Immunopathological Analysis of a Mouse Model of Arthritis-Associated Scleritis and Implications for Molecular Targeted Therapy for Severe Scleritis

Abstract

Scleritis involves inflammation of the sclera, which constitutes 75% of the wall of the eye. This pathology is often seen as an ocular lesion associated with systemic inflammatory diseases. Severe types of scleritis such as posterior scleritis require urgent immunosuppressive treatments, including molecularly targeted therapies to avoid permanent visual impairment. Which molecules should be selected as targets has remained unclear. To clarify the pathogenesis of scleritis and propose appropriate target molecules for therapy, we have established novel animal model of scleritis by modifying the Collagen-II Induced Arthritis (CIA) model. Immunization twice with collagen II emulsified with complete Freund's adjuvant (CFA) caused arthritis and scleritis. The clinical appearance resembled human diffuse scleritis. Histopathological analysis suggested that macrophages, plasma cells, deposition of immune complexes, and growth of blood and lymphatic vessels are involved in the pathogenesis of CIA-associated scleritis. In addition, we analysed the background diseases of posterior scleritis and responses to molecularly targeted therapies as a case series study. We inferred from both the animal model and case series study that targets should not be T cells, but factors inhibiting macrophage activity such as tumor necrosis factor (TNF) and interleukin (IL)-6, and molecules suppressing antibody-producing cells such as CD20 on B cells should be targeted by molecularly targeted therapies.

Keywords: CTLA4Ig; IL-6 inhibitors; TNF inhibitors; animal model; anti-CD20; biologic agent; collagen-II induced arthritis; molecular targeted therapy; posterior scleritis; scleritis.

Figure 1

Clinical appearance of arthritis and scleritis in CIA-scleritis model. DBA/1J mice were immunized intradermally at the back neck with bovine 200µg of CII emulsified with CFA. On day 21, the mice were boosted by intradermal injection with 200µg of bovine CII emulsified with CFA around the eye. Clinical appearance of arthritis (a) and scleritis (b) at 3weeks of the 2nd immunization is shown. Normal eye of DBA/1J mice is shown as control (c).

Figure 2

Infiltration of inflammatory cells into the sclera in CIA-scleritis model. The eyes of CIA-scleritis model were removed at 3–12 weeks after 2nd immunization. Cryostat sections of the eyes were stained with hematoxylin and eosin. Normal DBA/1J was used as control. “Scl” and “I-CB” denote sclera and Iris ciliary body, respectively. Original magnification, ×40 (a). The number of Infiltrating cells in scleral sections were counted (b). Data are the mean ± standard deviation of 3–6 eyes in each time point and were statistically compared with control (naïve) using the two-tailed Student’s t test (* p < 0.05, ** p < 0.001) (b). Arthritis was evaluated by Arthritis score for CIA model as below: 0 = Normal, 1 = Swelling of one digit, 2 = Swelling of two digits or more or swelling of the ankle or wrist, 3 = sever swelling of the entire paw (c).

Figure 3

Infiltration of CD11b⁺, CD4⁺, CD8⁺, CD11c⁺, B220⁺, and CD138⁺ cells into the sclera in CIA-scleritis model. The eyes of CIA-scleritis model were removed at 3–8 weeks after 2nd immunization. Cryostat sections of the eyes were stained with FITC- or PE-conjugated anti-CD4, CD8, CD11b, CD11c, B220, or CD138 mAb. Nuclei were stained with DAPI. Normal DBA/1J was used as control. “Scl” and “I-CB” denote sclera and iris ciliary body, respectively. Original magnification, ×40.

Figure 4

Expression of C3, IgM, IgG, CD31 and LYVE-1 in the sclera in CIA-scleritis model. The eyes of CIA-scleritis model were removed at 3–8 weeks after 2nd immunization. Cryostat sections of the eyes were stained with FITC-, PE-, or biotin-conjugated anti-C3, IgM, IgG, CD31, or LYVE-1 Ab. This was followed by staining with streptavidin-APC. Nuclei were stained with DAPI. Normal DBA/1J was used as control. “Scl” and “I-CB” denote sclera and iris ciliary body, respectively. Original magnification, ×40.

Figure 5

Case 1: Colour fundus photograph, OCT, and FFA of the right eye. (a) Colour fundus montage photograph of the right eye shows subretinal fluid and multiple scattered, discrete areas of choroiditis. (b) OCT of the right eye shows extensive SRD and a chorioretinal fold. (c) FFA of the right eye in the early phase shows subretinal granular hyperfluorescence. (d) FFA of the right eye in the late phase shows strong subretinal leakage and accumulation with extensive SRD.

Figure 6

Case 1: OCT of the right eye. (a) Posterior scleritis quickly disappeared after starting pulsed steroid therapy. (b) Posterior scleritis relapsed after tapering to 5 mg/day of PSL. (c) The dose of PSL was increased to 20 mg/day and 100 mg/day of CysA was added with once remission was achieved. (d) While taking 10 mg/day of PSL and 175 mg/day of CysA, scleritis relapsed. (e) After introducing ADA, the patient has remained in remission.

Figure 7

Case 2: Clinical appearance of anterior diffuse scleritis and OCT in the right eye. (a) Clinical appearance of anterior diffuse scleritis in the right eye at the first visit. (b) Scleritis persisted for about 6 months even after topical treatment of eye drops and SCTA. (c,d) One week after initiating CTLA4Ig, scleritis in the right eye worsened and macular oedema developed with an alleged paradoxical reaction. (e) After introducing GLM, scleritis rapidly disappeared within 1 month.

Figure 8

Case 2: Clinical appearance of anterior diffuse scleritis, FFA and OCT in the right eye (a–c) Scleritis relapsed, and the dose of GLM was doubled, but diffuse scleritis in the right eye did not go into remission. The patient also developed posterior scleritis in the right eye. (d,e) After bio-switching to SAR, scleritis rapidly disappeared. The patient remains in remission.

Figure 8

Case 2: Clinical appearance of anterior diffuse scleritis, FFA and OCT in the right eye (a–c) Scleritis relapsed, and the dose of GLM was doubled, but diffuse scleritis in the right eye did not go into remission. The patient also developed posterior scleritis in the right eye. (d,e) After bio-switching to SAR, scleritis rapidly disappeared. The patient remains in remission.

Figure 9

Case 3: Clinical appearance of anterior necrotizing scleritis, OCT, Colour fundus imaging, FFA, B-mode ultrasonography and MRI in the right eye. (a) Photograph of the anterior segment of the left eye at the first visit, showing necrotising scleritis and corneal marginal infiltration. (b) OCT shows the neuroepithelial layer as raised and wrinkled, with underlying fluid. (c) Colour fundus imaging shows papilloedema and extensive SRD including the fovea. (d) FFA shows retinal vasculitis consistent with SRD. (e) B-mode ultrasonography reveals increased scleral thickening (T-signatures) and extensive SRD. (f) Contrast-enhanced MRI shows contrast enhancement localised to the ocular wall of the left eye (yellow arrows).

Figure 10

Case 3: OCT and clinical appearance of anterior necrotizing scleritis in the right eye. (a) OCT after introduction of 30 mg of PSL, showing worsened SRD. (b) After four courses of steroid pulse therapy and RTX, scleritis temporarily resolved. (c) OCT at the time of reducing the PSL dose to 12.5 mg/day shows relapse of scleritis. (d) Anterior segment of the left eye after 250 mg/day PSL pulse therapy and SCTA, showing continued scleral thinning and melting.