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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 ., Competing Interests: MA, CB, CH, DS, EM No competing interests declared, (© 2024, Amason et al.)

Radiotherapy (RT) is commonly used to try to eliminate any remaining tumor cells following surgical resection of glioma. However, tumor recurrence is prevalent, highlighting the unmet medical need to develop therapeutic strategies to enhance the efficacy of RT in glioma. Focusing on the radiosensitizing potential of the currently approved drugs known to cross the blood-brain barrier can facilitate rapid clinical translation. Here, we assessed the role of catechol-O-methyltransferase (COMT), a key enzyme to degrade catecholamines and a drug target for Parkinson's disease, in glioma treatment. Analysis of The Cancer Genome Atlas data showed significantly higher COMT expression levels in both low-grade glioma and glioblastoma compared to normal brain tissues. Inhibition of COMT by genetic knockout or FDA-approved COMT inhibitors significantly sensitized glioma cells to RT in vitro and in vivo. Mechanistically, COMT inhibition in glioma cells led to mitochondria dysfunction and increased mitochondrial RNA release into the cytoplasm, activating the cellular antiviral double-stranded RNA sensing pathway and type I interferon (IFN) response. Elevated type I IFNs stimulated the phagocytic capacity of microglial cells, enhancing RT efficacy. Given the long-established safety record of the COMT inhibitors, these findings provide a solid rationale to evaluate them in combination with RT in patients with glioma. Significance: Inhibition of catechol-O-methyltransferase, a well-established drug target in Parkinson's disease, interferes with mitochondrial electron transport and induces mitochondrial double-stranded RNA leakage, activating type I interferon signaling and sensitizing glioma to radiotherapy., (©2024 The Authors; Published by the American Association for Cancer Research.)

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., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., (© 2024 Yu et al.)

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., (© 2023. Springer Nature Limited.)

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., (Copyright © 2022. Published by Elsevier Inc.)

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., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

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., (Copyright © 2022 the authors.)

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 T H 17 cell and neutrophil numbers in the conjunctiva. No significant change in conjunctival T H 2 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., (Copyright © 2021. Published by Elsevier Inc.)

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)., Competing Interests: M.T.M. reports grants for biomarker diagnostics from the Defense Advanced Research Projects Agency (DARPA), National Institutes of Health (NIH), Sanofi, and the Department of Veterans Affairs. T.W.B. reports grants from DARPA and is a consultant for Predigen. M.T.M., T.W.B., E.L.T., and C.W.W. report pending patents on molecular methods to diagnose and treat respiratory infections. E.L.T. reports grants on biomarker diagnostics from DARPA and the NIH/Antibacterial Resistance Leadership Group (ARLG) and an ownership stake in Predigen. G.S.G. reports an ownership stake in Predigen. C.W.W. reports grants for biomarker diagnostics from DARPA, NIH/ARLG, Predigen, and Sanofi and has received consultancy fees from bioMerieux, Roche, Biofire, Giner, and Biomeme., (© 2021 Elsevier Inc.)

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., (© 2021 by The American Society of Hematology.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., Competing Interests: RPM: Leica Microsystems (P). ANK: Leica Microsystems (P). JAI: Leica Microsystems (P, R), Carl Zeiss Meditec (P, R)., (© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

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.

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.

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

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., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Naturally occurring cell death is a fundamental developmental mechanism for regulating cell numbers and sculpting developing organs. This is particularly true in the nervous system, where large numbers of neurons and oligodendrocytes are eliminated via apoptosis during normal development. Given the profound impact of death upon these two major cell populations, it is surprising that developmental death of another major cell type-the astrocyte-has rarely been studied. It is presently unclear whether astrocytes are subject to significant developmental death, and if so, how it occurs. Here, we address these questions using mouse retinal astrocytes as our model system. We show that the total number of retinal astrocytes declines by over 3-fold during a death period spanning postnatal days 5-14. Surprisingly, these astrocytes do not die by apoptosis, the canonical mechanism underlying the vast majority of developmental cell death. Instead, we find that microglia engulf astrocytes during the death period to promote their developmental removal. Genetic ablation of microglia inhibits astrocyte death, leading to a larger astrocyte population size at the end of the death period. However, astrocyte death is not completely blocked in the absence of microglia, apparently due to the ability of astrocytes to engulf each other. Nevertheless, mice lacking microglia showed significant anatomical changes to the retinal astrocyte network, with functional consequences for the astrocyte-associated vasculature leading to retinal hemorrhage. These results establish a novel modality for naturally occurring cell death and demonstrate its importance for the formation and integrity of the retinal gliovascular network., Competing Interests: The authors have declared that no competing interests exist.

Whether complement dysregulation directly contributes to the pathogenesis of peripheral nervous system diseases, including sensory neuropathies, is unclear. We addressed this important question in a mouse model of ocular HSV-1 infection, where sensory nerve damage is a common clinical problem. Through genetic and pharmacologic targeting, we uncovered a central role for C3 in sensory nerve damage at the morphological and functional levels. Interestingly, CD4 T cells were central in facilitating this complement-mediated damage. This same C3/CD4 T cell axis triggered corneal sensory nerve damage in a mouse model of ocular graft-versus-host disease (GVHD). However, this was not the case in a T-dependent allergic eye disease (AED) model, suggesting that this inflammatory neuroimmune pathology is specific to certain disease etiologies. Collectively, these findings uncover a central role for complement in CD4 T cell-dependent corneal nerve damage in multiple disease settings and indicate the possibility for complement-targeted therapeutics to mitigate sensory neuropathies., Competing Interests: DR, JE, LL, GG, RM, DS, VP, DC No competing interests declared, (© 2019, Royer et al.)

Progressive rod-cone degeneration (PRCD) is a small protein residing in the light-sensitive disc membranes of the photoreceptor outer segment. Until now, the function of PRCD has remained enigmatic despite multiple demonstrations that its mutations cause blindness in humans and dogs. Here, we generated a PRCD knockout mouse and observed a striking defect in disc morphogenesis, whereby newly forming discs do not properly flatten. This leads to the budding of disc-derived vesicles, specifically at the site of disc morphogenesis, which accumulate in the interphotoreceptor matrix. The defect in nascent disc flattening only minimally alters the photoreceptor outer segment architecture beyond the site of new disc formation and does not affect the abundance of outer segment proteins and the photoreceptor's ability to generate responses to light. Interestingly, the retinal pigment epithelium, responsible for normal phagocytosis of shed outer segment material, lacks the capacity to clear the disc-derived vesicles. This deficiency is partially compensated by a unique pattern of microglial migration to the site of disc formation where they actively phagocytize vesicles. However, the microglial response is insufficient to prevent vesicular accumulation and photoreceptors of PRCD knockout mice undergo slow, progressive degeneration. Taken together, these data show that the function of PRCD is to keep evaginating membranes of new discs tightly apposed to each other, which is essential for the high fidelity of photoreceptor disc morphogenesis and photoreceptor survival., Competing Interests: The authors declare no conflict of interest.

In the eye immune defenses must take place in a plethora of differing microenvironments ranging from the corneal and conjunctival epithelia facing the external environment to the pigmented connective tissue of the uveal tract containing smooth muscle, blood vessels and peripheral nerves to the innermost and highly protected neural retina. The extravascular environment of the neural retina, like the brain parenchyma, is stringently controlled to maintain conditions required for neural transmission. The unique physiological nature of the neural retina can be attributed to the blood retinal barriers (BRB) of the retinal vasculature and the retinal pigment epithelium, which both tightly regulate the transport of small molecules and restrict passage of cells and macromolecules from the circulation into the retina in a similar fashion to the blood brain barrier (BBB). The extracellular environment of the neural retina differs markedly from that of the highly vascular, loose connective tissue of the choroid, which lies outside the BRB. The choroid hosts a variety of immune cell types, including macrophages, dendritic cells (DCs) and mast cells. This is in marked contrast to the neural parenchyma of the retina, which is populated almost solely by microglia. This review will describe the current understanding of the distribution, phenotype and physiological role of ocular immune cells behind or inside the blood-retinal barriers and those in closely juxtaposed tissues outside the barrier. The nature and function of these immune cells can profoundly influence retinal homeostasis and lead to disordered immune function that can lead to vision loss., (Copyright © 2018. Published by Elsevier Ltd.)

Microglia from different nervous system regions are molecularly and anatomically distinct, but whether they also have different functions is unknown. We combined lineage tracing, single-cell transcriptomics, and electrophysiology of the mouse retina and showed that adult retinal microglia shared a common developmental lineage and were long-lived but resided in two distinct niches. Microglia in these niches differed in their interleukin-34 dependency and functional contribution to visual-information processing. During certain retinal-degeneration models, microglia from both pools relocated to the subretinal space, an inducible disease-associated niche that was poorly accessible to monocyte-derived cells. This microglial transition involved transcriptional reprogramming of microglia, characterized by reduced expression of homeostatic checkpoint genes and upregulation of injury-responsive genes. This transition was associated with protection of the retinal pigmented epithelium from damage caused by disease. Together, our data demonstrate that microglial function varies by retinal niche, thereby shedding light on the significance of microglia heterogeneity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

With the new understanding that adult microglia in mice have embryonic origins and are maintained in situ throughout life, it has become pertinent to now understand how these unique cells differ from monocyte-derived macrophages. The latter are recruited into the neural retina (and elsewhere in CNS) in certain diseased states, such as in various forms of retinal degeneration. However, phenotypic markers expressed by microglia and monocyte-derived macrophages largely overlap, thereby making it technically challenging to distinguish the two cell types in disease. To address this problem in mice, we have established an in vivo fate mapping system that enables distinguishing these two cell types in retinal disease models. Our approach leverages the seminal work that originally developed Cx3cr1-CreER mice and is based on commercially available mouse strains. Here, we detail our protocol and how to apply this fate mapping method paired with flow cytometry (or immunohistochemistry) to faithfully distinguish and examine microglia vs. monocyte-derived macrophages in a mutually exclusive manner. This approach will henceforth empower new efforts to identify functional specializations of these two populations in the pathobiology of retinal degenerative diseases and possibly other conditions of the retina where monocyte recruitment is observed, such as in glaucoma, diabetic retinopathy, ischemia reperfusion, retinal detachment, and so on.

Severe, chronic eye allergy is an understudied, vision-threatening condition. Treatments remain limited. We used a mouse model of severe allergic eye disease (AED) to determine whether topical application of the pro-resolution mediator Resolvin D1 (RvD1) terminates the response. AED was induced by injection of ovalbumin (OVA) followed by topical challenge of OVA daily. RvD1 was applied topically prior to OVA. Clinical symptoms were scored. Eye washes were assayed for MUC5AC. After 7 days, eyes were removed and the number of goblet cells, T helper cell responses and presence of immune cells in draining lymph nodes and conjunctiva determined. Topical RvD1 treatment significantly reduced symptoms of AED. RvD1 did not alter the systemic type 2 immune response in the lymph nodes. AED increased the total amount of goblet cell mucin secretion, but not the number of goblet cells. RvD1 prevented this increase, but did not alter goblet cell number. Absolute numbers of CD4 + T cells, total CD11b + myeloid cells, eosinophils, neutrophils, and monocytes, but not macrophages increased in AED versus RvD1-treated mice. We conclude that topical application of RvD1 reduced the ocular allergic response by local actions in conjunctival immune response and a decrease in goblet cell mucin secretion.

Graft-versus-host disease (GVHD) is a major complication of hematopoietic stem cell transplantation (HCT). The tyrosine kinase SYK contributes to both acute and chronic GVHD development, making it an attractive target for GVHD prevention. Entospletinib (ENTO) is a second-generation highly selective SYK inhibitor with a high safety profile. Potential utility of ENTO as GVHD prophylaxis in patients was examined using a preclinical mouse model of eye and skin GVHD and ENTO-compounded chow. We found that early SYK inhibition improved blood immune cell reconstitution in GVHD mice and prolonged survival, with 60% of mice surviving to day +120 compared with 10% of mice treated with placebo. Compared with mice receiving placebo, mice receiving ENTO had dramatic improvements in clinical eye scores, alopecia scores, and skin scores. Infiltrating SYK+ cells expressing B220 or F4/80, resembling SYK+ cells found in lichenoid skin lesions of chronic GVHD patients, were abundant in the skin of placebo mice but were rare in ENTO-treated mice. Thus, ENTO given early after HCT safely prevented GVHD.

The number of neurons dedicated to vision itself is thought to be greater than the sum of the four other senses combined. Yet, little attention has been payed to the retina as compared to elsewhere in the central nervous system with respect to microglia, the macrophages of the neural parenchyma. Indeed, major advancements in the understanding of microglial ontogeny and maintenance in brain and spinal cord are now widely appreciated, whereas less notice has been given to the neural retina in this regard. The current Review covers topical concepts on adult microglia and perivascular macrophage ontogenies in the steady state retina, as well as parallels made with these macrophages in other areas of the central nervous system. The subject of recruited monocytes and their descendant monocyte-derived macrophages in degenerative diseases of the retina is also integrated into this Review. Key experiments that have led to the theories covered are highlighted throughout, as are the knowledge gaps that remain unresolved., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Meibomian glands (MGs) are sebaceous glands of the eyelid margin that secrete lipids needed to avert tear evaporation and to help maintain ocular surface homeostasis. Obstruction of MGs or other forms of MG dysfunction can promote chronic diseases of the ocular surface. Although chronic eyelid inflammation, such as allergic eye disease, is an associated risk factor for obstructive MG dysfunction, it is not clear whether inflammatory processes contribute to the pathophysiology of MG obstruction. We show that polymorphonuclear neutrophils (PMNs) promoted MG obstruction in a chronic inflammatory model of allergic eye disease in mice. Analysis of leukocytes in tears of patients with MG dysfunction showed an increase in PMN numbers compared to healthy subjects. Moreover, PMN numbers in tears positively correlated with clinical severity of MG dysfunction. Our findings point to a role for PMNs in the pathogenesis and progression of MG dysfunction., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Corneal transplantation (keratoplasty) is the most common type of tissue replacement in the world. The increased rate of graft rejection after keratoplasty is a central problem for repeated transplantations and in inflamed host corneas. It has been shown that apoptosis of grafted epithelium has a role in corneal allograft rejection. This study focused on the T-cell response triggered in BALB/c mice after allogeneic corneal transplantation with and without anti-apoptotic p35-transduced epithelium. To restrict p35 expression to the epithelial cells, modified allogeneic composite grafts were created. As a result, it was found that the proportion of alloreactive CD4 + T cells in postoperatively removed cervical lymph nodes was reduced in the p35-transduced group compared to the allogeneic control group. Diminished priming of the CD4 + T cells was supported by significantly decreased proliferation and lower interferon gamma secretion when compared to allogeneic engraftments. The reduced priming of CD4 + lymphocytes is the first confirmation of the functionality of p35 in the epithelium of corneal grafts to alter the development of the recipient's immune response. Thus, modification of allosensibilization seems to be a promising tool for reducing graft-mediated immune response following corneal transplantation.

Purpose: A large body of evidence supports a central role for complement activation in the pathobiology of age-related macular degeneration (AMD), including plasma complement component 5a (C5a). Interestingly, C5a is a chemotactic agent for monocytes, a cell type also shown to contribute to AMD. However, the role monocytes play in the pathogenesis of "dry" AMD and the pharmacologic potential of targeting C5a to regulate these cells are unclear. We addressed these questions via C5a blockade in a unique model of early/intermediate dry AMD and large panel flow cytometry to immunophenotype monocytic involvement., Methods: Heterozygous complement factor H (Cfh+/-) mice aged to 90 weeks were fed a high-fat, cholesterol-enriched diet (Cfh+/-∼HFC) for 8 weeks and were given weekly intraperitoneal injections of 30 mg/kg anti-C5a (4C9, Pfizer). Flow cytometry, retinal pigmented epithelium (RPE) flat mounts, and electroretinograms were used to characterize anti-C5a treatment., Results: Aged Cfh+/- mice developed RPE damage, sub-RPE basal laminar deposits, and attenuation of visual function and immune cell recruitment to the choroid that was accompanied by expression of inflammatory and extracellular matrix remodeling genes following 8 weeks of HFC diet. Concomitant systemic administration of an anti-C5a antibody successfully inhibited local recruitment of mononuclear phagocytes to the choroid-RPE interface but did not ameliorate these AMD-like pathologies in this mouse model., Conclusions: These results show that immunotherapy targeting C5a is not sufficient to block the development of the AMD-like pathologies observed in Cfh+/-∼HFC mice and suggest that other complement components or molecules/mechanisms may be driving "early" and "intermediate" AMD pathologies.

Ocular IgE-associated allergy ranges from mild disease (seasonal and perennial allergic conjunctivitis) to more chronic/severe and vision-threatening forms (atopic and vernal keratoconjunctivitis). Whereas mild forms of disease have been studied extensively, less is known about the more chronic forms. Our lab has helped to address this knowledge gap by developing and characterizing an allergen-induced, chronic/severe, IgE-associated model of ocular allergy referred to as the severe allergic eye disease (AED) model. It is distinct from previously described models that mimic the more mild forms, referred to in the literature as the allergic conjunctivitis (AC) model. The purpose of this method article is to detail the protocol to induce and characterize the AED model and directly compare these mice to the mild AC model. Troubleshooting and implications are also discussed.

The purpose of this study is to determine the effect of systemic blockade of Interleukin-6 (IL-6) on allosensitization, regulatory T cell frequencies and suppressive phenotype, and allograft survival rates in a mouse model of corneal transplantation. Allogeneic corneal transplantation was performed using C57BL/6 mice as donors and BALB/c mice as recipients. Graft recipients were injected daily with either anti-IL-6 antibody or an isotype control antibody (1.25 mg/ml) for the first 7 days and on alternate days thereafter until week 8 after transplantation. Allograft survival was evaluated for 8 weeks using Kaplan-Meier survival analysis. Draining lymph nodes (DLN) were harvested at week 3 after transplantation, and proliferation of isolated recipient T cells through direct and indirect pathways was determined using mixed lymphocyte reaction assay. Frequencies of CD4 + CD25 + Foxp3 + regulatory T cells, their expression of Foxp3, and frequencies of IFNy + CD4 + Th1 cells were determined in DLN using flow cytometry. Finally, CD4 + T cells were cultured with bone marrow-derived dendritic cells from either C57BL/6 or BALB/c mice in the presence of IL-6-blocking antibody to determine Treg induction through direct and indirect pathways, respectively. Treatment with anti-IL-6 antibody suppressed both the direct and indirect pathways of allosensitization in graft recipients and significantly improved allograft survival rates. Furthermore, in vivo blockade of IL-6 enhanced Foxp3 expression by Tregs in graft recipients undergoing rejection, but did not exert a significant effect on Treg frequencies. Finally, IL-6 neutralization in vitro enhanced the differentiation of Tregs from CD4 + T cells through both direct and indirect pathways. Our results demonstrate that systemic administration of IL-6-blocking antibody to corneal allograft recipients suppresses direct and indirect routes of allosensitization, is associated with increased expression of Foxp3 by Tregs, and improves allograft survival rates., Competing Interests: CONFLICT OF INTEREST STATEMENT The authors have no financial conflicts of interest.

Diabetic retinopathy (DR) is a complication secondary to diabetes and is the number one cause of blindness among working age individuals worldwide. Despite recent therapeutic breakthroughs using pharmacotherapy, a cure for DR has yet to be realized. Several clinical trials have highlighted the vital role dyslipidemia plays in the progression of DR. Additionally, it has recently been shown that activation of Liver X receptor (LXRα/LXRβ) prevents DR in diabetic animal models. LXRs are nuclear receptors that play key roles in regulating cholesterol metabolism, fatty acid metabolism and inflammation. In this manuscript, we show insight into DR pathogenesis by demonstrating an innovative signaling axis that unifies key metabolic regulators, Sirtuin 1 and LXR, in modulating retinal cholesterol metabolism and inflammation in the diabetic retina. Expression of both regulators, Sirtuin 1 and LXR, are significantly decreased in diabetic human retinal samples and in a type 2 diabetic animal model. Additionally, activation of LXR restores reverse cholesterol transport, prevents inflammation, reduces pro-inflammatory macrophages activity and prevents the formation of diabetes-induced acellular capillaries. Taken together, the work presented in this manuscript highlights the important role lipid dysregulation plays in DR progression and offers a novel potential therapeutic target for the treatment of DR., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

"Paucibacterial" levels of the normal eye surface have left immunologists wondering whether a true microbiome exists there. In this issue of Immunity, St. Leger et al. (2017) address this head-on, discovering a naturally existing commensal in mice that induces γδT cell-mediated protection from opportunistic infection., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Major advances in mononuclear phagocyte biology have been made but key questions pertinent to their roles in health and disease remain, including in the visual system. One problem concerns how dendritic cells can trigger immune responses from certain tightly regulated immune- privileged sites of the eye. Another, albeit separate, problem involves whether there are functional specializations for microglia versus monocytes in retinal neurodegeneration. In this Review, we examine novel insights in eye immune privilege and, separately, we discuss recent inroads concerning retinal degeneration. Both themes have been extensively studied in the visual system and show parallels with recent findings concerning mononuclear phagocytes in the central nervous system and in the periphery.

In this study, the role of CX3CR1 in the progression of diabetic retinopathy (DR) was investigated. The retinas of wild-type (WT), CX3CR1 null (CX3CR1 gfp/gfp , KO), and heterozygous (CX3CR1 +/gfp , Het) mice were compared in the presence and absence of streptozotocin (STZ)-induced diabetes. CX3CR1 deficiency in STZ-KO increased vascular pathology at 4 months of diabetes, as a significant increase in acellular capillaries was observed only in the STZ-KO group. CX3CR1 deficiency and diabetes had similar effects on retinal neurodegeneration measured by an increase in DNA fragmentation. Retinal vascular pathology in STZ-KO mice was associated with increased numbers of monocyte-derived macrophages in the retina. Furthermore, compared to STZ-WT, STZ-KO mice exhibited increased numbers of inflammatory monocytes in the bone marrow and impaired homing of monocytes to the spleen. The induction of retinal IL-10 expression by diabetes was significantly less in KO mice, and when bone marrow-derived macrophages from KO mice were maintained in high glucose, they expressed significantly less IL-10 and more TNF-α in response to LPS stimulation. These findings support that CX3CR1 deficiency accelerates the development of vascular pathology in DR through increased recruitment of proinflammatory myeloid cells that demonstrate reduced expression of anti-inflammatory IL-10., Key Messages: • CX3CR1 deletion in STZ-diabetic mice accelerated the onset of diabetic retinopathy (DR). • The early onset of DR was associated with increased retinal cell apoptosis. • The early onset of DR was associated with increased recruitment of bone marrow-derived macrophages to the retina. • Bone marrow-derived macrophages from CX3CR1 KO diabetic mice expressed more TNF-α and less IL-10. • The role of IL-10 in protection from progression of DR is highlighted.

In vivo imaging permits longitudinal study of ocular disease processes in the same animal over time. Two different in vivo optical imaging modalities - optical coherence tomography (OCT) and fluorescence - provide important structural and cellular data respectively about disease processes. In this Methods in Eye Research article, we describe and demonstrate the combination of these two modalities producing a truly simultaneous OCT and fluorescence imaging system for imaging of fluorescently labeled animal models. This system uses only a single light source to illuminate both modalities, and both share the same field of view. This allows simultaneous acquisition of OCT and fluorescence images, and the benefits of both techniques are realized without incurring increased costs in variability, light exposure, time, and post-processing effort as would occur when the modalities are used separately. We then utilized this system to demonstrate multi-modal imaging in a progression of samples exhibiting both fluorescence and OCT scattering beginning with resolution targets, ex vivo thy1-YFP labeled neurons in mouse eyes, and finally an in vivo longitudinal time course of GFP labeled myeloid cells in a mouse model of ocular allergy., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Fibrosis is a shared end-stage pathway to lung, liver, and heart failure. In the ocular mucosa (conjunctiva), fibrosis leads to blindness in trachoma, pemphigoid, and allergy. The indirect fibrogenic role of DCs via T cell activation and inflammatory cell recruitment is well documented. However, here we demonstrate that DCs can directly induce fibrosis. In the mouse model of allergic eye disease (AED), classical CD11b + DCs in the ocular mucosa showed increased activity of aldehyde dehydrogenase (ALDH), the enzyme required for retinoic acid synthesis. In vitro, CD11b + DC-derived ALDH was associated with 9- cis -retinoic acid ligation to retinoid x receptor (RXR), which induced conjunctival fibroblast activation. In vivo, stimulating RXR led to rapid onset of ocular mucosal fibrosis, whereas inhibiting ALDH activity in DCs or selectively depleting DCs markedly reduced fibrosis. Collectively, these data reveal a profibrotic ALDH-dependent pathway by DCs and uncover a role for DC retinoid metabolism.

Mucous membrane pemphigoid (MMP) is a systemic mucosal scarring disease, commonly causing blindness, for which there is no antifibrotic therapy. Aldehyde dehydrogenase family 1 (ALDH1) is upregulated in both ocular MMP (OMMP) conjunctiva and cultured fibroblasts. Application of the ALDH metabolite, retinoic acid (RA), to normal human conjunctival fibroblasts in vitro induced a diseased phenotype. Conversely, application of ALDH inhibitors, including disulfiram, to OMMP fibroblasts in vitro restored their functionality to that of normal controls. ALDH1 is also upregulated in the mucosa of the mouse model of scarring allergic eye disease (AED), used here as a surrogate for OMMP, in which topical application of disulfiram decreased fibrosis in vivo. These data suggest that progressive scarring in OMMP results from ALDH/RA fibroblast autoregulation, that the ALDH1 subfamily has a central role in immune-mediated ocular mucosal scarring, and that ALDH inhibition with disulfiram is a potential and readily translatable antifibrotic therapy.

Allergic eye disease, as in most forms of atopy, ranges in severity among individuals from immediate hypersensitivity to a severe and debilitating chronic disease. Dendritic cells play a key role in stimulating pathogenic T cells in allergen re-exposure, or secondary responses. However, molecular cues by dendritic cells underpinning allergic T cell response levels and the impact that this control has on consequent severity of allergic disease are poorly understood. Here, we show that a deficiency in thrombospondin-1, a matricellular protein known to affect immune function, has subsequent effects on downstream T cell responses during allergy, as revealed in an established mouse model of allergic eye disease. More specifically, we demonstrate that a thrombospondin-1 deficiency specific to dendritic cells leads to heightened secondary T cell responses and consequent clinical disease. Interestingly, whereas thrombospondin-1-deficient dendritic cells augmented activity of allergen-primed T cells, this increase was not recapitulated with naïve T cells in vitro. The role of dendritic cell-derived thrombospondin-1 in regulating secondary allergic T cell responses was confirmed in vivo, as local transfer of thrombospondin-1-sufficient dendritic cells to the ocular mucosa of thrombospondin-1 null hosts prevented the development of augmented secondary T cell responses and heightened allergic eye disease clinical responses. Finally, we demonstrate that topical instillation of thrombospondin-1-derived peptide reduces T cell activity and clinical progression of allergic eye disease. Taken together, this study reveals an important modulatory role of dendritic cell-derived thrombospondin-1 on secondary allergic T cell responses and suggests the possible dysregulation of dendritic cell-derived thrombospondin-1 expression as a factor in allergic eye disease severity., (© Society for Leukocyte Biology.)

The recent paradigm shift that microglia are yolk sac-derived, not hematopoietic-derived, is reshaping our knowledge about the isolated role of microglia in CNS diseases, including degenerative conditions of the retina. However, unraveling microglial-specific functions has been hindered by phenotypic overlap of microglia with monocyte-derived macrophages. The latter are differentiated from recruited monocytes in neuroinflammation, including retina. Here we demonstrate the use of fate mapping wherein microglia and monocyte-derived cells are endogenously labeled with different fluorescent reporters. Combining this method with 12-color flow cytometry, we show that these two populations are definitively distinguishable by phenotype in retina. We prove that retinal microglia have a unique CD45(lo) CD11c(lo) F4/80(lo) I-A/I-E(-) signature, conserved in the steady state and during retinal injury. The latter was observed in the widely used light-induced retinal degeneration model and corroborated in other models, including whole-body irradiation/bone-marrow transplantation. The literature contains conflicting observations about whether microglia, including in the retina, increase expression of these markers in neuroinflammation. We show that monocyte-derived macrophages have elevated expression of these surface markers, not microglia. Our resolution of such phenotypic differences may serve as a robust way to help characterize isolated roles of these cells in retinal neuroinflammation and possibly elsewhere in CNS.

Although sensory reinnervation occurs after injury in the peripheral nervous system, poor reinnervation in the elderly and those with diabetes often leads to pathology. Here we quantify sub-basal axon density in the central and peripheral mouse cornea over time after three different types of injury. The mouse cornea is highly innervated with a dense array of sub-basal nerves that form a spiral called the vortex at the corneal center or apex; these nerves are readily detected within flat mounted corneas. After anesthesia, corneal epithelial cells were removed using either a dulled blade or a rotating burr within an area demarcated centrally with a 1.5 mm trephine. A third wound type, superficial trephination, involved demarcating the area with the 1.5 mm trephine but not removing cells. By 7 days after superficial trephination, sub-basal axon density returns to control levels; by 28 days the vortex reforms. Although axon density is similar to control 14 days after dulled blade and rotating burr wounding, defects in axon morphology at the corneal apex remain. After 14 days, axons retract from the center leaving the sub-basal axon density reduced by 37.2 and 36.8% at 28 days after dulled blade and rotating burr wounding, respectively, compared with control. Assessment of inflammation using flow cytometry shows that persistent inflammation is not a factor in the incomplete reinnervation. Expression of mRNAs encoding 22 regeneration-associated genes involved in axon targeting assessed by QPCR reveals that netrin-1 and ephrin signaling are altered after wounding. Subpopulations of corneal epithelial basal cells at the corneal apex stop expressing ki67 as early as 7 days after injury and by 14 and 28 days after wounding, many of these basal cells undergo apoptosis and die. Although sub-basal axons are restored to their normal density and morphology after superficial trephination, sub-basal axon recovery is partial after debridement wounds. The increase in corneal epithelial basal cell apoptosis at the apex observed at 14 days after corneal debridement may destabilize newly reinnervated sub-basal axons and lead to their retraction toward the periphery.

Purpose of Review: Here, we explore an emerging theme in the literature, which is the role of dendritic cells in the causation of fibrosis. To fully appreciate this pathway to disease, we also review the most recent literature regarding dendritic cell biology as it pertains to ocular surface tissues. On the basis of this information, we propose a unifying hypothesis for how dendritic cells may cause conjunctival fibrosis in the allergy setting., Recent Findings: Work in models of airway remodeling and liver fibrosis has pointed to a potentially central role for dendritic cells in the pathobiology of fibrosis. Indeed, these cells are recognized as the most potent antigen-presenting cells, and as such activate T lymphocytes that are profibrotic under certain conditions. However, recent findings suggest a more direct role for dendritic cells, which opens up the possibility that a similar pathway may be relevant in the causation of conjunctival fibrosis, particularly in allergic eye disease., Summary: Conjunctival fibrosis is a serious clinical concern and is associated with chronic inflammation of the ocular surface tissue, such as in allergic eye disease. Dendritic cells are required in mediating allergic disease by activating pathologic T lymphocytes. Recent findings pointing to a central role for dendritic cell in fibrosis may, however, mean that these cells could also be contributing directly to conjunctival fibrosis. If so, furthering our understanding of dendritic cells could lead to the identification of novel and more effective therapeutic strategies to treat this disease.

Complement factor H (CFH) is a major susceptibility gene for age-related macular degeneration (AMD); however, its impact on AMD pathobiology is unresolved. Here, the role of CFH in the development of AMD pathology in vivo was interrogated by analyzing aged Cfh(+/-) and Cfh(-/-) mice fed a high-fat, cholesterol-enriched diet. Strikingly, decreased levels of CFH led to increased sub-retinal pigmented epithelium (sub-RPE) deposit formation, specifically basal laminar deposits, following high-fat diet. Mechanistically, our data show that deposits are due to CFH competition for lipoprotein binding sites in Bruch's membrane. Interestingly and despite sub-RPE deposit formation occurring in both Cfh(+/-) and Cfh(-/-) mice, RPE damage accompanied by loss of vision occurred only in old Cfh(+/-) mice. We demonstrate that such pathology is a function of excess complement activation in Cfh(+/-) mice versus complement deficiency in Cfh(-/-) animals. Due to the CFH-dependent increase in sub-RPE deposit height, we interrogated the potential of CFH as a previously unidentified regulator of Bruch's membrane lipoprotein binding and show, using human Bruch's membrane explants, that CFH removes endogenous human lipoproteins in aged donors. Thus, advanced age, high-fat diet, and decreased CFH induce sub-RPE deposit formation leading to complement activation, which contributes to RPE damage and visual function impairment. This new understanding of the complicated interactions of CFH in AMD-like pathology provides an improved foundation for the development of targeted therapies for AMD.

Purpose: The contribution of lymphangiogenesis (LA) to allergy has received considerable attention and therapeutic inhibition of this process via targeting VEGF has been considered. Likewise, certain inflammatory settings affecting the ocular mucosa can trigger pathogenic LA in the naturally avascular cornea. Chronic inflammation in allergic eye disease (AED) impacts the conjunctiva and cornea, leading to sight threatening conditions. However, whether corneal LA is involved is completely unknown. We addressed this using a validated mouse model of AED., Methods: Allergic eye disease was induced by ovalbumin (OVA) immunization and chronic OVA exposure. Confocal microscopy of LYVE-1-stained cornea allowed evaluation of corneal LA, and qRT-PCR was used to evaluate expression of VEGF-C, -D, and -R3 in these mice. Administration of VEGF receptor (R) inhibitor was incorporated to inhibit corneal LA in AED. Immune responses were evaluated by in vitro OVA recall responses of T cells, and IgE levels in the serum., Results: Confocal microscopy of LYVE-1-stained cornea revealed the distinct presence of corneal LA in AED, and corroborated by increased corneal expression of VEGF-C, -D, and -R3. Importantly, prevention of corneal LA in AED via VEGFR inhibition was associated with decreased T helper two responses and IgE production. Furthermore, VEGFR inhibition led a significant reduction in clinical signs of AED., Conclusions: Collectively, these data reveal that there is a distinct involvement of corneal LA in AED. Furthermore, VEGFR inhibition prevents corneal LA and consequent immune responses in AED.