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NMOSD IgG Impact Retinal Cells in Murine Retinal Explants

We are happy to announce that we have recently published some of my results in an interesting article! Check out a short summary and the most important results below:

Neuromyelitis optica spectrum disorders (NMOSD) are long-lasting diseases that affect the central nervous system. They are characterized by the body's immune system mistakenly attacking a protein called aquaporin-4. This usually leads to severe eye inflammation (optic neuritis) and spinal cord inflammation (transverse myelitis). While the disease typically worsens during relapses, recent studies have shown that there is also damage happening in the retina, even when there are no relapses. This damage might be caused by the immune system's attack directly on cells in the retina. In this research study, we looked at how retinas of mice reacted when exposed to the antibodies from NMOSD patients. We found that these antibodies caused inflammation in the retina and reduced the release of certain signaling molecules. This suggests that the antibodies from NMOSD patients can directly affect the retina, potentially leading to retinal damage, even when the disease isn't actively getting worse. This could mean that NMOSD is not just a disease of the nervous system but also a disease that directly affects the retina and thereby the eyesight of the patients.


RESULTS:


Figure 1. Retinal explant morphology, complement component transcription, and chemokine secretion during in vitro cultivation. (A) Retinal morphology of freshly fixed (ex vivo) and cultivated retinas of C57BL/6J mice was assessed using a HE staining to visualize cell nuclei, extracellular matrix, and cytoplasm, respectively. Retinal thinning was observed in all layers after one and three days of cultivation compared to uncultured retinas. Cell death increased during cultivation with no fluorescence detected in the ex vivo retina. Scale bar, 50 µm. (B) Retinal transcription of complement components changed depending on the cultivation time. C1qb, c3, and cfh mRNA expression significantly increased during cultivation. Aqp4 mRNA decreased, and no trend was detectable for c1s mRNA. Bars represent mean values (n = 3) ± SD. Compared to an uncultivated control: * p < 0.05, ** p < 0.01 (ordinary one-way ANOVA, Dunnett’s multiple comparisons test). (C) Supernatants of cultivated retinas were collected at one and three days of untreated retinal cultivation and analysed for chemokines using a multiplex cytokine assay. CXCL10 secretion increased during in vitro cultivation. CCL2, CCL3, CCL4, CCL5, and CXCL2 secretion remained unchanged. GCL: Ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer. Bars represent mean values (n = 6) ± SD. * p < 0.05 (multiple paired t-tests, Holm–Sidak method).



Figure 2. Human purified NMOSD IgG bound to the mouse retina. (A) AQP4-IgG reactivity was analysed in serum of controls (n = 5) and NMOSD patients (n = 5) as well as purified IgG pools (n = 1 each). Control serum samples and the control IgG pool tested negative for AQP4-IgG, while positive anti-AQP4 IgG titers were confirmed for NMOSD patient sera and the purified NMOSD IgG pool. Bars represent mean values ± SD. * p < 0.05. (unpaired t-test assuming Gaussian distribution). (B) The Coomassie staining of NMOSD patient serum before and after immunoaffinity purification was used to verify IgG purification. (C) Mouse retina sections were incubated with serum and purified IgG from controls and NMOSD patients. Human IgG binding was confirmed for serum and purified IgG from NMOSD patients, while no binding was observed for serum and IgG from controls. Scale bar, 50 μm.



Figure 3. NMOSD IgG-enhanced Müller cell gliosis in retinal explant cultures. (A) GFAP immunoreactivity (red) was assessed by immunohistochemistry staining in retinas treated with control IgG or NMOSD IgG pool. (B) Quantification of GFAP immunoreactivity after one and three days of treatment with respective IgG fractions in retinal explant culture demonstrated increased GFAP levels in NMOSD IgG pool-treated retinas after three days of cultivation compared to control IgG-treated retinas. Bars represent mean values ± SD. (control—one day (n = 3); three days (n = 2); NMOSD—one day (n = 3); three days (n = 3)). (C) Retinal aqp4 mRNA expression was analysed after one and three days of cultivation with control IgG or with NMOSD IgG pool (one day (n = 3), three days (n = 2)). The mRNA levels were compared to untreated, cultivated retinas. No significant changes in mRNA levels were observed due to different treatments. Bars represent mean values ± SD. (two-way ANOVA with Tukey’s multiple comparisons test).



Figure 4. Mouse retinal explants treated with NMOSD IgG showed decreased release of CCL2, CCL3, CCL4, and CXCL10. Chemokine levels were assessed in the supernatants of control IgG or NMOSD IgG-treated cultivated retinas. CCL2, CCL3, CCL4, and CXCL10 were significantly reduced after one day of cultivation with NMOSD IgG compared to control IgG-treated retinas. Bars represent mean values (n = 10–11) ± SD. * p < 0.05, ** p < 0.01 (two-way ANOVA with Sidak’s multiple comparisons test).



Figure 5. The effect of NMOSD IgG on complement mRNA expression in mouse retinal explants was investigated. The mRNA levels of complement components c1qb, c1s, c3, and cfh were analysed after one and three days of cultivation with control IgG (one day n = 4, three days n = 3) or with NMOSD IgG pool (one day n = 3, three days n = 2). The mRNA levels were compared to untreated, cultivated retinas. No significant changes in mRNA levels between the different antibody treatments were observed. Bars represent mean values ± SD (two-way ANOVA with Tukey’s multiple comparisons test).


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