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Mittal, Sharad

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Mittal

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Sharad

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Mittal, Sharad
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    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, Sunil
    Purpose 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.
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    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, Sunil
    Purpose 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.