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. 2019 Dec 11;21(1):278.
doi: 10.1186/s13075-019-2052-2.

Proposition of a novel animal model of systemic sclerosis induced by type V collagen in C57BL/6 mice that reproduces fibrosis, vasculopathy and autoimmunity

Walcy Rosolia Teodoro  1 , Zelita Aparecida de Jesus Queiroz  2 , Lais Araujo Dos Santos  2 , Sergio Catanozi  3 , Antonio Dos Santos Filho  2 , Cleonice Bueno  2 , Margarete B G Vendramini  2 , Sandra de Morais Fernezlian  4 , Esmeralda M Eher  4 , Percival D Sampaio-Barros  2 , Sandra Gofinet Pasoto  2 , Fernanda Degobbi T Q S Lopes  5 , Ana Paula Pereira Velosa  2 , Vera Luiza Capelozzi  4
Affiliations

Proposition of a novel animal model of systemic sclerosis induced by type V collagen in C57BL/6 mice that reproduces fibrosis, vasculopathy and autoimmunity

Walcy Rosolia Teodoro et al. Arthritis Res Ther. .

Abstract

Background: Type V collagen (Col V) has the potential to become an autoantigen and has been associated with the pathogenesis of systemic sclerosis (SSc). We characterized serological, functional, and histopathological features of the skin and lung in a novel SSc murine model induced by Col V immunization.

Methods: Female C57BL/6 mice (n = 19, IMU-COLV) were subcutaneously immunized with two doses of Col V (125 μg) emulsified in complete Freund adjuvant, followed by two intramuscular boosters. The control group (n = 19) did not receive Col V. After 120 days, we examined the respiratory mechanics, serum autoantibodies, and vascular manifestations of the mice. The skin and lung inflammatory processes and the collagen gene/protein expressions were analyzed.

Results: Vascular manifestations were characterized by endothelial cell activity and apoptosis, as shown by the increased expression of VEGF, endothelin-1, and caspase-3 in endothelial cells. The IMU-COLV mice presented with increased tissue elastance and a nonspecific interstitial pneumonia (NSIP) histologic pattern in the lung, combined with the thickening of the small and medium intrapulmonary arteries, increased Col V fibers, and increased COL1A1, COL1A2, COL3A1, COL5A1, and COL5A2 gene expression. The skin of the IMU-COLV mice showed thickness, epidermal rectification, decreased papillary dermis, atrophied appendages, and increased collagen, COL5A1, and COL5A2 gene expression. Anti-collagen III and IV and ANA antibodies were detected in the sera of the IMU-COLV mice.

Conclusion: We demonstrated that cutaneous, vascular, and pulmonary remodeling are mimicked in the Col V-induced SSc mouse model, which thus represents a suitable preclinical model to study the mechanisms and therapeutic approaches for SSc.

Keywords: Autoantibodies; Fibrosis; Mouse model; Systemic sclerosis; Type V collagen; Vascular.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Experimental design. The scheme shows the immunization protocol performed on C57BL/6 mice with type V collagen, detailing the dose, the period of inoculation of the antigen, and euthanasia of animals. FCA Freund’s complete adjuvant, FIA Freund’s incomplete adjuvant, SC subcutaneous, IM intramuscular
Fig. 2
Fig. 2
Changes in lung and skin morphology, dermal thickness, total lung collagen, and lung mechanics following the immunization with Col V in C57BL/6 mice and human SSc. C57BL/6 mice model was established by injection of Col V, as indicated as IMU-COLV, had similar changes to the traditional human SSc (a, b). The lung tissues were stained with H&E (a) and skin with Masson’s trichrome staining (b). Two independent individuals measured dermal thickness with Image Pro-Plus. Dermal thickness was shown to be significantly increased in Col V-immunized mice whereas control mice did not show an increase in dermal thickness (c). Lung total collagen content was measured with 4-hydroxyproline assay. Mice receiving Col V exhibited increased collagen content in the lung compared with control (d). Lung mechanics in mice immunized with Col V showed increased tissue elastance and resistance compared to control (e). Values are the means ± s.e (n = 38). P < 0.05
Fig. 3
Fig. 3
Changes in collagen fibers in the lung and skin lesions and immunohistochemical analysis of vascular injury in the lung following the Col V immunization in C57BL/6 mice. Immunofluorescence was performed to characterize collagen I (Col I), collagen III (Col III), and collagen V (Col V) in the lung (a) and skin (b). Mice receiving Col V exhibited increased deposition of Col I and Col V fibers in the lung and skin compared with control. The Picrosirius staining in skin samples showed total collagen in Col V-immunized mice and control (b). Immunohistochemical analysis was performed on the lung C57BL/6 mice receiving the treatment of Col V. Positive cells staining with endothelial markers VEGF and endothelin-1 and apoptosis marker caspase-3 were indicated by brown color (c)
Fig. 4
Fig. 4
Increased collagen fibers density in the lung and skin and increase expression of endothelial markers of vascular injury in mice following the immunization with Col V. Quantification of collagen I (Col I), collagen III (Col III), and collagen V (Col V) and endothelial markers in the lung (a) and Col I, Col III, and Col V in the skin (b) by Image Pro-Plus of mice immunized with Col V. The quantification of the Picrosirius staining showed collagen fine fibers increase in Col V-immunized mice compared with control. Data are mean ± s.e. (n = 38) P < 0.05
Fig. 5
Fig. 5
Quantitative qRT-PCR analysis of collagen I (COL1A1 and COL1A2), collagen III (COL3A1) and collagen V (COL5A1 and COL5A2) genes expression in the lung (a) and skin (c) tissues, and endothelial expression of VEGF, Endothelin-1 and Caspase-3 in lung tissue (b) from C57BL/6 mice immunized with Col V. Data represent mean ± s.e (n = 38) P < 0.05
Fig. 6
Fig. 6
ELISA assays showed elevated anti-collagen III, IV, and V antibodies (a) and immunofluorescence assays showed a higher levels of antinuclear antibodies (ANA) (b) in the serum from the mice immunized with Col V. The immunofluorescence shows ANA with a fine dotted and/or Golgi cytoplasmic pattern in serum from C57BL/6 mice immunized with Col V (b, arrow). Data represent the mean ± s.e. (n = 38). P < 0.05
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