Anti-Inflammatory (M2) Response Is Induced by a sp2-Iminosugar Glycolipid Sulfoxide in Diabetic Retinopathy

Abstract

Diabetic retinopathy (DR) is one of the most common complications of Diabetes Mellitus (DM) and is directly associated with inflammatory processes. Currently, neuro-inflammation is considered an early event in DR and proceeds via microglia polarization. A hallmark of DR is the presence of retinal reactive gliosis. Here we report the beneficial effect of (S_S,1R)-1-docecylsulfiny-5N,6O-oxomethylidenenojirimycin ((Ss)-DS-ONJ), a member of the sp²-iminosugar glycolipid (sp²-IGL) family, by decreasing iNOS and inflammasome activation in Bv.2 microglial cells exposed to pro-inflammatory stimuli. Moreover, pretreatment with (Ss)-DS-ONJ increased Heme-oxygenase (HO)-1 as well as interleukin 10 (IL10) expression in LPS-stimulated microglial cells, thereby promoting M2 (anti-inflammatory) response by the induction of Arginase-1. The results strongly suggest that this is the likely molecular mechanism involved in the anti-inflammatory effects of (S_S)-DS-ONJ in microglia. (S_S)-DS-ONJ further reduced gliosis in retinal explants from type 1 diabetic BB rats, which is consistent with the enhanced M2 response. In conclusion, targeting microglia polarization dynamics in M2 status by compounds with anti-inflammatory activities offers promising therapeutic interventions at early stages of DR.

Keywords: M2 response; diabetic retinopathy; immunomodulation; microglia; sp2-iminosugar glycolipids.

Figure 1

Effect of (S _S)-DS-ONJ in the cellular viability in Bv.2 microglial cells. (A) Chemical structures of the sp²-IGLs (S _S)-DS-ONJ, (S _R)-DS-ONJ and DSO₂-ONJ. (B) Bv.2 microglial cells were treated for 24 h with (S _S)-DS-ONJ (1–10 μM) and viability was determined by crystal violet staining. Colorimetric quantification results presented as mean ± SEM (n = 5 independent experiments). The fold changes are presented relative to basal values. *p ≤ 0.05 vs the basal values (two-way ANOVA followed by Bonferroni t-test.)

Figure 2

Protective effects of (S _S)-DS-ONJ against LPS-mediated iNOS activation and elevation of mRNA levels of pro-inflammatory cytokines in Bv.2 microglial cells. Bv.2 microglial cells were treated for 24 h with LPS (200 ng/mL) or LPS plus (S _S)-DS-ONJ (1–10 μM). (A) Colorimetric quantification of nitrites was performed. (B) mRNA of Nos2 was determined by qRT-PCR. (C) Protein extracts were analyzed by Western blot with the corresponding antibodies against iNOS and α-tubulin as loading control. Representative autoradiograms are shown. Blots were quantified by scanning densitometry and the results are presented as mean ± SEM. (D) Tnfa, Il1b, and Il6 mRNA values were determined by qRT-PCR. The results are presented as means ± SEM (n = 6 independent experiments). Fold changes are calculated relative to the basal value. *p ≤ 0.05 vs LPS treatment, ^†p ≤ 0.05 vs basal value (two-way ANOVA followed by Bonferroni t-test.).

Figure 3

Effects of (S _S)-DS-ONJ in the activation of MAPKs and NFκB-mediated signaling in LPS-stimulated Bv.2 microglial cells. Bv.2 microglial cells were stimulated with 200 ng/mL LPS in the absence, or presence, of 10 μM (S _S)-DS-ONJ for the indicated time periods. (A) Protein extracts were separated by SDS-PAGE, and analyzed by Western blot with antibodies against phosphorylated (p)-JNK, total JNK, phosphorylated (p)-p38α MAPK, total p38α MAPK, IκBα and α-tubulin. Representative autoradiograms are shown (n = 7 independent experiments). Blots were quantified with scanning densitometry, and the results are presented as mean ± SEM. The ratios between the indicated proteins and the fold changes relative to the basal values are shown. *p ≤0.05 vs LPS treatment (two-way ANOVA followed by Bonferroni t-test). (B) Confocal immunofluorescence assessment of the nuclear translocation of p65-NFκB in Bv.2 microglial cells following stimulation with LPS in the presence or absence of (S _S)-DS-ONJ. Activation of p65-NFκB nuclear translocation was defined by an increase in immunofluorescence of p65-NFκB (green channel) in the nuclear regions. Nuclear regions of Bv.2 microglial cells were determined by counterstaining nuclear DNA with DAPI (blue channel). White arrows indicate the p65-NFκB nuclear o cytoplasmic localization.

Figure 4

Protective effects of (S _S)-DS-ONJ against LPS-mediated inflammasome activation in Bv.2 microglial cells. (A) Nlrp3 mRNA was measured by qRT-PCR. (B) Protein extracts were analyzed by Western blot, with antibodies against NLRP3; α-tubulin was used as loading control. (C) Protein extracts were analyzed by Western blot, with antibodies against caspase-1; α-tubulin was used as loading control. Representative autoradiograms are shown (n = 6 independent experiments). Blots were quantified using scanning densitometry, and the results are presented as means ± SEM. The results are presented as means ± SEM (n = 6 independent experiments). Fold changes are calculated relative to the basal values. *p ≤ 0.05 vs LPS treatment, ^†p ≤ 0.05 vs basal values (two-way ANOVA followed by Bonferroni t-test).

Figure 5

The anti-inflammatory response mediated by HO-1 and IL-10. Bv.2 microglial cells were stimulated with 200 ng/mL LPS in the presence, or absence, of 10 μM (S _S)-DS-ONJ for 24 h. (A) Il10 mRNA determined by qRT-PCR. (B) Protein extracts were analyzed by Western blot with antibodies against HO-1 and α-tubulin (loading control). Representative autoradiograms are shown. Blots were quantified using scanning densitometry. (C) Arg1 mRNA determined by qRT-PCR. The results are presented as means ± SEM. (D) Protein extracts were analyzed using Western blot, with antibodies against Arginase-1; α-tubulin was a loading control. Representative autoradiograms are presented. Blots were quantified using scanning densitometry. The results are presented as means ± SEM. Fold changes are relative to the basal values. *p ≤ 0.05 vs LPS treatment, ^†p ≤ 0.05 vs the basal values (two-way ANOVA followed by Bonferroni t-test).

Figure 6

Reactive gliosis is present in BB rats from 6 weeks of age, and treatment with (S _S)-DS-ONJ reduced reactive gliosis. (A) Analysis of GFAP and Iba-1 in retinas from BB rats during DR progression. Protein concentrations in retinal extracts were analyzed by Western blot, with α-tubulin used as loading control. Representative autoradiograms are shown. Results are presented as means ± SEM (n = 5 retinas per condition). *p ≤ 0.05 BB vs WT matched at each age. (B) (upper panels) Retinas from 6 weeks old BB and WT rats. (lower panels) Retinal explants from 6 weeks old BB rats were treated for 24 h with (S _S)-DS-ONJ (20 μM) or treatment vehicle alone. Immunostaining for GFAP (green) was performed in whole retinas. Representative images are shown (n = 5 retinas per condition).

Figure 7

Schematic representation of the anti-neuro-inflammatory mechanisms triggered by (S _S)-DS-ONJ; 1) The pro-inflammatory environment activates NF-κB in microglia cells resulting in the transcription of IL-1β; 2) The diabetic status increases the formation of NLRP3 inflammasome, promotes the cleavage of pro-caspase-1 to its active form and increases IL-1β processing and secretion. The (S _S)-DS-ONJ compound could inhibit NF-κB as well as NLRP3; 3) (S_S)-DS-ONJ induces the expression of IL-10, HO-1 and arginase-1 driving a switch of microglia polarization towards the M2 stage to counteract inflammation in microglia cells.