Functional Loss of Terminal Complement Complex Protects Rabbits from Injury-Induced Osteoarthritis on Structural and Cellular Level

Abstract

The terminal complement complex (TCC) has been described as a potential driver in the pathogenesis of posttraumatic osteoarthritis (PTOA). However, sublytic TCC deposition might also play a crucial role in bone development and regeneration. Therefore, we elucidated the effects of TCC on joint-related tissues using a rabbit PTOA model. In brief, a C6-deficient rabbit breed was characterized on genetic, protein, and functional levels. Anterior cruciate ligament transection (ACLT) was performed in C6-deficient (C6^-/-) and C6-sufficient (C6^+/-) rabbits. After eight weeks, the progression of PTOA was determined histologically. Moreover, the structure of the subchondral bone was evaluated by µCT analysis. C6 deficiency could be attributed to a homozygous 3.6 kb deletion within the C6 gene and subsequent loss of the C5b binding site. Serum from C6^-/- animals revealed no hemolytic activity. After ACLT surgery, joints of C6^-/- rabbits exhibited significantly lower OA scores, including reduced cartilage damage, hypocellularity, cluster formation, and osteophyte number, as well as lower chondrocyte apoptosis rates and synovial prostaglandin E2 levels. Moreover, ACLT surgery significantly decreased the trabecular number in the subchondral bone of C6^-/- rabbits. Overall, the absence of TCC protected from injury-induced OA progression but had minor effects on the micro-structure of the subchondral bone.

Keywords: ACLT; C6; PTOA; TCC; complement activation; osteoarthritis; terminal complement complex.

Figure 1

Characterization of the mutated C6 gene. (A,B) Structure of the rabbit C6 protein according to transcript C6-201, ENSOCUT00000001933 (Ensemble release 84) with indication of the putative C5b binding site 2 and the stop codon caused by the mutation; TSP: thrombospondin type 1 repeat, LR: Low-density lipoprotein receptor class A repeat, MACPF: Membrane attack complex component/perforin domain, SCR: short consensus repeats, FIM: factor I modul. (C) Consequence of the genomic deletion in the C6 gene of C6-deficient rabbits on protein level. Serum of C6-deficient (C6-def.) and wildtype rabbits (WT) was resolved by 12% SDS-PAGE and coomassie-stained or used for western blot and detected with a C6-specific antibody; M: protein marker.

Figure 2

Functional testing of TCC in rabbit serum. (A) Hemolytic activity of rabbit serum derived from WT (n = 3), heterozygote (n = 8) and C6-deficient (n = 9) animals was determined by means of a CH50 assay; curves represent column means at different dilutions with standard deviation. (B) Anti-TCC immunocytochemical staining of sheep erythrocytes exposed to C6-sufficient (C6-suff.) and C6-deficient (C6-def.) serum, respectively.

Figure 3

Histopathologic assessment of medial condyle sections. (A) Exemplary images of SafO-stained medial condyles of both experimental groups. Cell clusters are indicated by red arrowheads; black asterisk indicates hypocellular region; red asterisk indicates irregular surface; the black bar represents 50 µm. (B–E) Corresponding statistical analysis of the overall score and single parameters of the histopathological assessment: (B) overall score, (C) proteoglycan content/ SafO staining intensities, (D) structure/ surface integrity, (E) hypocellularity, and (F) cluster formation. Data are charted as box plots with median and whiskers min to max; white boxes= control joint, grey boxes = ACLT-operated joints. Statistically significant differences between groups (n = 6 each) were depicted as: *: p ≤ 0.05, **: p ≤ 0.01, and ***: p ≤ 0.001, ****: p ≤ 0.0001.

Figure 4

TUNEL staining of medial condyle sections. (A) Exemplary images of TUNEL-stained cartilage. Images were taken from the superficial and transitional zone. TUNEL-positive cells (green) are exemplarily indicated by white arrowheads; the white bar represents 100 µm. (B) Corresponding statistical analysis of the apoptosis rate (percentage of TUNEL-positive chondrocytes). Data are charted as scattered bars with mean and SEM; white boxes = control joint, grey boxes = ACLT-operated joints. Statistically significant differences between groups (n = 3 each) were depicted as: **: p ≤ 0.01.

Figure 5

Evaluation of osteophyte formation and synovial inflammation. (A) Exemplary images of ACLT-operated joints after 8 weeks; macroscopic femoral osteophytes are indicated by a black circle. (B) Corresponding statistical analysis of macroscopically detectable osteophytes. (C) Exemplary images of Hematoxylin/ Eosin-stained synovial membrane of both experimental groups. Endothelial cells are indicated by black arrowheads; black asterisk indicates proliferation and hypertrophy of synoviocytes in lining layer; black star indicates lymphocyte aggregation; the black bar represents 50 µm. (D) Statistical analysis of the synovial score and (E) determination of synovial PGE2 concentrations. Data are charted as (B,D) box plots with median and whiskers min to max or (E) scattered bars with mean and SEM; white boxes/ bars= control joint, grey boxes/ bars= ACLT-operated joints. Statistically significant differences between groups (n = 6 each) were depicted as: *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, and ****: p ≤ 0.0001. Pictures in (A) were modified from [20].

Figure 6

Structural analysis of subchondral bone. (A) Exemplary image of µCT analysis after 8 weeks; reduction in cortical thickness is indicated by yellow arrowheads, reduction in BV/TV and Tb.N., respectively, is indicated by yellow asterisks. Statistical analysis of (B) cBMD, (C) Tb.N, (D) cTh, (E) BV/TV, (F) Tb.Th, and (G) Tb.Sp. Data are displayed as scattered bars with mean and SEM; white bars= control joint, grey bars= ACLT-operated joints. Statistically significant differences between groups (n = 6 each) were depicted as: *: p ≤ 0.05.