Mesenchymal stromal cells protect tissues from Th1 immune responses via IL-11 secretion.

Mesenchymal stromal cells (MSCs) have been shown to modulate the function of various subsets of T cells such as naïve CD4+ T cells and IFNγ+CD4+ Th1 cells; however, mechanisms underlying this regulation have not been fully deciphered. Our in vitro culture assays demonstrate that MSCs suppress the activation and function of CD4+ T cells by secreting interleukin 11, and neutralization of IL11 abrogates MSC-mediated suppression of CD4+ T cell function. Moreover, delayed-type, exogenous supplementation of IL11 significantly suppressed IFNγ+ expression by Th1 cells. Th1 and CD8+ cells play central roles in T cell-mediated tissue damage. Using a murine model of hypersensitivity response to study T cell-mediated tissue damage, we show that silencing IL11 in MSCs significantly abates the capacity of MSCs to suppress the generation of IFNγ-secreting CD4+ and CD8+ cells, failing to prevent T cell-mediated tissue inflammation and tissue damage.

Effector T Cells Promote Fibrosis in Corneal Transplantation Failure.

Purpose: To evaluate whether fibrosis contributes to corneal transplant failure and to determine whether effector CD4+ T cells, the key immune cells in corneal transplant rejection, play a direct role in fibrosis formation. Methods: Allogeneic corneal transplantation was performed in mice. Graft opacity was evaluated by slit-lamp biomicroscopy, and fibrosis was assessed by in vivo confocal microscopy. Expression of alpha-smooth muscle actin (α-SMA) in both accepted and failed grafts was assessed by real-time PCR and immunohistochemistry. Frequencies of graft-infiltrating CD4+ T cells, neutrophils, and macrophages were assessed using flow cytometry. In vitro, MK/T-1 corneal fibroblasts were co-cultured with activated CD4+CD25- effector T cells isolated from corneal transplant recipient mice, and α-SMA expression was quantified by real-time PCR and ELISA. Neutralizing antibody was used to evaluate the role of interferon gamma (IFN-γ) in promoting α-SMA expression. Results: The majority of failed grafts demonstrated clinical signs of fibrosis which became most evident at week 6 after corneal transplantation. Failed grafts showed higher expression of α-SMA as compared to accepted grafts. Flow cytometry analysis showed a significant increase in CD4+ T cells in failed grafts compared to accepted grafts. Co-culture of activated CD4+CD25- effector T cells with corneal fibroblasts led to an increase in α-SMA expression by fibroblasts. Inhibition of IFN-γ in culture significantly suppressed this increase in α-SMA expression as compared to immunoglobulin G control. Conclusions: Fibrosis contributes to graft opacity in corneal transplant failure and is mediated at least in part by effector CD4+ T cells via IFN-γ.

Interleukin-11 Suppresses Ocular Surface Inflammation and Accelerates Wound Healing.

Purpose: Regulation of inflammation is critical for achieving favorable outcomes in wound healing. In this study, we determine the functional role and mechanism of action of IL-11, an immunomodulatory cytokine, in regulating inflammatory response at the ocular surface. Methods: Corneal injury was induced by mechanical removal of the epithelium and anterior stroma using an AlgerBrush II. Transcript and protein levels of IL-11 in injured cornea were quantified using real-time PCR and ELISA analysis. Corneal inflammation was assessed by measuring frequencies of total CD45+ inflammatory cells, CD11b+Ly6G+ polymorphonuclear cells (neutrophils), and CD11b+Ly6G- mononuclear cells (macrophages, monocytes) at the ocular surface using flow cytometry. To assess the effect of IL-11 on innate immune cell function, cell activation marker and inflammatory cytokines including major histocompatibility complex (MHC) class II, myeloperoxidase (MPO), TNFα, and inducible nitric oxide synthase (iNOS) were measured following recombinant IL-11 treatment (1 µg/mL). Injured corneas were topically treated with IL-11 (1 µg/mL), and wound healing was evaluated using corneal fluorescein staining. Results: Corneal injury resulted in increased levels of IL-11 in the cornea, particularly in the stroma. Neutrophils and CD11b+ mononuclear cells (macrophages, monocytes) substantially expressed IL-11 receptor. Interestingly, IL-11 significantly downregulated the activation of immune cells, as evidenced by the lower expression of MHC II and TNFα by CD11b+ mononuclear cells and lower levels of MPO by neutrophils. Topical administration of IL-11 to injured corneas led to faster wound healing and better retention of tissue architecture. Conclusions: Our findings demonstrate IL-11 is a key modulator of ocular surface inflammation and provide novel evidence of IL-11 as a potential therapeutic to control inflammatory damage and accelerate wound repair following injury.

Mesenchymal Stromal Cells Suppress T-Cell-Mediated Delayed-Type Hypersensitivity via ALCAM-CD6 Interaction.

Mounting evidence suggests mesenchymal stromal cells (MSCs) suppress CD4+ T-cell activation, but whether MSCs directly regulate activation and expansion of allogeneic T cells has not been fully deciphered. Here, we identified that both human and murine MSCs constitutively express ALCAM, a cognate ligand for CD6 receptors on T cells, and investigated its immunomodulatory function using in vivo and in vitro experiments. Our controlled coculture assays demonstrated that ALCAM-CD6 pathway is critical for MSCs to exert its suppressive function on early CD4+CD25- T-cell activation. Moreover, neutralizing ALCAM or CD6 results in the abrogation of MSC-mediated suppression of T-cell expansion. Using a murine model of delayed-type hypersensitivity response to alloantigen, we show that ALCAM-silenced MSCs lose the capacity to suppress the generation of alloreactive IFNγ-secreting T cells. Consequently, MSCs, following ALCAM knockdown, failed to prevent allosensitization and alloreactive T-cell-mediated tissue damage.

Increased activity of lacrimal gland mast cells are associated with corneal epitheliopathy in aged mice.

The lacrimal gland undergoes significant structural and functional deterioration with aging. Marked with increased inflammation and fibrosis, the aged lacrimal gland is unable to perform its protective function. As a result, the ocular surface becomes highly susceptible to various ocular surface pathologies, including corneal epitheliopathy. We and others have previously shown that mast cells mediate tissue inflammation by recruiting other immune cells. However, despite their well-known characteristics of secreting various inflammatory mediators, whether mast cells contribute to the immune cell aggregation and activation, and acinar dystrophy of the aged lacrimal gland has not been investigated. Here, we demonstrate the role of mast cells in age-related lacrimal gland pathophysiology using mast cell-deficient (cKitw-sh) mice. Our data demonstrated a significant increase in mast cell frequencies and immune cell infiltration in the lacrimal gland of aged mice. Interestingly, mast cell deficiency resulted in a substantial reduction in inflammation and preservation of lacrimal gland structure, suggesting that mast cells mediate the aging process of the lacrimal gland.

IL-36γ Augments Ocular Angiogenesis by Promoting the Vascular Endothelial Growth Factor-Vascular Endothelial Growth Factor Receptor Axis.

Prevention of inflammatory angiogenesis is critical for suppressing chronic inflammation and inhibiting inflammatory tissue damage. Angiogenesis is particularly detrimental to the cornea because pathologic growth of new blood vessels can lead to marked vision impairment and even loss of vision. The expression of proinflammatory cytokines by injured tissues exacerbates the inflammatory cascade, including angiogenesis. IL-36 cytokine, a subfamily of the IL-1 superfamily, consists of three proinflammatory agonists, IL-36α, IL-36ß, and IL-36γ, and an IL-36 receptor antagonist (IL-36Ra). Data from the current study indicate that human vascular endothelial cells constitutively expressed the cognate IL-36 receptor. The current investigation, for the first time, characterized the direct contribution of IL-36γ to various angiogenic processes. IL-36γ up-regulated the expression of vascular endothelial growth factors (VEGFs) and their receptors VEGFR2 and VEGFR3 by human vascular endothelial cells, suggesting that IL-36γ mediates the VEGF-VEGFR signaling by endothelial cells. Moreover, by using a naturally occurring antagonist IL-36Ra in a murine model of inflammatory angiogenesis, this study demonstrated that blockade of endogenous IL-36γ signaling results in significant retardation of inflammatory angiogenesis. The current investigation on the proangiogenic function of IL-36γ provides novel evidence of the development of IL-36γ-targeting strategies to hamper inflammatory angiogenesis.

Non-immune and immune functions of interleukin-36γ suppress epithelial repair at the ocular surface.

Regulation of innate inflammation is critical for maintaining tissue homeostasis and barrier function, especially in those interfacing the external environments such as the skin and cornea. Expression of pro-inflammatory cytokines by injured tissues has been shown to exacerbate the inflammatory cascade, causing tissue damage. Interleukin 36, a subfamily of the IL-1 superfamily, consists of three pro-inflammatory agonists-IL36α, IL36ß, and IL36γ and an IL36 receptor antagonist (IL36Ra). The current investigation, for the first time, reports that IL36γ is the primary agonist expressed by the corneal epithelium, which is significantly upregulated following corneal injury. The function of IL36γ on non-immune cells, in addition to innate inflammatory cells, in regulating tissue homeostasis has not been well investigated. Using a loss-of-function approach via neutralizing antibody treatment, our data demonstrate that blocking endogenously expressed IL36γ in epithelial cells promotes rapid re-epithelialization in in vitro wound closure assay. Finally, by utilizing a naturally occurring antagonist IL36Ra in a well-established murine model of ocular injury, our study demonstrates that inhibition of IL36γ accelerates epithelial regeneration and suppresses tissue inflammation. Given rapid wound healing is critical for re-establishing normal tissue structure and function, our investigation on the function of IL36γ provides evidence for the development of novel IL36γ-targeting strategies to promote tissue repair.

Mesenchymal stem cells augment regulatory T cell function via CD80-mediated interactions and promote allograft survival.

Mesenchymal stem cells (MSCs) and regulatory T cells (Tregs) both have been shown to modulate the alloimmune response and promote transplant survival. Mounting evidence suggests that MSCs augment Treg function, but the mechanisms underlying this phenomenon have not been fully deciphered. Here, we identified that MSCs express substantial levels of CD80 and evaluated its immunoregulatory function using in vivo and in vitro experiments. Our in vitro culture assays demonstrated that MSCs induce expression of FoxP3 in Tregs in a contact-dependent manner, and the blockade of CD80 abrogates this FoxP3 induction and Treg-mediated suppression of T cell proliferation. Moreover, supplementation of soluble CD80 significantly upregulated FoxP3 expression. Using a well-characterized murine model of corneal transplantation, we show that silencing CD80 in MSCs diminishes the capacity of MSCs to promote selective graft infiltration of Tregs, promote FoxP3 expression and upregulate suppressive function of Tregs. Consequently, MSCs, following CD80 knockdown, failed to promote corneal allograft survival.

Ocular surface mast cells promote inflammatory lymphangiogenesis.

Mast cells, sentinel immune cells, are most abundantly expressed in vascularized tissues that interface the external environment, such as the skin and ocular surface. Our previous reports have shown mast cells reside closely with vascular endothelial cells and mediate the pathogenic angiogenic response. However, the contribution of mast cells and their underlying mechanisms on lymphangiogenesis have not been fully deciphered. Using a murine model of inflammatory corneal angiogenesis, we observed adjacent migration of activated mast cells with new lymph vessel growth. Our in vitro co-culture assays demonstrate that mast cells express high levels of of VEGF-D and directly promote lymphatic endothelial cell tube formation and proliferation. Moreover, our loss-of-function approaches, using mast cell knockout mice and cromolyn-mediated mast cell inhibition, showed mast cell deficiency suppresses the induction of inflammatory lymphangiogenesis and VEGF-D expression at the ocular surface following corneal tissue insult. Our findings suggest blockade of mast cells as a potential therapeutic strategy to inhibit pathological lymphangiogenesis.

Suppression of lipopolysaccharide-induced corneal opacity by hepatocyte growth factor.

Keratitis induced by bacterial toxins, including lipopolysaccharide (LPS), is a major cause of corneal opacity and vision loss. Our previous study demonstrates hepatocyte growth factor (HGF) promotes epithelial wound healing following mechanical corneal injury. Here, we investigated whether HGF has the capacity to suppress infectious inflammatory corneal opacity using a new model of LPS-induced keratitis. Keratitis, induced by two intrastromal injections of LPS on day 1 and 4 in C57BL/6 mice, resulted in significant corneal opacity for up to day 10. Following keratitis induction, corneas were topically treated with 0.1% HGF or PBS thrice daily for 5 days. HGF-treated mice showed a significantly smaller area of corneal opacity compared to PBS-treated mice, thus improving corneal transparency. Moreover, HGF treatment resulted in suppression of α-SMA expression, compared to PBS treatment. HGF-treated corneas showed normalized corneal structure and reduced expression of pro-inflammatory cytokine, demonstrating that HGF restores corneal architecture and immune quiescence in corneas with LPS-induced keratitis. These findings offer novel insight into the potential application of HGF-based therapies for the prevention and treatment of infection-induced corneal opacity.

Ocular redness - I: Etiology, pathogenesis, and assessment of conjunctival hyperemia.

The translucent appearance of the conjunctiva allows for immediate visualization of changes in the circulation of the conjunctival microvasculature consisting of extensive branching of superficial and deep arterial systems and corresponding drainage pathways, and the translucent appearance of the conjunctiva allows for immediate visualization of changes in the circulation. Conjunctival hyperemia is caused by a pathological vasodilatory response of the microvasculature in response to inflammation due to a myriad of infectious and non-infectious etiologies. It is one of the most common contributors of ocular complaints that prompts visits to medical centers. Our understanding of these neurogenic and immune-mediated pathways has progressed over time and has played a critical role in developing targeted novel therapies. Due to a multitude of underlying etiologies, patients must be accurately diagnosed for efficacious management of conjunctival hyperemia. The diagnostic techniques used for the grading of conjunctival hyperemia have also evolved from descriptive and subjective grading scales to more reliable computer-based objective grading scales.

Ocular redness - II: Progress in development of therapeutics for the management of conjunctival hyperemia.

Conjunctival hyperemia is one of the most common causes for visits to primary care physicians, optometrists, ophthalmologists, and emergency rooms. Despite its high incidence, the treatment options for patients with conjunctival hyperemia are restricted to over-the-counter drugs that provide symptomatic relief due to short duration of action, tachyphylaxis and rebound redness. As our understanding of the immunopathological pathways causing conjunctival hyperemia expands, newer therapeutic targets are being discovered. These insights have also contributed to the development of animal models for mimicking the pathogenic changes in microvasculature causing hyperemia. Furthermore, this progress has catalyzed the development of novel therapeutics that provide efficacious, long-term relief from conjunctival hyperemia with minimal adverse effects.

Spatial Distribution of Mast Cells Regulates Asymmetrical Angiogenesis at the Ocular Surface.

Mast cells, historically known for their function as effector cells in the induction of allergic diseases, reside in all vascularized tissues of the body, particularly, in proximity to blood and lymphatic vessels. Despite being neighboring sentinel cells to blood vessels, whether the spatial distribution of mast cells regulates the degree of angiogenesis remains to be investigated. Herein, an asymmetrical distribution of mast cells was shown at the murine ocular surface, with the higher number of mast cells distributed along the nasal limbus of the cornea compared with the temporal side. Using a well-characterized murine model of suture-induced corneal neovascularization, insult to the nasal side was shown to result in more extensive angiogenesis compared with that to the temporal side. To directly assess the impact of the spatial distribution of mast cell on angiogenesis, neovascularization was induced in mast cell-deficient mice (cKitw-sh). Unlike the wild-type (C57BL/6) mice, cKitw-sh mice did not show disproportionate growth of corneal blood vessels following the temporal and nasal insult. Moreover, cromolyn-mediated pharmacologic blockade of mast cells at the ocular surface attenuated the asymmetrical nasal and temporal neovascularization, suggesting that spatial distribution of mast cells significantly contributes to angiogenic response at the ocular surface.

Pigment Epithelium-Derived Factor Enhances the Suppressive Phenotype of Regulatory T Cells in a Murine Model of Dry Eye Disease.

Pigment epithelium-derived factor (PEDF) is a widely expressed 50-kDa glycoprotein belonging to the serine protease inhibitor family, with well-established anti-inflammatory functions. Recently, we demonstrated the immunoregulatory role played by PEDF in dry eye disease (DED) by suppressing the maturation of antigen-presenting cells at the ocular surface following exposure to the desiccating stress. In this study, we evaluated the effect of PEDF on the immunosuppressive characteristics of regulatory T cells (Tregs), which are functionally impaired in DED. In the presence of PEDF, the in vitro cultures prevented proinflammatory cytokine (associated with type 17 helper T cells)-induced loss of frequency and suppressive phenotype of Tregs derived from normal mice. Similarly, PEDF maintained the in vitro frequency and enhanced the suppressive phenotype of Tregs derived from DED mice. On systemically treating DED mice with PEDF, moderately higher frequencies and significantly enhanced suppressive function of Tregs were observed in the draining lymphoid tissues, leading to the efficacious amelioration of the disease. Our results demonstrate that PEDF promotes the suppressive capability of Tregs and attenuates their type 17 helper T-cell-mediated dysfunction in DED, thereby playing a role in the suppression of DED.

Characterization of Clinical and Immune Responses in an Experimental Chronic Autoimmune Uveitis Model.

Autoimmune uveitis is a sight-threatening intraocular inflammatory disease. For >30 years, the mouse model of experimental autoimmune uveitis has been employed to investigate disease mechanisms and test immunotherapeutic approaches. However, inflammation in this model is self-limited, and does not replicate the chronic, insidious nature prevalent in the human disease. Herein, a robust and reliable model of chronic autoimmune uveitis was developed and characterized in two strains of wild-type mice by modifying interphotoreceptor retinoid-binding protein dose and peptide fragments from conventional experimental autoimmune uveitis models. In both of these murine strains, immunization with our modified protocols resulted in a slowly progressive uveitis, with retinal scars and atrophy observed in the chronic stage by fundoscopy. Optical coherence tomography demonstrated decreased retinal thickness in chronic autoimmune uveitis mice, and electroretinography showed significantly reduced amplitudes of dark-adapted a- and b-waves and light-adapted b-waves. Histologic examination revealed prominent choroiditis with extensive retinal damage. Flow cytometry analysis showed substantially increased numbers of CD44hiIL-17+IFN-γ- memory T-helper 17 (Th17) cells in the retina, cervical lymph nodes, inguinal lymph nodes, and spleen. These data establish new modified protocols for inducing chronic uveitis in wild-type mice, and demonstrate a predominant memory Th17 cell response, suggesting an important role for memory Th17 cells in driving chronic inflammation in autoimmune uveitis.

Activation of ocular surface mast cells promotes corneal neovascularization.

PURPOSE: Mast cells, historically known for their effector function in the induction of allergic diseases, reside in all vascularized tissues of the body in particular proximity to blood and lymphatic vessels. As neighboring sentinel cells to blood vessels, mast cells have been associated with angiogenesis. Here we assess the direct contribution of mast cells to neovascularization at the ocular surface. METHODS: Corneal neovascularization was induced by placing a single figure-of-eight intrastromal suture 1 mm from the limbus in mast cell-deficient (cKitW-sh), C57BL/6, and Balb/c mice. Corneas were harvested at 6 h post-suture to quantify cKit+FcεR1+ mast cells using flow cytometry and tear wash was collected within 6 h to measure ß-hexosaminidase and tryptase. Neovascularization was assessed using slit-lamp biomicroscope and immunohistochemistry analysis of corneas harvested on day 4 post-suture. To investigate the effects of mast cells on blood vessel growth, mast cells were co-cultured with vascular endothelial cells (VECs), and tube formation and proliferation of VECs were measured. 2% cromolyn was administered locally to inhibit mast cell activation in vivo. RESULTS: Placement of corneal suture activates ocular surface mast cells, which infiltrate into the cornea adjacent to new vessels. Mast cell-deficient mice develop significantly fewer new vessels following suture placement. Mast cells directly promote VEC proliferation and tube formation, partly through secreting high levels of VEGF-A. Pharmacological inhibition of mast cell activation results in significantly less corneal neovascularization. CONCLUSION: Our data demonstrate that ocular surface mast cells are critical to corneal neovascularization, suggesting mast cells as a potential therapeutic target in the treatment of corneal neovascularization.

Steroid-eluting contact lenses for corneal and intraocular inflammation.

Ocular inflammation is one of the leading causes of blindness worldwide, and steroids in topical ophthalmic solutions (e.g. dexamethasone eye drops) are the mainstay of therapy for ocular inflammation. For many non-infectious ocular inflammatory diseases, such as uveitis, eye drops are administered as often as once every hour. The high frequency of administration coupled with the side effects of eye drops leads to poor adherence for patients. Drug-eluting contact lenses have long been sought as a potentially superior alternative for sustained ocular drug delivery; but loading sufficient drug into contact lenses and control the release of the drug is still a challenge. A dexamethasone releasing contact lens (Dex-Lens) was previously developed by encapsulating a dexamethasone-polymer film within the periphery of a hydrogel-based contact lens. Here, we demonstrate safety and efficacy of the Dex-Lens in rabbit models in the treatment of anterior ocular inflammation. The Dex-Lens delivered drug for 7 days in vivo (rabbit model). In an ocular irritation study (Draize test) with Dex-Lens extracts, no adverse events were observed in normal rabbit eyes. Dex-Lenses effectively inhibited suture-induced corneal neovascularization and inflammation for 7 days and lipopolysaccharide-induced anterior uveitis for 5 days. The efficacy of Dex-Lenses was similar to that of hourly-administered dexamethasone eye drops. In the corneal neovascularization study, substantial corneal edema was observed in rabbit eyes that received no treatment and those that wore a vehicle lens as compared to rabbit eyes that wore the Dex-Lens. Throughout these studies, Dex-Lenses were well tolerated and did not exhibit signs of toxicity. Dexamethasone-eluting contact lenses may be an option for the treatment of ocular inflammation and a platform for ocular drug delivery. STATEMENT OF SIGNIFICANCE: Inflammation of the eye can happen either on the ocular surface (i.e. the cornea) or inside the eye, both of which can result in loss of vision or even blindness. Ocular inflammation is normally treated by steroid eye drops. Depending on the type and severity of inflammation, patients may have to take drops every hour for days at a time. Such severe dosing regimen can lead to patients missing doses. Also, more than 95% drug in an eye drop never goes inside the eye. Here we present a contact lens that release a steroid (dexamethasone) for seven days at a time. It is much more efficient than eye drops and a significant improvement since once worn, the patient will avoid missing doses.

Epithelium-derived IL-33 activates mast cells to initiate neutrophil recruitment following corneal injury.

PURPOSE: Neutrophils play a critical role in defending against threats such as microbial infection, yet their activation during innate immune response incites collateral damage to healthy tissues. We have previously shown that corneal injury induces mast cells to express the neutrophil chemoattractant CXCL2. Here we delineate the mechanism of injury-induced, non-IgE-mediated mast cell activation at the ocular surface. METHODS: Corneal injury was induced by mechanical removal of the epithelium and anterior stroma in mast cell deficient (cKitW-sh) and C57BL/6 mice using Algerbrush II. Corneas were analyzed for frequencies of total CD45+ inflammatory cells, CD11b+Ly6G+ neutrophils, and cKit+FcεR1+ mast cells using flow cytometry. Mast cells were stimulated with different inflammatory factors known to increase during corneal injury (IL-33, IL-1ß, IL-36γ, IL-6, SDF1α and Substance P) and assessed for the secretion of ß-hexosaminidase, tryptase and CXCL2 using ELISA. IL-33 neutralizing antibody (1 mg/ml) was administered locally for mast cell inhibition in vivo. RESULTS: Mast cell deficient mice failed to recruit early neutrophils to the injured corneas. IL-33 stimulation upregulated CXCL2 secretion by mast cells. Corneal injury resulted in amplified expression of IL-33 at the cornea and epithelium was identified as its primary source. Topical neutralization of IL-33 at the ocular surface inhibited mast cell activation, limited neutrophil infiltration, and reduced corneal inflammatory haze, normalizing tissue architecture following ocular injury. CONCLUSIONS: These data implicate IL-33 in mast cell activation and early neutrophil recruitment in non-allergic inflammation, suggesting IL-33 as a potential therapeutic target in inflammatory disorders of the ocular surface.

Pigment Epithelium-derived Factor secreted by corneal epithelial cells regulates dendritic cell maturation in dry eye disease.

PURPOSE: In this study, we quantify Pigment Epithelium-derived Factor (PEDF) secreted by corneal epithelial cells and evaluate its immunomodulatory functions in a murine model of dry eye disease (DED). METHODS: We induced DED in female C57BL/6 mice using a controlled environment chamber for 14 days. We quantified mRNA expression of Serpinf1 gene and PEDF protein synthesis by corneal epithelial cells (CEpCs) using RT-PCR and ELISA. CEpCs from normal or DED mice were cultured with IFNγ-stimulated-dendritic cells (DCs) for 24 h, and expression of MHC-II and CD86 by DCs was determined using flow cytometry. Next, we either added recombinant PEDF (rPEDF) or anti-PEDF antibody to co-culture, and DC expression of the above maturation markers was quantified. Lastly, we treated DED mice with either topical rPEDF, anti-PEDF Ab or murine serum albumin (MSA), and DC maturation, expression of pro-inflammatory cytokines, and DED severity were investigated. RESULTS: Serpinf1 mRNA expression and PEDF protein production levels by CEpCs were upregulated in DED. CEpCs from DED mice exhibited an enhanced suppressive effect on the expression of MHC-II and CD86 by DCs, compared to normal mice. This effect was abolished by blocking endogenous PEDF with anti-PEDF Ab or enhanced by supplementing with rPEDF. Treatment with anti-PEDF antibody blocked the effect of endogenous-PEDF and increased DC maturation, expression of pro-inflammatory cytokines in conjunctivae, and exacerbated disease severity in DED mice. Conversely, topical rPEDF enhanced the suppressive effect of endogenous PEDF on DC maturation, decreased expression of pro-inflammatory cytokines in conjunctivae, and reduced disease severity. CONCLUSIONS: The results from our study elucidate the role of PEDF in impeding DC maturation, and suppression of ocular surface inflammation, explicating a promising therapeutic potential of PEDF in limiting the corneal epitheliopathy as a consequence of DED.

Neutrophil L-Plastin Controls Ocular Paucibacteriality and Susceptibility to Keratitis.

Why ocular mucosa is paucibacterial is unknown. Many different mechanisms have been suggested but the comprehensive experimental studies are sparse. We found that a deficiency in L-plastin (LCP1), an actin bundling protein, resulted in an ocular commensal overgrowth, characterized with increased presence of conjunctival Streptococcal spp. The commensal overgrowth correlated with susceptibility to P. aeruginosa-induced keratitis. L-plastin knock-out (KO) mice displayed elevated bacterial burden in the P. aeruginosa-infected corneas, altered inflammatory responses, and compromised bactericidal activity. Mice with ablation of LPL under the LysM Cre (LysM. CreposLPLfl/fl ) and S100A8 Cre (S100A8.CreposLPLfl/fl ) promoters had a similar phenotype to the LPL KOs mice. In contrast, infected CD11c.CreposLPLfl/fl mice did not display elevated susceptibility to infection, implicating the myeloid L-plastin-sufficient cells (e.g., macrophages and neutrophils) in maintaining ocular homeostasis. Mechanistically, the elevated commensal burden and the susceptibility to infection were linked to defects in neutrophil frequencies at steady state and during infection and compromised bactericidal activities upon priming. Macrophage exposure to commensal organisms primed neutrophil responses to P. aeruginosa, augmenting PMN bactericidal capacity in an L-plastin dependent manner. Cumulatively, our data highlight the importance of neutrophils in controlling ocular paucibacteriality, reveal molecular and cellular events involved in the process, and suggest a link between commensal exposure and resistance to infection.