This preprint is under consideration at Journal of Neuroinflammation. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Small-Molecule Antagonist of VLA-4 (GW559090) Attenuated Neuro-Inflammation by Targeting Th17 Cell Trafficking across the Blood-Retinal Barrier in Experimental Autoimmune Uveitis
Virginia Calder
University College London
Corresponding Author
ORCiD: https://orcid.org/0000-0003-1422-2926
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: The integrin VLA-4 (α4β1) plays an important role in leukocyte trafficking. This study investigated the efficacy of a novel topical α4β1 integrin inhibitor (GW559090, GW) in a mouse model for non-infectious posterior uveitis (experimental autoimmune uveitis; EAU) and its effect on intraocular leukocyte subsets.
Methods: Mice (female; B10.RIII or C57Bl/6; aged 6-8 weeks) were immunized with specific interphotoreceptor retinoid-binding protein (IRBP) peptides to induce EAU. Topically administered GW (3, 10, and 30 mg/ml) were given twice daily either therapeutically once disease was evident, or prophylactically, and compared with vehicle-treated (Veh) and 0.1% dexamethasone-treated (Dex) controls. Mice were sacrificed at peak disease. The retinal T cell subsets were investigated by immunohistochemistry and immunofluorescence staining. The immune cells within the retina, blood and draining lymph nodes (dLNs) were phenotyped by flow cytometry. The effect of GW559090 on non-adherent, adherent and migrated CD4+ T cell subsets across a central nervous system (CNS) endothelium was further assayed in vitro and quantitated by flow cytometry.
Results: There was a significant reduction in clinical and histological scores in GW10 and Dex treated groups as compared to controls either administered therapeutically or prophylactically. There were fewer CD45+ leukocytes infiltrating the retinae and vitreous fluids in the treated GW10 group (P < 0.05). Immunofluorescence staining and flow cytometry data identified decreased levels of retinal Th17 cells (P ≤ 0.001) in the GW10 treated eyes, leaving systemic T cell subsets unaffected. In addition, fewer Ly6C+ inflammatory monocyte/macrophages (P = 0.002) and dendritic cells (P = 0.017) crossed the BRB following GW10 treatment. In vitro migration assays confirmed that Th17 cells were selectively suppressed by GW559090 by adhering to endothelial monolayers.
Conclusions: This α4β1 integrin inhibitor may exert a modulatory effect in EAU progression by selectively blocking Th17 cell migration across the blood-retinal barrier without affecting systemic CD4+ T cell subsets. Local α4β1 integrin-directed inhibition could be clinically relevant in treating a Th17-dominant form of uveitis.
Keywords
Integrin (α4β1, VLA-4), uveitis, experimental autoimmune uveitis, leukocyte migration, Th17 cells, inflammatory monocytes/macrophages
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
- 20200725a4b1PaperFigureDraftsFig1.jpg
Supplementary Fig.1 a Representative gating strategy to identify CD4+ T cell subsets in the retina for Fig.3. CD4+ T cells were first gated for single cells and live cells, and then for their expression of CD4. Within the CD4+ cell region, Th1 and Th17 were distinguished by intracellular expression of IFNγ and IL-17A respectively, within the FoxP3- region. b Representative gating strategy to identify myeloid cell subsets within the retina for Fig 4. Single live cells were gated for CD45 firstly, CD11b and CD11c, then CD64, Ly6G, and finally Ly6C.
- 20200725a4b1PaperFigureDraftsFig2.jpg
Supplementary Fig. 2 Representative flow cytometry figure depicting VLA-4 expression in different CD4+ T cell subsets in healthy control (Ctrl) and EAU eye.
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Revision
Version 1
Posted 24 Sep, 2020
No community comments so far
Editorial decision: Major Revision
On 26 Oct, 2020
Review #2 received
Received 21 Oct, 2020
Review #1 received
Received 14 Oct, 2020
Reviewer #2 agreed
On 09 Oct, 2020
Reviewer #1 agreed
On 29 Sep, 2020
Reviewers invited
Invitations sent on 21 Sep, 2020
First submitted
On 20 Sep, 2020
Editor assigned
On 20 Sep, 2020
Submission checks complete
On 19 Sep, 2020
Editor invited
On 19 Sep, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Learn more about our company.
This preprint is under consideration at Journal of Neuroinflammation. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Small-Molecule Antagonist of VLA-4 (GW559090) Attenuated Neuro-Inflammation by Targeting Th17 Cell Trafficking across the Blood-Retinal Barrier in Experimental Autoimmune Uveitis
Virginia Calder
University College London
Corresponding Author
ORCiD: https://orcid.org/0000-0003-1422-2926
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Revision
Version 1
Posted 24 Sep, 2020
No community comments so far
Editorial decision: Major Revision
On 26 Oct, 2020
Review #2 received
Received 21 Oct, 2020
Review #1 received
Received 14 Oct, 2020
Reviewer #2 agreed
On 09 Oct, 2020
Reviewer #1 agreed
On 29 Sep, 2020
Reviewers invited
Invitations sent on 21 Sep, 2020
First submitted
On 20 Sep, 2020
Editor assigned
On 20 Sep, 2020
Submission checks complete
On 19 Sep, 2020
Editor invited
On 19 Sep, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: The integrin VLA-4 (α4β1) plays an important role in leukocyte trafficking. This study investigated the efficacy of a novel topical α4β1 integrin inhibitor (GW559090, GW) in a mouse model for non-infectious posterior uveitis (experimental autoimmune uveitis; EAU) and its effect on intraocular leukocyte subsets.
Methods: Mice (female; B10.RIII or C57Bl/6; aged 6-8 weeks) were immunized with specific interphotoreceptor retinoid-binding protein (IRBP) peptides to induce EAU. Topically administered GW (3, 10, and 30 mg/ml) were given twice daily either therapeutically once disease was evident, or prophylactically, and compared with vehicle-treated (Veh) and 0.1% dexamethasone-treated (Dex) controls. Mice were sacrificed at peak disease. The retinal T cell subsets were investigated by immunohistochemistry and immunofluorescence staining. The immune cells within the retina, blood and draining lymph nodes (dLNs) were phenotyped by flow cytometry. The effect of GW559090 on non-adherent, adherent and migrated CD4+ T cell subsets across a central nervous system (CNS) endothelium was further assayed in vitro and quantitated by flow cytometry.
Results: There was a significant reduction in clinical and histological scores in GW10 and Dex treated groups as compared to controls either administered therapeutically or prophylactically. There were fewer CD45+ leukocytes infiltrating the retinae and vitreous fluids in the treated GW10 group (P < 0.05). Immunofluorescence staining and flow cytometry data identified decreased levels of retinal Th17 cells (P ≤ 0.001) in the GW10 treated eyes, leaving systemic T cell subsets unaffected. In addition, fewer Ly6C+ inflammatory monocyte/macrophages (P = 0.002) and dendritic cells (P = 0.017) crossed the BRB following GW10 treatment. In vitro migration assays confirmed that Th17 cells were selectively suppressed by GW559090 by adhering to endothelial monolayers.
Conclusions: This α4β1 integrin inhibitor may exert a modulatory effect in EAU progression by selectively blocking Th17 cell migration across the blood-retinal barrier without affecting systemic CD4+ T cell subsets. Local α4β1 integrin-directed inhibition could be clinically relevant in treating a Th17-dominant form of uveitis.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5