Elevation of granulocyte colony stimulating factor in human AMD donor RPE-choroid
Elevation of granulocyte colony stimulating factor in human AMD donor RPE-choroid
Purpose: choroidal inflammation, complement deposition, and accumulation of C-reactive protein (CRP) are involved in age-related macular degeneration (AMD) pathology. The pro-inflammatory signals that regulate immune cell recruitment in the choroid of patients with AMD remain to be determined. We performed cytokine profiling of human AMD and age-matched control donor tissue to identify inflammatory molecules upregulated in AMD tissue.
Methods: protein was isolated from 25 AMD and 21 control donor RPE/choroid macular punches. Total protein was quantified, and 50 µg assayed for expression of 40 cytokines using an inflammation array. We validated the elevated expression of granulocyte colony stimulating factor (G-CSF) protein by ELISA in a second cohort of 22 control and 26 AMD donors. To identify an AMD associated stressor responsible for upregulating G-CSF we assayed for changes in G-CSF protein secretion in RPE/choroid organ cultures treated with the monomeric (m)CRP, an inflammatory protein elevated in AMD.
Results: using a multiplex array, we identified elevated G-CSF protein in the choroid of AMD donors compared to age-matched non-AMD controls. Differential expression of G-CSF was confirmed via ELISA in an independent cohort of samples (P = 0.01). The mCRP, which is deposited in AMD choroids, increased G-CSF protein secretion in RPE/choroid organ cultures. Single nuclei RNA sequencing identified choroidal endothelial cells and fibroblasts as the primary cell types responsible for increased G-CSF secretion in response to mCRP. The G-CSF receptor is expressed primarily by choroidal macrophages and dendritic cells and anti-G-CSFR colocalizes with anti-CD45 and anti-CD68 in human donor choroid tissue.
Conclusions: elevated G-CSF expression in AMD donor tissue as a result of increased levels of mCRP may be involved in immune cell recruitment in AMD contributing to inflammatory stress in the choroid.
C-reactive protein (CRP), age-related macular degeneration (AMD), cytokine, granulocyte colony stimulating factor (G-CSF), inflammation
Mulfaul, Kelly
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Khan, Adnan H.
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Schwarte, Samantha G.
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Voigt, Andrew P.
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Moore, Rachel F.
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Potempa, Lawrence A.
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Wang, Kai
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Scheetz, Todd E.
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Stone, Edwin M.
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Tucker, Budd A.
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Mullins, Robert F.
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6 December 2024
Mulfaul, Kelly
eb0881cd-ec81-4975-a953-4a787150baa8
Khan, Adnan H.
97374057-d7e7-4849-ac94-c125ba1cc360
Schwarte, Samantha G.
0a06e45c-f5d8-4e3e-bcae-395a57856fb2
Voigt, Andrew P.
1675c6c3-6729-465e-99da-471cced0fa7a
Moore, Rachel F.
0cdfb3b5-5fdd-4f70-8070-5759803a8961
Potempa, Lawrence A.
a986f0a4-d7d4-4b83-8777-c32513b2ec04
Wang, Kai
507ac082-3082-448d-985c-e40fe90d30cc
Scheetz, Todd E.
b3725b17-a60f-4451-a092-44021b8af440
Stone, Edwin M.
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Tucker, Budd A.
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Mullins, Robert F.
3178c5eb-7b57-411f-9e0f-4312b890d26a
Mulfaul, Kelly, Khan, Adnan H., Schwarte, Samantha G., Voigt, Andrew P., Moore, Rachel F., Potempa, Lawrence A., Wang, Kai, Scheetz, Todd E., Stone, Edwin M., Tucker, Budd A. and Mullins, Robert F.
(2024)
Elevation of granulocyte colony stimulating factor in human AMD donor RPE-choroid.
Investigative Ophthalmology & Visual Science, 65 (14), [15].
(doi:10.1167/iovs.65.14.15).
Abstract
Purpose: choroidal inflammation, complement deposition, and accumulation of C-reactive protein (CRP) are involved in age-related macular degeneration (AMD) pathology. The pro-inflammatory signals that regulate immune cell recruitment in the choroid of patients with AMD remain to be determined. We performed cytokine profiling of human AMD and age-matched control donor tissue to identify inflammatory molecules upregulated in AMD tissue.
Methods: protein was isolated from 25 AMD and 21 control donor RPE/choroid macular punches. Total protein was quantified, and 50 µg assayed for expression of 40 cytokines using an inflammation array. We validated the elevated expression of granulocyte colony stimulating factor (G-CSF) protein by ELISA in a second cohort of 22 control and 26 AMD donors. To identify an AMD associated stressor responsible for upregulating G-CSF we assayed for changes in G-CSF protein secretion in RPE/choroid organ cultures treated with the monomeric (m)CRP, an inflammatory protein elevated in AMD.
Results: using a multiplex array, we identified elevated G-CSF protein in the choroid of AMD donors compared to age-matched non-AMD controls. Differential expression of G-CSF was confirmed via ELISA in an independent cohort of samples (P = 0.01). The mCRP, which is deposited in AMD choroids, increased G-CSF protein secretion in RPE/choroid organ cultures. Single nuclei RNA sequencing identified choroidal endothelial cells and fibroblasts as the primary cell types responsible for increased G-CSF secretion in response to mCRP. The G-CSF receptor is expressed primarily by choroidal macrophages and dendritic cells and anti-G-CSFR colocalizes with anti-CD45 and anti-CD68 in human donor choroid tissue.
Conclusions: elevated G-CSF expression in AMD donor tissue as a result of increased levels of mCRP may be involved in immune cell recruitment in AMD contributing to inflammatory stress in the choroid.
Text
i1552-5783-65-14-15_1733470335.62769
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Accepted/In Press date: 12 November 2024
Published date: 6 December 2024
Keywords:
C-reactive protein (CRP), age-related macular degeneration (AMD), cytokine, granulocyte colony stimulating factor (G-CSF), inflammation
Identifiers
Local EPrints ID: 497036
URI: http://eprints.soton.ac.uk/id/eprint/497036
ISSN: 0146-0404
PURE UUID: 74e02ce0-c170-41af-b1a7-04b81467c519
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Date deposited: 10 Jan 2025 17:42
Last modified: 22 Aug 2025 02:26
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Contributors
Author:
Kelly Mulfaul
Author:
Samantha G. Schwarte
Author:
Andrew P. Voigt
Author:
Rachel F. Moore
Author:
Lawrence A. Potempa
Author:
Kai Wang
Author:
Todd E. Scheetz
Author:
Edwin M. Stone
Author:
Budd A. Tucker
Author:
Robert F. Mullins
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