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. 2015 Apr 15;194(8):4029-38.
doi: 10.4049/jimmunol.1401922. Epub 2015 Mar 9.

Anti-IL-17 therapy restricts and reverses late-term corneal allorejection

Xiao-Tang Yin  1 , Stephanie Zobell  1 , Jason G Jarosz  1 , Patrick M Stuart  2
Affiliations

Anti-IL-17 therapy restricts and reverses late-term corneal allorejection

Xiao-Tang Yin et al. J Immunol. .

Abstract

Corneal allograft rejection has been described as a Th1-mediated process involving IFN-γ production. However, recent evidence also implicated IL-17 as being involved in acute corneal allograft responses. Our data support that IL-17 is involved in early acute corneal allograft acceptance. However, we decided to extend these studies to include a later phase of rejection in which there is a peak of IL-17 production that is >15-fold higher than that seen during acute rejection and occurs >45 d postengraftment at the onset of late-term rejection. We demonstrate that neutralizing IL-17A at this time significantly reduced corneal graft rejection. Surprisingly, when corneal grafts that are undergoing this later phase of rejection are treated with anti-IL-17A, there is a reversal of both opacity and neovascularization. Compared with the early phase of rejection, the cellular infiltrate is significantly less, with a greatly reduced presence of Gr-1(+) neutrophils and a relative increase in CD4(+) T cells and macrophages. We went on to identify that the cells expressing IL-17 were CD4(+) IL-17(+) T cells and, somewhat surprisingly, IL-17(+) F4/80(+) macrophages within the rejecting corneal allografts. Taken together, these findings describe a distinct late phase of corneal allograft rejection that is likely mediated by Th17 cells; therapeutic neutralization of IL-17A reverses this rejection. This further suggests that IL-17 might serve as an excellent therapeutic target to reduce this form of corneal allograft rejection.

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Conflict of interest statement

The authors have no conflict of interest with this work.

Figures

Figure 1
Figure 1
Multiplex cytokine analysis of engrafted corneas. The corneas of BALB/c mice engrafted with B6 allogeneic corneas were removed at various time points including time points shortly following corneal engraftment when only a few corneas show any evidence of inflammation (3, 5 and 7 days post-engraftment), during subclinical rejection and time points when clinical rejection is apparent and corneal lysates were made. These corneal lysates were analyzed with a multiplex cytokine array. Average cytokine concentrations determined from at least four mice/time point are reported as pg/ml (y axis). The cytokines measured were IL-17 (A), IL-6 (B) and IFN- γ (C) and values indicate mean ± SEM of IL-17, IL-6 and IFN- γ.
Figure 2
Figure 2
Neutralization of IL-17A during acute corneal allograft decreases allograft survival. BALB/c mice were engrafted with B6 corneas and then injected S.C. with either control Ig (n=12) or anti-IL-17A (n=12) and observed for 30 days. Mice receiving anti-IL-17A displayed reduced corneal allograft survival (P<0.01).
Figure 3
Figure 3
Late-term corneal allograft rejection appears to be very similar to that seen in acutely rejected corneal allografts. Eyes of mice were photographed using a vertical slit-lamp during acute allografts rejection (A), allograft acceptance (B), or late-term allograft rejection (C).
Figure 4
Figure 4
Prophylactic neutralization of IL-17A beginning at Day 40 significantly increased corneal allograft survival. Mice bearing clear corneas at Day 40 were treated with anti-mouse IL-17A mAb (n=6) or control isotype IgG (n=10) at a dose of 30mg/kg, twice weekly for two weeks by SC injection. Corneal allograft survival for mice treated with anti-IL-17A during late-term rejection were enhanced compared to mice treated with control antibody (p<0.05).
Figure 5
Figure 5
Therapeutic neutralization of IL-17A during late-term rejection for mice demonstrating rejection of donor corneas reversed both opacity (A) and neovascularization (B). Representative mice displaying the corneas appearance at the start of treatment and then after treatment were photographed using a vertical slit-lamp (C). Mice were treated with anti-IL-17A antibody or control antibody by twice weekly injections for three weeks beginning at the point when grafted corneas were displaying signs of late-term rejection (first photo of C). Treatment was initiated between day 45 and day 60 post-grafting depending on when the corneal allograft showed signs of rejection. The subsequent data is expressed as a consequence of when treatment began. Results are expressed as mean opacity (A) or mean neovascularization (B) ± SEM. The opacity was compared using ANOVA analysis and asterisk indicates significant differences (P< 0.01–0.001). Neovascularization was analyzed by unpaired 2-tailed Student t test indicating significant differences between the two groups (p<0.05).
Figure 6
Figure 6
Histological sections indicate that acute corneal allograft rejection has significantly greater numbers of inflammatory cells than corneas from mice undergoing late-term corneal allograft rejection. Hematoxylin and eosin stain of corneal sections are from acute rejection (day 17 post-engraftment), late-term rejection (day 65 post-engraftment), accepted corneal allograft (day post-engraftment), and an ungrafted B6 cornea (B). (original magnification × 20).
Figure 7
Figure 7
Inflammatory infiltrate from acute and late-term rejection are not equivalent. Rejected corneas were removed at days 18 (acute rejection, n=4) or day 65 (late-term rejection, n=6) and disaggregated into single-cell suspensions and stained with anti-CD45. The CD45+ cells were gated and further analyzed for staining with antibodies directed against CD3, CD4, Gr-1, CD11b, and F4/80 surface markers. Cells were analyzed by flow cytometry. Late-term rejected corneas had a much smaller infiltrate of Gr-1+ neutrophils than did acutely rejected corneas (p< 0.01).
Figure 8
Figure 8
Inflammatory infiltrate from acute and late-term rejection do not express equivalent numbers of IL-17 expressing cells. Rejected corneas were removed at days 18 (acute rejection, n=5) or days 55 – 65 (late-term rejection, n=5) and disaggregated into single-cell suspensions and stained with anti-CD45. The CD45+ cells were gated and further analyzed for staining with antibodies directed against IL-17A and IFNγ. Cells were analyzed by flow cytometry. Late-term rejected corneas contained a much greater number of IL-17A expressing cells than did acutely rejected corneas (p< 0.05).
Figure 9
Figure 9
IL-17A is produced by CD4+ T cells. Sections were stained with CD4, Gr-1 or CD11b and each were then stained for IL-17A. DAPI was used to counterstain the nuclei. Results are only shown for CD4 (100X) as Gr-1 and CD11b did not demonstrate convincing co-localization of IL-17A.
Figure 10
Figure 10
IL-17A is produced by F4/80+ macrophages. Sections were stained with F4/80 and then stained for IL-17A. DAPI was used to counterstain the nuclei. Results are only shown for F4/80 (40X).
Figure 11
Figure 11
Flow cytometric analysis confirms that both CD4+ and F4/80+ cells produce IL-17A and that no cells express both at the same time. A. Cells isolated from the indicated corneas were stained with CD45, then CD4 and permeabilized and further stained for IL-17A and IFN-γ. Figure displays a series of representative dot plots where the CD45+CD4+F4/80− cells were analyzed for IL-17A and IFN-γ expression. Figure displays a representative dot-plot of cells gated first for CD45+ cells and then those gated for CD4+ cells and assayed for IL-17A and IFN-γ expression. B. Representative plot of corneal inflammatory cells that were analyzed for CD45 and IL-17A expression to visualize the CD45+ cells that express IL-17A. C. The IL-17A+ cells shown in part B were further analyzed for CD4 and F4/80 expression. This demonstrates that there are both CD4+ T cells that express IL-17A and F4/80+ cells that express IL-17A. D. Graphic representation of cells isolated from corneas undergoing late-term rejection that were stained with surface markers for CD4, F4/80 and CD1d tetramer and then permeabilized and stained with either IL-17A or IFN-γ. Data show that no CD4+ cells were producing both IL-17A and IFN-γ, they also demonstrate significantly more CD4+IL-17A+ cells than CD4+IFNγ+ cells (P<0.01). No F4/80+IFNγ+ or CD1d tetramer+ cells were detected.
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