Sex differences in long-term effects of collagen-induced arthritis in middle-aged mice

Abstract

Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory disorder with high prevalence among middle-aged women. Collagen-induced arthritis (CIA) is the most widely used animal model of RA, however, sex differences and long-term effects of CIA in mice are poorly described in the literature. Aim: Therefore, the present study aimed to analyze the long-term effects of CIA on the joints of middle-aged mice of both sexes and to describe potential sex differences. Materials and methods: CIA was induced in middle-aged DBA/1J mice by immunization with bovine type II collagen and complete Freund's adjuvant. Saline was administered to control mice. Arthritis score assessment, plethysmometry, and thermal imaging of the joints were performed weekly for 15 weeks. Locomotor activity, micro-computed tomography, joint histology and biochemical analyses were performed at the end of the experiment. Results: Our results indicate a similar prevalence of arthritis in both sexes of mice-67% (8/12) of females and 89% (8/9) males with an earlier onset in males (day 14 vs. day 35). After the arthritis scores peaked on day 56 for males and day 63 for females, they steadily declined until the end of the experiment on day 105. A similar dynamics was observed in paw volume and temperature analyzing different aspects of joint inflammation. Long-term consequences including higher proteinuria (by 116%), loss of bone density (by 33.5%) and joint damage in terms of synovial hyperplasia as well as bone and cartilage erosions were more severe in CIA males compared to CIA females. There were no significant differences in locomotor activity between CIA mice and CTRL mice of any sex. Conclusion: This is the first study to describe the long-term effects of the CIA model in terms of sex differences in DBA/1J mice. Our results indicate sex differences in the dynamics, but not in the extent of arthritis. An earlier onset of arthritis and more severe consequences on joints, bones and kidneys were found in males. The underlying immune pathomechanisms responsible for the limited duration of the arthritis symptoms and the opposite sex difference in comparison to RA patients require further investigation.

Keywords: aging; animal model limitations; autoantibodies; autoimmune disease; disease severity; murine models; sex disparities; synovial inflammation.

FIGURE 1

Incidence of arthritis signs followed by the dynamics of arthritis score. Representative pictures of the development of the arthritis signs, including redness, swelling and thickness of CIA mice paws compared to CTRL mice paws (taken on day 56, representing a score 4 for the front paw and a score 2 for the back paw). (A)—Incidence of arthritis signs for front and back paws according to the sex of mice. (B)—Dynamics of arthritis scores for the front and back paws. (C)—Dynamics of arthritis scores for front and back paws in the area under the curve between male and female CIA mice. (D)—Incidence of arthritis for front paws according to the sex of mice. (E) - Dynamics of arthritis scores for the front paws. (F)—Dynamics of arthritis scores for front paws in the area under the curve between male and female CIA mice. (G) Incidence of arthritis signs for back paws according to the sex of mice. (H) Dynamics of arthritis scores for the back paws. (I)—Dynamics of arthritis scores for back paws in the area under the curve between male and female CIA mice. [CTRL-females (n = 5), CIA-females (n = 12), CTRL-males (n = 5), CIA-males (n = 9)]. (p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.05 = #, p < 0.01 = ##, p < 0.001 = ###, p < 0.05 = +, p < 0.01 = ++, p < 0.001 = +++).

FIGURE 2

Dynamics of paw temperatures in the CIA model. (A)—Representative thermal images illustrating the temperature differences in the paws between a CTRL mouse and a CIA mouse with active arthritis. (B)—Dynamics of the temperature of paws regarding the sex and group of mice. (C)—Differences in the area under the curve of the temperature of paws during the experiment according to the sex and groups of mice. [CTRL-females (n = 5), CIA-females (n = 12), CTRL-males (n = 5), CIA-males (n = 9)). (p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.05 = #, p < 0.01 = ##, p < 0.001 = ###, p < 0.05 = +, p < 0.01 = ++, p < 0.001 = +++).

FIGURE 3

Long-term effects of the CIA model on the bones and locomotion. (A)—Analysis of periarticular bone density based on micro-CT. (B)—Results of the locomotor activity regarding the sex and the group of mice. (C)—Representative pictures showing the bone density as a long-term effect of the CIA model (p < 0.05 = #, p < 0.01 = ##, p < 0.001 = ###). (D)—Correlation between micro-CT density and locomotor activity (R2 = 0.172; p < 0.05). [CTRL-females (n = 5), CIA-females (n = 9), CTRL-males (n = 5), CIA-males (n = 9)].

FIGURE 4

A correlation analysis was performed to show the relationship between arthritis scores and paw swelling, which was assessed using plethysmometry. (A)—Correlation of arthritis scores and swelling of front paws in CIA females mice. (B)—Correlation of arthritis scores and swelling of back paws in CIA females mice. (C)—Correlation of arthritis scores and swelling of front paws in CIA male mice. (D)—Correlation of arthritis scores and swelling of back paws in CIA male mice. [CIA-females (n = 12), CIA-males (n = 9)].

FIGURE 5

(A)—Quantification of proteinuria according to the sex and group of mice. [CTRL-females (n = 5), CIA-females (n = 12), CTRL-males (n = 4), CIA-males (n = 9)]. (B)—Quantification of plasma estradiol concentration in CTRL and CIA female mice. [CTRL-females (n = 5), CIA-females (n = 9)]. (C)—Quantification of plasma testosterone concentration in CTRL and CIA male mice. [CTRL-males (n = 4), CIA-males (n = 7)]. (p < 0.05 = #, p < 0.01 = ##, p < 0.001 = ###).

FIGURE 6

(A–C)—Representative pictures of histological evaluation of paw in a Ctrl female. (D–F)—Representative pictures of histological evaluation of paw in a CIA female. A histologically normal layer of hyaline cartilage in the superficial bone joints in control females [(A–C) black arrows], while in CIA females, an atrophic residual layer of hyaline cartilage with some residual disintegrating pinkish tissue [(D) red arrow], including a preserved layer of hyaline cartilage with compensatory hyperplasia of thinned, variably sized groups of chondrocytes was observed [(E, F) blue arrows]. HE: 200x.

FIGURE 7

(A–C)—Representative pictures of histological evaluation of paw in a CTRL male. (D–F)—Representative pictures of histological evaluation of paw in a CIA female. In control males, in the joints of long and short bone, a 5/5 continuous, equally thick layer of normal hyaline cartilage was observed [(A–C) black arrows]. In CIA males, an atrophic residual discontinuous layer of hyaline cartilage with individual chondrocytes [(D) red arrow], cartilage erosions [(D) blue arrow], or only some residual thin cartilage with disintegrating pinkish material [(E) red arrow], and focally cartilage erosions with disintegrated tissue was detected [(F) blue arrow]. IIE: 200x.

FIGURE 8

(A)—Inflammatory cells infiltration in joints of front paws according to the sex and group of mice. (B)—Inflammatory cells infiltration in joints of back paws according to the sex and group of mice. (C)—Synovial hyperplasia in joints of front paws according to the sex and group of mice. (D)—Synovial hyperplasia in joints of back paws according to the sex and group of mice. (E)—Cartilage and bone erosion in joints of front paws according to the sex and group of mice. (F)—Cartilage and bone erosion in joints of back paws according to the sex and group of mice. [CTRL-females (n = 5), CIA-females (n = 12), CTRL-males (n = 4), CIA-males (n = 7)]. (p < 0.01 = **, p < 0.001 = ***, p < 0.01 = ##).