Person: Chauhan, Sunil
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Chauhan
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Sunil
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Chauhan, Sunil
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Publication T Cell–Derived Granulocyte-Macrophage Colony-Stimulating Factor Contributes to Dry Eye Disease Pathogenesis by Promoting CD11b+ Myeloid Cell Maturation and Migration(The Association for Research in Vision and Ophthalmology, 2017) Dohlman, Thomas H.; Ding, Julia; Dana, Reza; Chauhan, SunilPurpose Growing evidence suggests that granulocyte-macrophage colony-stimulating factor (GM-CSF) contributes to T helper 17 (Th17) cell–associated immunoinflammatory diseases. The purpose of this study was to evaluate the effect of T cell–derived GM-CSF on CD11b+ myeloid cell function in dry eye disease (DED). Methods: In a murine model of DED, quantitative real-time PCR and ELISA were used to measure GM-CSF expression at the ocular surface, and flow cytometry was used to enumerate GM-CSF producing Th17 cells. A granulocyte-macrophage colony-stimulating factor neutralizing antibody was used topically in vivo and in an in vitro culture system to evaluate the role of GM-CSF in recruiting and maturing CD11b+ cells. Clinical disease severity was evaluated after topical administration of GM-CSF neutralizing antibody. Results: In dry eye disease, GM-CSF is significantly upregulated at the ocular surface and the frequency of GM-CSF producing Th17 cells is significantly increased in the draining lymph nodes. In vitro neutralization of GM-CSF from CD4+ T cells derived from DED mice suppresses major histocompatibility complex II expression by CD11b+ cells and CD11b+ cell migration. Topical neutralization of GM-CSF in a murine model of DED suppresses CD11b+ maturation and migration, as well as Th17 cell induction, yielding a reduction in clinical signs of disease. Conclusions: T helper 17 cell–derived GM-CSF contributes to DED pathogenesis by promoting CD11b+ cell activation and migration to the ocular surface.Publication Amelioration of Murine Dry Eye Disease by Topical Antagonist to Chemokine Receptor 2(American Medical Association (AMA), 2009) Goyal, Sunali; Chauhan, Sunil; Zhang, Qiang; Dana, RezaObjective To determine the effect of a topical antagonist to the chemokine receptor 2 (CCR2) in a murine model of dry eye disease. Methods The effects of a topical CCR2 antagonist and a vehicle control treatment were studied in murine dry eyes. A controlled environment chamber induced dry eye by exposing mice to high-flow desiccated air. Corneal fluorescein staining and enumeration of corneal CD11b+ and conjunctival CD3+ T cells were performed in the different groups. Real-time polymerase chain reaction was performed to quantify expression of different inflammatory cytokine transcripts in the cornea and conjunctiva. Results Eyes receiving the formulation containing CCR2 antagonist showed a significant decrease in corneal fluorescein staining and decreased infiltration of corneal CD11b+ cells and conjunctival T cells compared with the vehicle-treated and untreated dry eye groups. The CCR2 antagonist also significantly decreased messenger RNA expression levels of interleukins 1α and 1β in the cornea, and tumor necrosis factor α and interleukin 1β in the conjunctiva. Conclusion Topical application of CCR2 antagonist is associated with significant improvement in dry eye disease and is reflected by a decrease in inflammation at the clinical, molecular, and cellular levels. Clinical Relevance Topical application of CCR2 antagonist may hold promise as a therapeutic modality in dry eye disease.Publication Role of CCR7 in Facilitating Direct Allosensitization and Regulatory T-Cell Function in High-Risk Corneal Transplantation(Association for Research in Vision and Ophthalmology (ARVO), 2010) Jin, Yiping; Chauhan, Sunil; Saban, Daniel R.; Dana, RezaPurpose. Chemokine receptor 7 (CCR7) is a key homing molecule for immune cell trafficking, including corneal antigen-presenting cell (APC) migration from the inflamed cornea to draining lymph nodes (LNs). Here, the authors investigated the effect of CCR7-facilitated donor APC trafficking on allosensitization, regulatory T-cell (Treg) function, and graft survival in corneal transplantation. Methods. CCR7−/− or wild-type (WT) allogeneic corneal grafts were transplanted onto the neovascularized high-risk recipient beds. Two weeks later, the frequency of directly alloprimed host T cells was measured by the IFN-γ ELISPOT assay. Treg function was tested by a coculture suppression assay and an IFN-γ ELISPOT assay. Kaplan-Meier analysis was performed to evaluate graft survival. Results. The recipients of CCR7−/− grafts had fewer migrated donor APCs and lower frequency of IFN-γ–producing T cells in the draining LNs. However, there was no statistically significant difference in transplant survival between recipients of CCR7−/− and those of WT grafts. Tregs from the CCR7−/− graft recipient group showed reduced regulatory potential for the suppression of proliferation of naive T cells and direct alloprimed T cells and expressed lower Foxp3 levels. In vitro studies confirmed that mature CCR7+ major histocompatibility complex class II+ CD86+ graft-derived dendritic cells were critical for Treg function. Conclusions. Not only is CCR7-mediated donor-derived APC trafficking to the draining LNs important in the initiation of host T-cell priming, it is crucial for Treg-mediated tolerance.Publication Blockade of Prolymphangiogenic Vascular Endothelial Growth Factor C in Dry Eye Disease(American Medical Association (AMA), 2012) Goyal, Sunali; Chauhan, Sunil; Dana, RezaObjective To determine if blocking prolymphangiogenic factors like VEGF-C would suppress alloimmunity in dry eye disease (DED) using a murine model. Methods The effects of intraperitoneal injections of 400 μg of anti-VEGF-C antibody (treated group) and intraperitoneal normal saline (untreated group) were studied in murine dry eyes induced by exposing mice to high-flow desiccated air in the Controlled Environment Chamber (CEC). Growth of lymphatic vessels and infiltration of macrophages was evaluated by immunohistochemistry using CD31 (pan-endothelial marker), LYVE -1(lymphatic endothelial marker) and CD11b (monocytes/macrophages marker). Real time PCR was performed to quantify expression of different inflammatory cytokine transcripts in the conjunctiva and lymph nodes, and vascular endothelial growth factors and their receptors (VEGF-A, C, D/R2, R3) in the cornea. Results Blocking VEGF-C led to significant reduction in lymphatic caliber (P=0.025) and lymphatic area (P=0.006) in the corneas of DED mice. In addition to significantly decreasing (P=0.005) CD11b+ cells, anti-VEGF-C treatment significantly decreased transcript levels of VEGF-C (P=0.002), VEGF-D (P=0.014) and VEGFR-3 (P=0.023) in the corneas of treated group. Significant decrease in expression of inflammatory cytokines in the conjunctiva (IL1-α, P= 0.003; IL1-β, P= 0.025 and IL-6, P= 0.005) and lymph nodes (IFN-γ, P= 0.008; and IL-17, P= 0.003) was also seen with anti-VEGF-C treatment. Conclusions Treatment with anti-VEGF-C led to significant improvement in DED reflected by decrease in inflammation at the clinical, molecular, and cellular levels.Publication A Novel Pro-Angiogenic Function for Interferon-Y–Secreting Natural Killer Cells(Association for Research in Vision and Ophthalmology (ARVO), 2014) Lee, HyunSoo; Schlereth, Simona L.; Park, Eun Y.; Emami-Naeini, Parisa; Chauhan, Sunil; Dana, RezaPurpose. To explore the function of natural killer (NK) cells in inflammatory angiogenesis in mice. Methods. To study ocular angiogenic responses we used the cornea BFGF micropellet and the laser-induced choroidal neovascularization (CNV) mouse models (C57BL/6). To deplete NK cells in these models, we injected an anti-NK1.1 antibody or an isotype antibody as a control. Corneas or choroids were immunohistochemically stained for blood vessels (CD31), macrophages (F4/80), or CNV (isolectin-IB4). Vascular endothelial growth factors (VEGF), IFN-γ, or TNF-α levels were measured by real-time quantitative PCR (qPCR) or flow cytometry. A coculture assay of macrophages, NK cells, and human umbilical vein endothelial cells (HUVECs) was analyzed morphometrically to examine the ability of NK cells to induce angiogenesis in vitro. Results. Our data demonstrate that in vivo depletion of NK cells leads to a significant reduction of corneal angiogenesis and CNV. Furthermore, NK cell depletion reduces macrophage infiltration into the cornea and mRNA expression levels of VEGF-A, VEGF-C, and VEGFR3 at day 7 after micropellet insertion. In the laser-induced CNV model, our data show that NK cell depletion leads to decreased areas of CNV and significantly reduced mRNA expression of VEGFs and IFN-γ in the choroid. An in vitro coculture assay shows an IFN-γ–dependent increase in VEGF expression levels, thereby increasing endothelial cell proliferation. Conclusions. Our findings demonstrate a novel pro-angiogenic function for NK cells, indicating that IFN-γ–secreting NK cells can induce angiogenesis by promoting enhanced VEGF expression by macrophages.Publication PTK7+Mononuclear Cells Express VEGFR2 and Contribute to Vascular Stabilization by Upregulating Angiopoietin-1Significance(Ovid Technologies (Wolters Kluwer Health), 2015) Chauhan, Sunil; Lee, Hyung Keun; Lee, Hyun Soo; Park, Eun Young; Jeong, Eunae; Dana, RezaObjective In angiogenesis, circulating mononuclear cells are recruited to vascular lesions; however, the underlying mechanisms are poorly understood. Approach and Results Here, we characterize the functional role of protein tyrosine kinase 7 (PTK7)-expressing CD11b+ mononuclear cells in vitro and in vivo using a mouse model of angiogenesis. While the frequencies of PTK7+CD11b+ cells in the bone marrow remained similar after vascular endothelial growth factor (VEGF)-A induced neovascularization, we observed an 11-fold increase in the cornea. Importantly, VEGF-A–induced chemotaxis of PTK7+ cells was mediated by VEGF receptor (VEGFR) 2. In a co-culture with endothelial cells, PTK7+CD11b+ cells stabilized the vascular network for 2 weeks by expressing high levels of angiopoietin-1 (ANG-1). The enhanced vascular stability was abolished by knockdown of ANG-1 in PTK7+CD11b+ cells and could be restored by ANG-1 treatment. Conclusions We conclude that PTK7 expression in perivascular mononuclear cells induces VEGFR2 and ANG-1 expression, and thus contributes to vascular stabilization in angiogenesis.Publication Mast Cells Initiate the Recruitment of Neutrophils Following Ocular Surface Injury(The Association for Research in Vision and Ophthalmology, 2018) Sahu, Srikant K.; Mittal, Sharad; Foulsham, William; Li, Mingshun; Sangwan, Virender S.; Chauhan, SunilPurpose The purpose of this study was to investigate the contribution of mast cells to early neutrophil recruitment during ocular inflammation. Methods: In a murine model of corneal injury, the epithelium and anterior stroma were removed using a handheld motor brush. Cromolyn sodium (2% in PBS) eye drops were administered topically for mast cell inhibition. In vitro, bone marrow–derived mast cells were cultured alone or with corneal tissue. The frequencies of CD45+ inflammatory cells, CD11b+Ly6G+ neutrophils, and ckit+FcεR1+ mast cells in the cornea were assessed by flow cytometry. mRNA expression of CXCL2 was evaluated by real-time PCR and protein expression by ELISA. β-Hexosaminidase assays were performed to gauge mast cell activation. Results: Neutrophil infiltration of the cornea was observed within 1 hour of injury, with neutrophil frequencies increasing over subsequent hours. Concurrent expansion of mast cell frequencies at the cornea were observed, with mast cell activation (assessed by β-hexosaminidase levels) peaking at 6 hours after injury. Evaluation of CXCL2 mRNA and protein expression levels demonstrated augmented expression by injured corneal tissue relative to naïve corneal tissue. Mast cells were observed to constitutively express CXCL2, with significantly higher expression of CXCL2 protein compared with naïve corneal tissue. Culture with harvested injured corneas further amplified CXCL2 expression by mast cells. In vivo, mast cell inhibition was observed to decrease CXCL2 expression, limit early neutrophil infiltration, and reduce inflammatory cytokine expression by the cornea. Conclusions: Our data suggest that mast cell activation after corneal injury amplifies their secretion of CXCL2 and promotes the initiation of early neutrophil recruitment.Publication Mesenchymal Stromal Cells Inhibit Neutrophil Effector Functions in a Murine Model of Ocular Inflammation(The Association for Research in Vision and Ophthalmology, 2018) Mittal, Sharad; Mashaghi, Alireza; Amouzegar, Afsaneh; Li, Mingshun; Foulsham, William; Sahu, Srikant K.; Chauhan, SunilPurpose Neutrophil-secreted effector molecules are one of the primary causes of tissue damage during corneal inflammation. In the present study, we have investigated the effect of stromal cells in regulating neutrophil expression of tissue-damaging enzymes, myeloperoxidase (MPO), and N-elastase (ELANE). Methods: Bone marrow–purified nonhematopoietic mesenchymal stromal cells and formyl-methionyl-leucyl-phenylalanine–activated neutrophils were cocultured in the presence or absence of Transwell inserts for 1 hour. Neutrophil effector molecules, MPO and ELANE, were quantified using ELISA. In mice, corneal injury was created by mechanical removal of the corneal epithelium and anterior stroma approximating one third of total corneal thickness, and mesenchymal stromal cells were then intravenously injected 1 hour post injury. Corneas were harvested to evaluate MPO expression and infiltration of CD11b+Ly6G+ neutrophils. Results: Activated neutrophils cocultured with mesenchymal stromal cells showed a significant 2-fold decrease in secretion of MPO and ELANE compared to neutrophils activated alone (P < 0.05). This suppressive effect was cell–cell contact dependent, as stromal cells cocultured with neutrophils in the presence of Transwell failed to suppress the secretion of neutrophil effector molecules. Following corneal injury, stromal cell–treated mice showed a significant 40% decrease in MPO expression by neutrophils and lower neutrophil frequencies compared to untreated injured controls (P < 0.05). Reduced MPO expression by neutrophils was also accompanied by normalization of corneal tissue structure following stromal cell treatment. Conclusions: Mesenchymal stromal cells inhibit neutrophil effector functions via direct cell–cell contact interaction during inflammation. The current findings could have implications for the treatment of inflammatory ocular disorders caused by excessive neutrophil activation.Publication Chronic Dry Eye Disease is Principally Mediated by Effector Memory Th17 Cells(2013) Chen, Yihe; Chauhan, Sunil; Lee, Hyun Soo; Saban, Daniel R.; Dana, RezaRecent experimental and clinical data suggest that there is a link between dry eye disease (DED) and T cell-mediated immunity. However, whether these immune responses are a consequence or cause of ocular surface inflammation remains to be determined. Thus far, only models of acute DED have been used to derive experimental data. This is in contrast to clinical DED which usually presents as a chronic disease. In the present study, using a murine model of chronic DED, it was established that the chronic phase of the disease is accompanied by Th17 responses at the ocular surface, and that a significant memory T cell population can be recovered from chronic DED. This memory response is predominantly mediated by Th17 cells. Moreover, adoptive transfer of this memory T cell population was shown to induce more severe and rapidly progressing DED than did the adoptive transfer of its effector or naïve counterparts. Not only do these results clearly demonstrate that effector memory Th17 cells are primarily responsible for maintaining the chronic and relapsing course of DED, but they also highlight a potentially novel therapeutic strategy for targeting memory immune responses in patients with DED.Publication Expression of the chemokine decoy receptor D6 mediates dendritic cell function and promotes corneal allograft rejection(Molecular Vision, 2013) Hajrasouliha, Amir R.; Sadrai, Zahra; Lee, Hyung K.; Chauhan, Sunil; Dana, RezaPurpose To identify the role of chemokine receptor 6 (D6) expression by dendritic cells (DCs) and its role in corneal transplant immunity. Methods: Flow cytometry analysis was used to assess the expression level of the D6 chemokine receptor in different leukocyte populations and DC maturation following lipopolysaccharides (LPS) stimulation of bone marrow–derived DCs isolated from wild-type (WT) or D6−/− mice (C57BL/6 background). Mixed-lymphocyte reactions and delayed-type hypersensitivity assays were performed with bone marrow–derived DCs from WT or D6−/− mice to evaluate T-cell alloreactivity. Adoptive transfer experiments with T cells from WT or D6−/− hosts with BALB/c corneal allografts were performed. Graft opacity was assessed over an 8-week period, and graft survival was plotted using Kaplan–Meier survival curves. Results: Expression of the D6 chemokine receptor was significantly higher in DCs compared to other leukocyte subpopulations, including neutrophils, lymphocytes, and monocytes/macrophages. LPS challenge of D6−/− bone marrow–derived DCs elicited significantly lower levels of major histocompatibility complex II and costimulatory molecules (CD40, CD80, and CD86) compared to WT bone marrow–derived DCs, indicating the role of the D6 chemokine receptor in DC maturation. Further, DCs isolated from D6−/− mice induced less T-cell proliferation (p≤0.001) and interferon-gamma production in T cells of draining lymph nodes compared to WT mice following corneal transplantation (p≤0.001). Moreover, adoptively transferred T cells from D6−/− corneal transplanted mice to WT mice led to impaired graft rejection, compared to the hosts that received T cells from the WT transplanted mice. Conclusions: We demonstrated D6 chemokine receptor expression by DCs and identified its critical function in multiple aspects of DC biology, including maturation and consequent elicitation of alloreactive T-cell responses that are responsible for corneal allograft rejection.