Abstract: P420
Type: Poster Sessions
Abstract Category: Pathology and pathogenesis of MS - Experimental models
Background: In MS, chronic activation of innate immune cells of the central nervous system (CNS) is thought to be a significant contributor to neuronal loss and disease progression. However it is unclear if innate immune activation drives or is a consequence of neuronal dysfunction and death. Furthermore, even if innate immune cells do contribute to injury, the relative importance of resident vs. infiltrating cells is uncertain. The retina exhibits neuronal injury and shows innate immune activation, and is devoid of myelin. This may permit to study an anatomically separate “gray matter” structure, capable of disentangling the effects of innate immune activation from directly proximate damage to myelin as is seen in the cortex. It also provides a means to study the direct localized effect of innate immune activation on neuronal function and health.
Objective: To characterize the timing of innate immune activation in the retina during EAE.
Methods: We used confocal scanning laser ophthalmoscopy (CSLO) to monitor changes in the density and morphology of retinal innate immune cells in 6 adult CX3CR1 +/- GFP mice during two months after direct immunization against MOG35-55. We scored EAE severity daily. Mice were then sacrificed and surviving ganglion cells (GC) were counted through Brn3a staining of retinal flat-mounts. In a separate study, we performed single-cell RNAseq of retinal CD11b+ cells sorted by flow cytometry 6 days after immunization (n=25 immunized vs. 25 healthy controls), using canonical markers to distinguish between microglia, macrophages, NK cells and circulating monocytes.
Results: Density of retinal CX3CR1+ cells increased progressively during the first weeks after immunization, reaching a two-fold change compared to baseline levels around day 30, slowly decreasing thereafter. Morphology changes were consistent with immune activation. Mice with more severe EAE had a higher peak of retinal CX3CR1+ cell density (r2 = 0.48; P=0.003), which was also associated with worse GC loss (r2= 0.29; P=0.031). Transcriptomic profiling revealed activation of retinal microglia and macrophages at day 6 after immunization, before any clinical signs were observed.
Conclusions: Retinal innate immune activation is local to the site of neuronal loss, and precedes clinical onset in EAE. Flow cytometry and single-cell RNAseq can be used to distinguish cell subsets and identify pathways that are activated during EAE and that contribute to neuronal loss.
Disclosure: Cruz-Herranz A: Research support from the National Multiple Sclerosis Society (Postdoctoral Fellowship), UCSF Program for Breakthrough Biomedical Research (Postdoctoral Independent Research Award), and the Conrad N. Hilton Foundation (Pilot Innovator Award).
Kim K: nothing to disclose
Cantó E: nothing to disclose
Cordano C: Research support from the Fondazione Italiana Sclerosi Multipla (Research Fellowship).
Timmons G: nothing to disclose
Sin JH: nothing to disclose
Devereux M: nothing to disclose
Baker N: nothing to disclose
Michel B: nothing to disclose
Schubert R: Research support from the American Academy of Neurology-National Multiple Sclerosis Society (Clinician Scientist Development Award)
Rani L: nothing to disclose
Baranzini S: Research support from the National Institutes of Health (R01 NS088155)
Green AJ: Research support from the National Multiple Sclerosis Society (Harry Weaver Neurosciences Scholar Award), Novartis, UCSF CTSI, That Man May See, and the Conrad N. Hilton Foundation (Pilot Innovator Award); personal fees from Inception Sciences and Mylan Pharmaceuticals; philanthropic support from the Rachleff Family and the Robert Dale Family. He also reports serving on an end point adjudication committee for Biogen and Medimmune. He serves on trial steering committees for Novartis and Scientific Advisory Board for Bionure
Abstract: P420
Type: Poster Sessions
Abstract Category: Pathology and pathogenesis of MS - Experimental models
Background: In MS, chronic activation of innate immune cells of the central nervous system (CNS) is thought to be a significant contributor to neuronal loss and disease progression. However it is unclear if innate immune activation drives or is a consequence of neuronal dysfunction and death. Furthermore, even if innate immune cells do contribute to injury, the relative importance of resident vs. infiltrating cells is uncertain. The retina exhibits neuronal injury and shows innate immune activation, and is devoid of myelin. This may permit to study an anatomically separate “gray matter” structure, capable of disentangling the effects of innate immune activation from directly proximate damage to myelin as is seen in the cortex. It also provides a means to study the direct localized effect of innate immune activation on neuronal function and health.
Objective: To characterize the timing of innate immune activation in the retina during EAE.
Methods: We used confocal scanning laser ophthalmoscopy (CSLO) to monitor changes in the density and morphology of retinal innate immune cells in 6 adult CX3CR1 +/- GFP mice during two months after direct immunization against MOG35-55. We scored EAE severity daily. Mice were then sacrificed and surviving ganglion cells (GC) were counted through Brn3a staining of retinal flat-mounts. In a separate study, we performed single-cell RNAseq of retinal CD11b+ cells sorted by flow cytometry 6 days after immunization (n=25 immunized vs. 25 healthy controls), using canonical markers to distinguish between microglia, macrophages, NK cells and circulating monocytes.
Results: Density of retinal CX3CR1+ cells increased progressively during the first weeks after immunization, reaching a two-fold change compared to baseline levels around day 30, slowly decreasing thereafter. Morphology changes were consistent with immune activation. Mice with more severe EAE had a higher peak of retinal CX3CR1+ cell density (r2 = 0.48; P=0.003), which was also associated with worse GC loss (r2= 0.29; P=0.031). Transcriptomic profiling revealed activation of retinal microglia and macrophages at day 6 after immunization, before any clinical signs were observed.
Conclusions: Retinal innate immune activation is local to the site of neuronal loss, and precedes clinical onset in EAE. Flow cytometry and single-cell RNAseq can be used to distinguish cell subsets and identify pathways that are activated during EAE and that contribute to neuronal loss.
Disclosure: Cruz-Herranz A: Research support from the National Multiple Sclerosis Society (Postdoctoral Fellowship), UCSF Program for Breakthrough Biomedical Research (Postdoctoral Independent Research Award), and the Conrad N. Hilton Foundation (Pilot Innovator Award).
Kim K: nothing to disclose
Cantó E: nothing to disclose
Cordano C: Research support from the Fondazione Italiana Sclerosi Multipla (Research Fellowship).
Timmons G: nothing to disclose
Sin JH: nothing to disclose
Devereux M: nothing to disclose
Baker N: nothing to disclose
Michel B: nothing to disclose
Schubert R: Research support from the American Academy of Neurology-National Multiple Sclerosis Society (Clinician Scientist Development Award)
Rani L: nothing to disclose
Baranzini S: Research support from the National Institutes of Health (R01 NS088155)
Green AJ: Research support from the National Multiple Sclerosis Society (Harry Weaver Neurosciences Scholar Award), Novartis, UCSF CTSI, That Man May See, and the Conrad N. Hilton Foundation (Pilot Innovator Award); personal fees from Inception Sciences and Mylan Pharmaceuticals; philanthropic support from the Rachleff Family and the Robert Dale Family. He also reports serving on an end point adjudication committee for Biogen and Medimmune. He serves on trial steering committees for Novartis and Scientific Advisory Board for Bionure