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. 2023 May:230:109461.
doi: 10.1016/j.exer.2023.109461. Epub 2023 Apr 5.

Mustard gas exposure instigates retinal Müller cell gliosis

Binapani Mahaling  1 , Nishant R Sinha  2 , Sibabalo Sokupa  1 , Utkarsh Reddy Addi  1 , Rajiv R Mohan  2 , Shyam S Chaurasia  3
Affiliations

Mustard gas exposure instigates retinal Müller cell gliosis

Binapani Mahaling et al. Exp Eye Res. 2023 May.

Abstract

Sulfur mustard (SM) is a chemical warfare agent (CWA) that causes severe eye pain, photophobia, excessive lacrimation, corneal and ocular surface defects, and blindness. However, SM's effects on retinal cells are relatively meager. This study investigated the role of SM toxicity on Müller glial cells responsible for cellular architecture, inner blood-retinal barrier maintenance, neurotransmitter recycling, neuronal survival, and retinal homeostasis. Müller glial cells (MIO-M1) were exposed to SM analog, nitrogen mustard (NM), at varying concentrations (50-500 μM) for 3 h, 24 h, and 72 h. Müller cell gliosis was evaluated using morphological, cellular, and biochemical methods. Real-time cellular integrity and morphological evaluation were performed using the xCELLigence real-time monitoring system. Cellular viability and toxicity were measured using TUNEL and PrestoBlue assays. Müller glia hyperactivity was calculated based on glial fibrillary acidic protein (GFAP) and vimentin immunostaining. Intracellular oxidative stress was measured using DCFDA and DHE cell-based assays. Inflammatory markers and antioxidant enzyme levels were determined by quantitative real-time PCR (qRT-PCR). AO/Br and DAPI staining further evaluated DNA damage, apoptosis, necrosis, and cell death. Inflammasome-associated Caspase-1, ASC, and NLRP3 were studied to identify mechanistic insights into NM toxicity in Müller glial cells. The cellular and morphological evaluation revealed the Müller glia hyperactivity after NM exposure in a dose- and time-dependent manner. NM exposure caused significant oxidative stress and enhanced cell death at 72 h. A significant increase in antioxidant indices was observed at the lower concentrations of NM. Mechanistically, we found that NM-treated MIO-M1 cells increased caspase-1 levels that activated NLRP3 inflammasome-induced production of IL-1β and IL-18, and elevated Gasdermin D (GSDMD) expression, a crucial component actuating pyroptosis. In conclusion, NM-induced Müller cell gliosis via increased oxidative stress results in caspase-1-dependent activation of the NLRP3 inflammasome and cell death driven primarily by pyroptosis.

Keywords: Caspase 1; Cell death; Gliosis; Mustard gas; Müller glial cells; NLRP3; Nitrogen mustard; Oxidative stress; Pyroptosis; Retina; Sulfur mustard.

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Figures

Figure 1.
Figure 1.
Nitrogen mustard (NM) initiates morphological alterations in Müller glia in a dose- and time-dependent manner. (A) Phase contrast microscopy images of NM treated Müller glia at 3h (first column), 24h (second column) and 72h (third column) timepoints. (B) Quantification of rounded cells per total number of cells at 3h time point. (C) Quantification of rounded cells per total number of cells at 24h time point. (D) Quantification of rounded cells per total number of cells at 72h time point. All the results were presented as mean ±SD. *, p<0.05, ***, p<0.001, and ****p<0.0001.
Figure 2.
Figure 2.
Nitrogen mustard (NM) affects cell viability, migration, and adhesion in Müller glia. (A-C) Percentage cell viability at 3h, 24h and 72h time points measured by Presto Blue assay. (D) Normalized cell index determined by xCELLigence real-time cell monitoring system, (E) Percentage cell adhesion was calculated from normalized cell index. All the results were presented as mean±SD. **, p<0.01, and ****, p<0.0001.
Figure 3.
Figure 3.
Nitrogen mustard (NM) induces oxidative stress in Müller glia as determined by DCF immunocytochemistry and TBARS assay. (A) Fluorescent microscopy images of NM treated Müller glia at 3h (first column), 24h (second column) and 72h (third column). (B) Quantification of percentage change fluorescence intensity at 3h, 24h and 72h time points. (C) Measurement of lipid peroxidation determined by TBARS assay at 24h. All the results were presented as mean ±SD. *, p<0.05, ***, p<0.001, ****, p<0.0001.
Figure 4.
Figure 4.
Nitrogen mustard (NM) induces oxidative stress in Müller glia as determined by DHE immunocytochemistry and nitrotyrosine staining. (A) Fluorescent microscopic images of NM treated Müller glia at 3h (first column), 24h (second column) and 72h (third column). (B) Quantification of percentage change fluorescence intensity at 3h, 24h and 72h time points. (C) Nitrotyrosine immunostaining in NM treated cells. (D) Quantification of nitrotyrosine positive cells per total number of cells at 24h time point. Red arrow indicated nitrotyrosine positive cells. All the results were presented as mean ±SD. *, p<0.05, ***, p<0.001, and ****, p<0.0001.
Figure 5.
Figure 5.
Nitrogen mustard (NM) refines antioxidant system in Müller glia. Gene expression for antioxidant enzymes in MIO-M1 cells was analyzed by RT-qPCR after 24h of NM (0, 50, 125, 250 and 500μM) treatments. (A) catalase, (B) MnSOD, (C) Gpx-1, (D) GR, (E) GCLC, and (F) Txnrd-1. β-actin was used as the house keeping gene. All the results were presented as mean ±SD. *, p<0.05, **, p<0.01, ***, p<0.001, and ****, p<0.0001.
Figure 6.
Figure 6.
Nitrogen mustard (NM) stimulates Müller cell gliosis and inflammatory milieu. (A) Fluorescence microscopy images of GFAP and vimentin staining in NM treated MIO-M1 cells at 0, 50, 125, 250 and 500μM concentrations for 24h. Gene expression level analyzed for (B) TNFα, (C) Cox-2, (D) IL-6, (E) MCP-1, (F) IL-8, and (G) VEGF by RT-qPCR in NM treated MIO-M1 cells. n = 6. All the results were presented as mean ±SD. *, p<0.05, ***, p<0.001, and ****, p<0.0001.
Figure 7.
Figure 7.
Nitrogen mustard (NM) causes cell death in Müller glia in a dose- and time-dependent manner. (A) Fluorescent microscopic images of NM treated Müller glia at 3h (first column), 24h (second column) and 72h (third column). Red color stains for EtBr and Green for acridine orange staining. Solid arrow represents living cells, dotted arrow shows necrotic cells, star represents early apoptotic cells, heart indicates late apoptotic cells, and triangle shiws fragmented DNA. (B) Quantification of EtBr positive cells per total number of cells at 3h, 24h and 72 h time points. (C) TUNEL staining in NM treated MIO-M1 cells. (D) Quantification of TUNEL positive cells per total number of cells at 24h time point. (E) DAPI staining in NM treated MIO-M1 cells and (F) Quantification of ruptured and swollen nuclei as compared to total number of nuclei at 24h time point. Star represented apoptotic cells, white arrow indicated swollen nucleus and yellow arrow represented fragmented nucleus. All the results were presented as mean ±SD. *, p<0.05, **, p<0.01, ***, p<0.001, and ****, p<0.0001.
Figure 8.
Figure 8.
NM activates caspase-1 to actuate NLRP3 inflammasome and pyroptosis in Müller glia. (A) Percentage Caspase activity in MIO-M1 cells treated with NM at different concentrations and collected after 24h. (B) Gene expression of inflammasome related genes, caspase-1, NLRP3, ASC, IL-1β, and IL-18 was analyzed by RT-qPCR after 24h of NM (0, 50, 125, 250 and 500μM) treatments. β-actin was used as the house keeping gene. (C) Immunocytochemistry of NLRP3 inflammasome-related proteins in MIO-M1 cells treated with different concentrations of NM at 24h time point. (D) Quantification of caspase-1, NLRP3, and ASC positive cells per total number of cells at 24h time point. (C) Quantification of caspase-1 positive cells per total number of cells at 24h time point. (D) Quantification of NLRP3 positive cells per total number of cells at 24h time point. (E) GSDMD expression in MIO-M1 cells treated with different concentrations of NM. (F) Quantification of GSDMD positive cells per total number of cells at 24h time point. (G-J) All the results were presented as mean ±SD. *, p<0.05, **, p<0.01, ***, p<0.001, and ****, p<0.0001.
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