuloma model in mice with an environmental bacterium called Chromobacterium violaceum. Granuloma formation not only successfully walls off, but also clears, the infection. The infected lesion can arise from a single bacterium that replicates despite the presence of a neutrophil swarm. Bacterial replication ceases when macrophages organize around the infection and form a granuloma. This granuloma response is accomplished independently of adaptive immunity that is typically required to organize granulomas. The C. violaceum-induced granuloma requires at least two separate defense pathways, gasdermin D and iNOS, to maintain the integrity of the granuloma architecture. This innate granuloma successfully eradicates C. violaceum infection. Therefore, this C. violaceum-induced granuloma model demonstrates that innate immune cells successfully organize a granuloma and thereby resolve infection by an environmental pathogen.

Microglia Preserve Visual Function in a Mouse Model of Retinitis Pigmentosa with Rhodopsin-P23H Mutant.

Most forms of outer retinal degenerative diseases involve the ectopic accumulation of microglia/macrophages in the subretinal space, including retinitis pigmentosa. However, their role in the loss of photoreceptor function during retinal degeneration remains unknown. Here, we examined the effect of conditional microglial depletion on photoreceptor numbers and visual function in mice with the rhodopsin P23H mutation, a dominant form of retinitis pigmentosa in humans. We found that microglial depletion led to an elevated level of rhodopsin and increased photoreceptor layer thickness. However, overall electrophysiological functions of the retina were reduced with microglial depletion. Therefore, these results identify an essential role of microglia specially in preserving visual function in outer retinal degeneration.

Microglia at Sites of Atrophy Restrict the Progression of Retinal Degeneration via Galectin-3 and Trem2 Interactions.

Degenerative diseases of the outer retina, including age-related macular degeneration (AMD), are characterized by atrophy of photoreceptors and retinal pigment epithelium (RPE). In these blinding diseases, macrophages are known to accumulate ectopically at sites of atrophy, but their ontogeny and functional specialization within this atrophic niche remain poorly understood, especially in the human context. Here, we uncovered a transcriptionally unique profile of microglia, marked by galectin-3 upregulation, at atrophic sites in mouse models of retinal degeneration and in human AMD. Using disease models, we found that conditional deletion of galectin-3 in microglia led to defects in phagocytosis and consequent augmented photoreceptor death, RPE damage and vision loss, suggestive of a protective role. Mechanistically, Trem2 signaling orchestrated the migration of microglial cells to sites of atrophy, and there, induced galectin-3 expression. Moreover, pharmacologic Trem2 agonization led to heightened protection, but only in a galectin-3-dependent manner, further signifying the functional interdependence of these two molecules. Likewise in elderly human subjects, we identified a highly conserved population of microglia at the transcriptomic, protein and spatial levels, and this population was enriched in the macular region of postmortem AMD subjects. Collectively, our findings reveal an atrophy-associated specialization of microglia that restricts the progression of retinal degeneration in mice and further suggest that these protective microglia are conserved in AMD.

An innate granuloma eradicates an environmental pathogen using Gsdmd and Nos2.

Granulomas often form around pathogens that cause chronic infections. Here, we discover a novel granuloma model in mice. Chromobacterium violaceum is an environmental bacterium that stimulates granuloma formation that not only successfully walls off but also clears the infection. The infected lesion can arise from a single bacterium that replicates in the presence of a neutrophil swarm. Bacterial replication ceases when macrophages organize around the infection and form a granuloma. This granuloma response is accomplished independently of adaptive immunity that is typically required to organize granulomas. The C. violaceum -induced granuloma requires at least two separate defense pathways, gasdermin D and iNOS, to maintain the integrity of the granuloma architecture. These innate granulomas successfully eradicate C. violaceum infection. Therefore, this new C. violaceum -induced granuloma model demonstrates that innate immune cells successfully organize a granuloma and thereby eradicate infection by an environmental pathogen.

Meibomian Gland Dysfunction: A Route of Ocular Graft-Versus-Host Disease Progression That Drives a Vicious Cycle of Ocular Surface Inflammatory Damage.

PURPOSE: To investigate the role of aggressive meibomian gland dysfunction (MGD) in the immune pathogenesis of ocular graft-vs-host disease (GVHD). METHODS: In mice, an allogeneic GVHD model was established by transferring bone marrow (BM) and purified splenic T cells from C57BL/6J mice into irradiated C3-SW.H2b mice (BM+T). Control groups received BM only. Mice were scored clinically across the post-transplantation period. MGD severity was categorized using the degree of atrophy on harvested lids. Immune disease was analyzed using flow cytometry of tissues along with fluorescent tracking of BM cells onto the ocular surface. In humans, parameters from 57 patients with ocular GVHD presenting to the Duke Eye Center were retrospectively reviewed. MGD was categorized using the degree of atrophy on meibographs. Immune analysis was done using high-parameter flow cytometry on tear samples. RESULTS: Compared with BM only, BM+T mice had higher systemic disease scores that correlated with tear fluid loss and eyelid edema. BM+T had higher immune cell infiltration in the ocular tissues and higher CD4+-cell cytokine expression in draining lymph nodes. BM+T mice with worse MGD scores had significantly worse corneal staining. In patients with ocular GVHD, 96% had other organs affected. Patients with ocular GVHD had abnormal parameters on dry eye testing, high matrix metalloproteinase-9 positivity (92%), and abundance of immune cells in tear samples. Ocular surface disease signs were worse in patients with higher MGD severity scores. CONCLUSIONS: Ocular GVHD is driven by a systemic, T-cell-dependent process that causes meibomian gland damage and induces a robust form of ocular surface disease that correlates with MGD severity. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

Microglia Drive Pockets of Neuroinflammation in Middle Age.

During aging, microglia produce inflammatory factors, show reduced tissue surveillance, altered interactions with synapses, and prolonged responses to CNS insults, positioning these cells to have profound impact on the function of nearby neurons. We and others recently showed that microglial attributes differ significantly across brain regions in young adult mice. However, the degree to which microglial properties vary during aging is largely unexplored. Here, we analyze and manipulate microglial aging within the basal ganglia, brain circuits that exhibit prominent regional microglial heterogeneity and where neurons are vulnerable to functional decline and neurodegenerative disease. In male and female mice, we demonstrate that VTA and SNc microglia exhibit unique and premature responses to aging, compared with cortex and NAc microglia. This is associated with localized VTA/SNc neuroinflammation that may compromise synaptic function as early as middle age. Surprisingly, systemic inflammation, local neuron death, and astrocyte aging do not appear to underlie these early aging responses of VTA and SNc microglia. Instead, we found that microglial lysosome status was tightly linked to early aging of VTA microglia. Microglial ablation/repopulation normalized VTA microglial lysosome swelling and suppressed increases in VTA microglial density during aging. In contrast, CX3CR1 receptor KO exacerbated VTA microglial lysosome rearrangements and VTA microglial proliferation during aging. Our findings reveal a previously unappreciated regional variation in onset and magnitude of microglial proliferation and inflammatory factor production during aging and highlight critical links between microglial lysosome status and local microglial responses to aging.SIGNIFICANCE STATEMENT Microglia are CNS cells that are equipped to regulate neuronal health and function throughout the lifespan. We reveal that microglia in select brain regions begin to proliferate and produce inflammatory factors in late middle age, months before microglia in other brain regions. These findings demonstrate that CNS neuroinflammation during aging is not uniform. Moreover, they raise the possibility that local microglial responses to aging play a critical role in determining which populations of neurons are most vulnerable to functional decline and neurodegenerative disease.

In vivo quantitative analysis of anterior chamber white blood cell mixture composition using spectroscopic optical coherence tomography.

Anterior uveitis is the most common form of intraocular inflammation, and one of its main signs is the presence of white blood cells (WBCs) in the anterior chamber (AC). Clinically, the true composition of cells can currently only be obtained using AC paracentesis, an invasive procedure to obtain AC fluid requiring needle insertion into the AC. We previously developed a spectroscopic optical coherence tomography (SOCT) analysis method to differentiate between populations of RBCs and subtypes of WBCs, including granulocytes, lymphocytes and monocytes, both in vitro and in ACs of excised porcine eyes. We have shown that different types of WBCs have distinct characteristic size distributions, extracted from the backscattered reflectance spectrum of individual cells using Mie theory. Here, we further develop our method to estimate the composition of blood cell mixtures, both in vitro and in vivo. To do so, we estimate the size distribution of unknown cell mixtures by fitting the distribution observed using SOCT with a weighted combination of reference size distributions of each WBC type calculated using kernel density estimation. We validate the accuracy of our estimation in an in vitro study, by comparing our results for a given WBC sample mixture with the cellular concentrations measured by a hemocytometer and SOCT images before mixing. We also conducted a small in vivo quantitative cell mixture validation pilot study which demonstrates congruence between our method and AC paracentesis in two patients with uveitis. The SOCT based method appears promising to provide quantitative diagnostic information of cellular responses in the ACs of patients with uveitis.

Mucosal-associated invariant T cell responses differ by sex in COVID-19.

BACKGROUND: Sexual dimorphisms in immune responses contribute to coronavirus disease 2019 (COVID-19) outcomes, but the mechanisms governing this disparity remain incompletely understood. METHODS: We carried out sex-balanced sampling of peripheral blood mononuclear cells from hospitalized and non-hospitalized individuals with confirmed COVID-19, uninfected close contacts, and healthy control individuals for 36-color flow cytometry and single-cell RNA sequencing. FINDINGS: Our results revealed a pronounced reduction of circulating mucosal-associated invariant T (MAIT) cells in infected females. Integration of published COVID-19 airway tissue datasets suggests that this reduction represented a major wave of MAIT cell extravasation during early infection in females. Moreover, MAIT cells from females possessed an immunologically active gene signature, whereas cells from males were pro-apoptotic. CONCLUSIONS: Our findings uncover a female-specific protective MAIT cell profile, potentially shedding light on reduced COVID-19 susceptibility in females. FUNDING: This work was supported by NIH/NIAID (U01AI066569 and UM1AI104681), the Defense Advanced Projects Agency (DARPA; N66001-09-C-2082 and HR0011-17-2-0069), the Veterans Affairs Health System, and Virology Quality Assurance (VQA; 75N93019C00015). The content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health. COVID-19 samples were processed under Biosafety level 2 (BSL-2) with aerosol management enhancement or BSL-3 in the Duke Regional Biocontainment Laboratory, which received partial support for construction from NIH/NIAID (UC6AI058607).

Meibomian gland dysfunction is suppressed via selective inhibition of immune responses by topical LFA-1/ICAM antagonism with lifitegrast in the allergic eye disease (AED) model.

PURPOSE: The etiology of meibomian gland dysfunction (MGD) is incompletely understood, despite being a common ophthalmic condition and an area of unmet medical need. It is characterized by an insufficiency in glandular provision of specialized lipids (meibum) to the tear film and is a major cause of dry eye. Work in the allergic eye disease (AED) mouse model has revealed an immunopathogenic role in MGD causation, now raising interest in the applicability of immunomodulatory therapies. As such, we herein ask whether inhibition of lymphocyte function associated antigen (LFA)-1/intracellular adhesion molecules (ICAM)-1 signaling via topical lifitegrast administration has a therapeutic effect on MGD in AED mice. METHODS: Mice were induced with AED by i.p. injection of ovalbumin (OVA) mixed with alum and pertussis toxin, followed 2 weeks later by once daily topical OVA challenges for 7 days. Mice were treated topically with 5% lifitegrast ophthalmic solution or vehicle (PBS) 30 min prior to challenge. We developed a clinical ranking method to assess MGD severity, and also scored clinical allergy. Conjunctivae and draining lymph nodes were collected for flow cytometry. RESULTS: Topical lifitegrast significantly inhibited clinical MGD severity, which was associated with diminished pathogenic TH17 cell and neutrophil numbers in the conjunctiva. No significant change in conjunctival TH2 cells or eosinophils, and only marginal differences in ocular allergy were observed. CONCLUSIONS: In AED mice, lifitegrast inhibited MGD severity marked by a reduction in select immune populations in the conjunctiva. Our findings warrant future examination of lifitegrast in the treatment of patients with forms of MGD.

Dysregulated transcriptional responses to SARS-CoV-2 in the periphery.

SARS-CoV-2 infection has been shown to trigger a wide spectrum of immune responses and clinical manifestations in human hosts. Here, we sought to elucidate novel aspects of the host response to SARS-CoV-2 infection through RNA sequencing of peripheral blood samples from 46 subjects with COVID-19 and directly comparing them to subjects with seasonal coronavirus, influenza, bacterial pneumonia, and healthy controls. Early SARS-CoV-2 infection triggers a powerful transcriptomic response in peripheral blood with conserved components that are heavily interferon-driven but also marked by indicators of early B-cell activation and antibody production. Interferon responses during SARS-CoV-2 infection demonstrate unique patterns of dysregulated expression compared to other infectious and healthy states. Heterogeneous activation of coagulation and fibrinolytic pathways are present in early COVID-19, as are IL1 and JAK/STAT signaling pathways, which persist into late disease. Classifiers based on differentially expressed genes accurately distinguished SARS-CoV-2 infection from other acute illnesses (auROC 0.95 [95% CI 0.92-0.98]). The transcriptome in peripheral blood reveals both diverse and conserved components of the immune response in COVID-19 and provides for potential biomarker-based approaches to diagnosis.

BAFF promotes heightened BCR responsiveness and manifestations of chronic GVHD after allogeneic stem cell transplantation.

Patients with chronic graft-versus-host disease (cGVHD) have increased B cell-activating factor (BAFF) levels, but whether BAFF promotes disease after allogeneic bone marrow transplantation (allo-BMT) remains unknown. In a major histocompatibility complex-mismatched model with cGVHD-like manifestations, we first examined B-lymphopenic µMT allo-BMT recipients and found that increased BAFF levels in cGVHD mice were not merely a reflection of B-cell number. Mice that later developed cGVHD had significantly increased numbers of recipient fibroblastic reticular cells with higher BAFF transcript levels. Increased BAFF production by donor cells also likely contributed to cGVHD, because BAFF transcript in CD4+ T cells from diseased mice and patients was increased. cGVHD manifestations in mice were associated with high BAFF/B-cell ratios and persistence of B-cell receptor (BCR)-activated B cells in peripheral blood and lesional tissue. By employing BAFF transgenic (Tg) mice donor cells, we addressed whether high BAFF contributed to BCR activation in cGVHD. BAFF increased NOTCH2 expression on B cells, augmenting BCR responsiveness to surrogate antigen and NOTCH ligand. BAFF Tg B cells had significantly increased protein levels of the proximal BCR signaling molecule SYK, and high SYK protein was maintained by BAFF after in vitro BCR activation or when alloantigen was present in vivo. Using T cell-depleted (BM only) BAFF Tg donors, we found that BAFF promoted cGVHD manifestations, circulating GL7+ B cells, and alloantibody production. We demonstrate that pathologic production of BAFF promotes an altered B-cell compartment and augments BCR responsiveness. Our findings compel studies of therapeutic targeting of BAFF and BCR pathways in patients with cGVHD.

The cornea IV immunology, infection, neovascularization, and surgery chapter 1: Corneal immunology.

PURPOSE: of Review: This review offers an informed and up-to-date insight on the immune profile of the cornea and the factors that govern the regulation of such a unique immune environment. SUMMARY: The cornea is a unique tissue that performs the specialized task of allowing light to penetrate for visual interpretation. To accomplish this, the ocular surface requires a distinct immune environment that is achieved through unique structural, cellular and molecular factors. Not only must the cornea be able to fend off invasive infectious agents but also control the inflammatory response as to avoid collateral, and potentially blinding damage; particularly of post-mitotic cells such as the corneal endothelium. To combat infections, both innate and adaptive arms of the inflammatory immune response are at play in the cornea. Dendritic cells play a critical role in coordinating both these responses in order to fend off infections. On the other side of the spectrum, the ocular surface is also endowed with a variety of anatomic and physiologic components that aid in regulating the immune response to prevent excessive, potentially damaging, inflammation. This attenuation of the immune response is termed immune privilege. The balance between pro and anti-inflammatory reactions is key for preservation of the functional integrity of the cornea. RECENT FINDINGS: The understanding of the molecular and cellular factors governing corneal immunology and its response to antigens is a growing field. Dendritic cells in the normal cornea play a crucial role in combating infections and coordinating the inflammatory arms of the immune response, particularly through coordination with T-helper cells. The role of neuropeptides is recently becoming more highlighted with different factors working on both sides of the inflammatory balance.

Disease-Specific Expression of Conjunctiva Associated Lymphoid Tissue (CALT) in Mouse Models of Dry Eye Disease and Ocular Allergy.

Conjunctiva-associated tissue (CALT) is assumed to play a crucial role in the immune system of the ocular surface. Its function in several ocular surface diseases (OSD) is still not fully understood. This study investigates the function of CALT in mouse models of dry-eye disease and ocular allergy. Since antigen-presentation is the central similarity in the pathologies, this study focuses on antigen-presentation in CALT Morphology and the expression of CALT, which was investigated in mice after induction of dry-eye, ocular allergy, topical antigen-stimulation, and after local depletion of phagocytic cells. Antigen uptake was investigated after the application of fluorescent ovalbumin (OVA). OSD influences the appearance and morphology of CALT in a disease-dependent manner. Ocular allergy leads to an increase and dry-eye disease to a decrease in number and size of CALT. The development of CALT is dependent on the presence of APCs. Professional APCs are present in CALT, and soluble antigen is transported into the follicle. CALT appearance is disease-specific and indicative of differing functions. Although the specific involvement of CALT in OSD needs further study, the existence of functional APCS and antigen-uptake supports the hypothesis that CALT is an immunological key player at the ocular surface.

Microglia versus Monocytes: Distinct Roles in Degenerative Diseases of the Retina.

Unlike in the healthy mammalian retina, macrophages in retinal degenerative states are not solely comprised of microglia but may include monocyte-derived recruits. Recent studies have applied transgenics, lineage-tracing, and transcriptomics to help decipher the distinct roles of these two cell types in the diseasesettings of inherited retinal degenerations and age-related macular degeneration.Literature discussed here focuses on the ectopic presence of both macrophage types in the extracellular site surrounding the outer aspect ofphotoreceptor cells (i.e.,the subretinal space), which is crucially involved in the pathobiology. From these studies we propose a working model in which perturbed photoreceptor states cause microglial dominant migration to the subretinal space as a protective response, whereas the abundant presence ofmonocyte-derived cells there instead drives and accelerates pathology. The latter, we propose, is underpinned by specific genetic and nongenetic determinants that lead to a maladaptive macrophage state.

The Immunological Basis of Dry Eye Disease and Current Topical Treatment Options.

Homeostasis of the lacrimal functional unit is needed to ensure a well-regulated ocular immune response comprising innate and adaptive phases. When the ocular immune system is excessively stimulated and/or immunoregulatory mechanisms are disrupted, the balance between innate and adaptive phases is dysregulated and chronic ocular surface inflammation can result, leading to chronic dry eye disease (DED). According to the Tear Film and Ocular Surface Society Dry Eye Workshop II definition, DED is a multifactorial disorder of the ocular surface characterized by impairment and loss of tear homeostasis (hyperosmolarity), ocular discomfort or pain, and neurosensory abnormalities. Dysregulated ocular immune responses result in ocular surface damage, which is a further contributing factor to DED pathology. Several therapeutics are available to break the vicious circle of DED and prevent chronic disease and progression, including immunosuppressive agents (steroids) and immunomodulators (cyclosporine and lifitegrast). Given the chronic inflammatory nature of DED, each of these agents is commonly used in clinical practice. In this study, we review the immunopathology of DED and the molecular and cellular actions of current topical DED therapeutics to inform clinical decision making.

scDAPA: detection and visualization of dynamic alternative polyadenylation from single cell RNA-seq data.

MOTIVATION: Alternative polyadenylation (APA) plays a key post-transcriptional regulatory role in mRNA stability and functions in eukaryotes. Single cell RNA-seq (scRNA-seq) is a powerful tool to discover cellular heterogeneity at gene expression level. Given 3' enriched strategy in library construction, the most commonly used scRNA-seq protocol-10× Genomics enables us to improve the study resolution of APA to the single cell level. However, currently there is no computational tool available for investigating APA profiles from scRNA-seq data. RESULTS: Here, we present a package scDAPA for detecting and visualizing dynamic APA from scRNA-seq data. Taking bam/sam files and cell cluster labels as inputs, scDAPA detects APA dynamics using a histogram-based method and the Wilcoxon rank-sum test, and visualizes candidate genes with dynamic APA. Benchmarking results demonstrated that scDAPA can effectively identify genes with dynamic APA among different cell groups from scRNA-seq data. AVAILABILITY AND IMPLEMENTATION: The scDAPA package is implemented in Shell and R, and is freely available at https://scdapa.sourceforge.io. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Identification of a Unique Subretinal Microglia Type in Retinal Degeneration Using Single Cell RNA-Seq.

As the resident macrophages of central nervous system, microglia reside in the plexiform and nerve fiber layers of the retina. In degenerative diseases, monocyte-derived macrophages can be recruited to the retina, and histopathology shows abnormal accumulation of macrophages subretinally. However, due to lack of known markers, recruited cells and resident microglia are phenotypically indistinguishable, leaving a major knowledge gap about their potentially independent roles. Here, we used single cell RNA-seq and analyzed over 10,000 immune cells of mouse retinas from normal control and light damage-induced retinal degeneration. We observed ten major macrophage clusters. Moreover, combining trajectory analysis and in situ validation allowed us to pinpoint that subretinal phagocytes are microglia-derived and express high levels of Gal3, Cd68, and Lpl but not P2ry12. Hence, we have identified novel subretinal macrophage markers indicative of their origin and phenotype, which may be useful in other degeneration models and human specimens.