Ocular Graft-versus-Host Disease in a Chemotherapy-Based Minor-Mismatch Mouse Model Features Corneal (Lymph-) Angiogenesis

Abstract

Ocular graft-versus-host disease (oGVHD) is a fast progressing, autoimmunological disease following hematopoietic stem cell transplantation, leading to severe inflammation of the eye and destruction of the lacrimal functional unit with consecutive sight-threatening consequences. The therapeutic "window of opportunity" is narrow, and current treatment options are limited and often insufficient. To achieve new insights into the pathogenesis and to develop new therapeutic approaches, clinically relevant models of oGVHD are desirable. In this study, the ocular phenotype was described in a murine, chemotherapy-based, minor-mismatch GVHD model mimicking early-onset chronic oGVHD, with corneal epitheliopathy, inflammation of the lacrimal glands, and blepharitis. Additionally, corneal lymphangiogenesis was observed as part of oGVHD pathogenesis for the first time, thus opening up the investigation of lymphangiogenesis as a potential therapeutic and diagnostic tool.

Keywords: blepharitis; chemotherapy; lymphangiogenesis; oGVHD; ocular graft-versus-host-disease; pre-clinical model.

Figure 1

Systemic phenotype after chemotherapy and either syngeneic (B6-B6) or allogeneic (129s/B6) BMT, respectively. GVHD score contained weight loss, fur and skin condition, posture, and overall activity. (A) Allogeneic transplanted mice demonstrated a significant GVHD burden. Exemplary macroscopic images showing typical hunched back (upper right photo) and scaled skin of allogeneic GVHD mice (lower right photo; see arrows) compared to baseline. (B) Flow cytometry analysis of eye-draining lymph nodes. Compared to syngeneic controls allogeneic mice showed a significantly higher percentage of CD8+ T cells and CD4+ T cells from day 14 after BMT. (* p ≤ 0.05, *** p < 0.0001).

Figure 2

Ocular phenotype after chemotherapy and either syngeneic (B6-B6) or allogeneic (129-B6) BMT, respectively. (A) Corneal epitheliopathy increased in allogeneic mice 2 weeks after BMT compared to baseline and was significantly worse compared to syngeneic controls at each time point after BMT. Syngeneic mice developed no epitheliopathy after BMT. (B) Tear production remained almost constant in both groups until the end of the observation period at d28 after BMT. A minor increase of tears was observed in allogeneic mice compared to baseline 7 days after BMT and compared to syngeneic controls at days 7 and 14 after BMT. (C,D) Blepharitis score (including lid edema and loss of fur, see (D) exemplary photos) was significantly increased in allogeneic mice compared to syngeneic mice three weeks after BMT. Blepharitis score remained at the baseline level in syngeneic mice. (E,F) Immunohistochemistry of frozen tissue sections of eyelids at day 21 after BMT. Allogeneic mice present distinct inflammatory infiltration of CD8+ (E) and CD4+ (F) T cells in the lids and conjunctival tissues (see inlays) (tConj/bConj = tarsal/bulbar conjunctiva; * p ≤ 0.05, ** p < 0.001, *** p < 0.0001).

Figure 3

Effect of chemotherapy conditioning followed by either syngeneic (B6-B6) or allogeneic (129s-B6) BMT on extra-orbital lacrimal glands. (A) Flow cytometry analysis of ELG tissue. A significant infiltration with CD45+ leucocytes was observed in both groups compared to naïve mice. Allogeneic mice developed more pronounced CD45+ infiltration compared to syngeneic control mice. In allogeneic mice, the numbers of CD8+ and CD4+ cells were significantly increased compared to syngeneic mice. (B) Gating strategy. After excluding dead cells and doublets, viable cells were gated on CD45+CD3+ cells, and the number of CD8+ and CD4+ T cells was calculated. (C) Immunohistochemistry of frozen sections. The number of positive cells was counted manually and calculated per mm². The results confirmed significant infiltration with CD8+ and CD4+ T cells in ELG tissue of allogeneic mice. In allogeneic mice, a higher number of CD8+ and CD4+ cells per mm² was found at d14 and d21 after BMT (* p ≤ 0.05).

Figure 4

Corneal hem- and lymphangiogenesis in oGVHD. (A) Exemplary corneal flat-mount samples of naïve, syngeneic, and allogeneic transplanted mice 21 days after BMT (CD31 = green, Lyve1 = red; bar = 1 mm). (B) Average area covered by Lyve1+ lymphatics and CD31+ blood vessels in percent of the total cornea. 21–28 days after BMT, a significant increase of lymphatics was observed in allogeneic mice compared to syngeneic control. No differences in corneal blood vessels were observed. (C) Corneal hem- and lymphangiogenesis was correlated to the systemic GVHD score and the corneal fluorescein score (FL). (D,E) VEGF-C expression in the cornea (D) and conjunctiva (E) normalized to naïve controls. In the cornea, but not in the conjunctiva, the VEGF-C expression was increased in allogeneic mice compared to syngeneic control 14 days after BMT (* p ≤ 0.05).