Animal Models in Systemic Sclerosis: An update

Abstract

Purpose of review:

Systemic sclerosis (SSc) is a multisystem autoimmune connective tissue disease characterized by early inflammation followed by excessive fibrosis in the skin and internal organs. Enhancing our comprehension of SSc pathogenesis is essential to develop effective therapeutic strategies. Animal models that mimic one or more aspects of SSc have been proven to be a valuable resource for investigating disease mechanisms. This review aims to provide an updated overview of the existing SSc animal models and the potentially relevant pathways to SSc pathogenesis.

Recent findings:

This review focuses on the most recently generated and investigated animal models, which delve into novel pathways beyond existing models or employ genetic technologies to gain a deeper understanding of SSc pathogenesis including activation of early type I interferon (IFN) signaling pathway, immune cell function and pulmonary artery hypertension (PAH).

Summary:

While no single animal model can fully replicate SSc, a combination of different models can offer valuable insights into the pathways involved in the onset and advancement of the SSc. These insights can prove animal models as a crutial pre-clinical tool for developing effective treatments for SSc.

Introduction

Systemic sclerosis (SSc) is a complex autoimmune disease of unknown etiology that is characterized by excessive fibrosis with inflammation in the skin and internal organs, along with the presence of autoantibodies and vascular damage (1–4). Since fibrosis is a multi-cellular and multi-organ disease, there is currently no perfect in vitro or microbial models for studying the pathogenic processes of SSc outside of the living animal models as the primary option. To enhance our understanding of SSc pathogenesis and foster the development of new treatments, researchers have extensively relied on various animal models. In this manuscript, we provide an overview of currently known mouse models for SSc (Table 1.), highlight their impact on inflammatory response and fibrosis signaling, and discuss how these animal models provide a critical tool for studying disease pathogenesis and treatment interventions.

Table 1:

Currently known animal models in systemic sclerosis discussed in this review.

Model	Induction	Species	Methods	Time	skin	Lung	Kidney	ANAs	Phenotype	References
Bleomycin model	Bleomycin	mouse	s.c. injection (low does; 10ug/injection)	daily, 3–4 wks	Yes	Yes	N/A	N/A	Inflammation; fibrosis	5
			s.c. injection (high does; 100ug/injection)	daily, 3 wks	Yes	Yes	Yes	N/A	Inflammation; fibrosis	6
HOCI model	HOCl	mouse	s.c. injection (low does)	5 days/wk; 6 wks	Yes	Yes	Yes	anti-topo I Abs;	Inflammation; fibrosis;	25; 26
Tsk1/+ model	transgenic	mouse	Fibrillin-1 gene duplication	6 wks and older	Yes	Yes	N/A	anti-topo-I Abs anti-RNA pol I Abs	skin fibrosis; lung emphysema	34; 35; 36
Tsk2/+ model	transgenic	mouse	mutagen-induced mutation in chromosome 1	4 wks	Yes	N/A	N/A	anti-topo-I Abs; anti-centromere Ab	skin fibrosis;	9; 37;38;39;
sclGVHD model	transplantation model	mouse	spleen cells transplant to rag-2 mouse	21 days	Yes	Yes	Yes	N/A	Inflammation; fibrosis	9; 40; 41
Fra-2 transgenic mouse model	transgenic	mouse	fra-2 gene under H2Kb promoter	9 wks	Yes	Yes	N/A	N/A	fibrosis; vasculopathy; PAH	42; 43;45
TNF-tg mouse	transgenic	mouse	TNF gene	3 months	No	Yes	N/A	N/A	fibrosis; PAH	48
Fli1&Klf5 deficient mouse model	transgenic	mouse	Klf5 +/− & Fli1+/−	3 months	Yes	Yes	Yes	N/A	Inflammation; fibrosis; vasculopathy;	49; 50; 51; 52
Xenotransplant mouse model	transplantation	mouse	pDCs transplant to NOD SCID mouse & bleomycin	3 wks	Yes	N/A	N/A	N/A	skin inflammation; fibrosis	54
Topoisomerase I mouse model	Human topo I with CFA	mouse	s.c. injection (75 units/injection)	4 times/2 wks	Yes	Yes	N/A	anti-topo-I Abs	Inflammation; fibrosis; anti-topo-I Abs	56

Bleomycin induced animal model of systemic sclerosis

The bleomycin induced skin and lung fibrosis models effectively mimic both early inflammatory and the late fibrotic stages seen in SSc or SSc-ILD. The skin model represents an inflammation-driven dermal fibrosis, where bleomycin initially leads to inflammation at an early stage followed by TGFβ -mediated extracellular matrix (ECM) deposition (5, 6). Type I interferon (IFN) pathway activation has been implicated in SSc pathogenesis (7, 8). Bleomycin mouse model also recapitulates the type I IFN signaling activation pattern present in SSc patients. Global gene expression datafrom human SSc and mice skin samples from bleomycin injected animal model demonstrated a prominent cluster of interferon genes (9). A recent study demonstrated that mice treated with IFN-α2 (IFNA2) encoded adeno-associated virus (AAV) in combination with s.c. bleomycin for 4 weeks, skin fibrosis was strongly accelerated with increased dermal thickness, Endothelial-to-mesenchymal transition (EndoMT) process and pro-fibrotic factors (10).

Interferon regulatory factors (IRFs) are crutial transcriptional factors that regulate type I IFNs and IFN-inducible genes, as well as innate and adaptive immunity in SSc (11–15). Studies on Irf5 ^−/− mice showed genetically abnormal thinner collagen bundles and fibrils in the dermis, and attenuated skin and lung fibrosis after bleomycin administration (16). Interestingly, IFN-γ mRNA levels were significantly upregulated and IL-4, IL-6 mRNA levels were decreased in the bleomycin treated Irf5^−/− mice (16). IRF7 is activated by TLR3/7/9 and leads to the secretion of a large amount of type I IFN and has been implicated in SSc pathogenesis (17, 18). Irf7^−/− mice exhibited attenuated dermal inflammation and fibrosis in response to bleomycin(19). Type I IFN levels were reduced in the pDCs from Irf7^−/− mice (20). This was consistent with findings showing TLR7 and TLR9 induced a high level of type I IFN in plasmacytoid dendritic cells (pDCs) (21–23). Moreover, Irf8^−/− mouse monocyte-derived macrophages displayed an M2 phenotype and significant upregulation of profibrotic factors and extracellular matrix components. Myeloid cell specific Irf8 conditional knockout mice displayed increased levels of profibrotic genes and skin fibrosis by bleomycin challenges (24).

Altogether, these animal studies highlight the bleomycin induced skin and lung fibrosis model as an excellent in vivo tool. It offers valuable insights into both innate and adaptive immune pathways specifically type I IFN signaling as well as fibrosis signaling pathways for our understanding of early disease development in SSc.

Hypochlorous Acid (HOCl) induced animal model of systemic sclerosis

Reactive oxygen species (ROSs) play a critical role in the pathogenesis and development of SSc. ROSs are continuously produced by SSc fibroblasts and over expression of ROS lead to fibroblasts proliferation and overproduction of collagen. HOCl belongs to the group of ROS generating substances, which directly contribute to the development of fibrosis, autoimmune processes, and vascular damage (25). Subcutaneous injections of agents generating HOCl or HOCl, induced skin and internal organ fibrosis including lung and kidney. Compared with bleomycin animal model, HOCl-injected mice displayed diffuse skin and lung fibrosis, and renal involvement, along with the production of serum anti-DNA topoisomerase 1 autoantibodies (anti-Topo-1). All these features are similar to diffuse cutaneous SSc (dcSSc) in humans (26). Tgf-β, Smad3, Tlr-4, Nf-κB, and Stat3 were increased in the fibrotic skin tissues of the HOCl-induced animal model. These fibrotic mediators were also increased in the lung tissues of HOCl treated mice but not in the s.c. injection of bleomycin model (25). Furthermore, a striking increase of CD4+ & CD8+ T cells and CD19+ B cells was predominantly found in the dermal layer in both of the bleomycin and HOCl-induced mice but not in the lung tissues in the intradermal injection bleomycin. Cytokines including IL-1β, IL-6, IL-17, IL-33, and TNF-α were also increased in HOCl-induced mice (25, 26). Another interesting study utilized adult Wistar rats to administer subcutaneous injection of 5% NaClO three times a week for 6 consecutive weeks (27). NaOCl breaks down to form HOCL (28). After 6 weeks of treatments, this animal revealed significant lung abnormalities. The perivascular inflammatory cell infiltration and microvessel wall thickening were presented in this animal model (27). Taken together, HOCl induced animal model decapsulates specific oxidation in the autoantibody production and is a useful tool for studying pathogenesis of oxidant stress ROS pathways and autoantibodies in SSc.

Tight Skin 1 (Tsk1/+) mouse model of systemic sclerosis

Duplication of the Fbn1 gene encoding fibrillin-1 in mouse leads to development of tight-skin mouse 1(Tsk1/+). These mice have thickened skin firmly bound to the subcutaneous and deep muscular tissue, and spontaneously develop hypodermal fibrosis in the absence of inflammation which is considered a skin fibrosis animal model for SSc (29, 30). Because of lacking prominent inflammatory features, Tsk1/+ mouse model is frequently used as a second model of SSc for studying fibrotic features in conditions lacking inflammation. Of note, Tsk1/+ mouse model is also frequently used for generating multiple congenic mouse model with specific targeted gene. Interestingly, a study demonstrated that lacking Irf7 gene in type I IFN signaling pathway with Tsk1/+ mouse model had decreased dermal and hypodermal thickness and dermal fibrosis with attenuated collagen and profibrotic genes (19). Another study targeting TLR4 signaling by its inhibitor treatment demonstrated activation of TLR4 signaling pathway in the Tsk1/+ mouse model (31, 32). Activation of TGFβ by MFG-E8 might contribute to the fibrosis signaling pathway in Tsk1/+ mouse model (33). Tsk1/+ mice have SSc related autoantibodies including anti-topoisomerase I antibodies (anti-topo-I Abs) and high titers of anti-RNA polymerase I antibodies (anti-pol I Abs) (34, 35). CD19 dependent signaling pathway in B cells may contribute to the development of systemic autoimmunity and TGFβ driven skin fibrosis in Tsk1/+ mice (36).

Tight skin 2 (Tsk2/+) mouse model of systemic sclerosis

The tight skin-2 (Tsk2/+) mouse is a mutagen-induced mutation localized on the proximal arm of mouse chromosome 1(37). Tsk2/+ mice harbor a deleterious coding mutation in Col3a1, leading to an amino acid change (C33S) in the N-terminal region of the protein procollagen III amino terminal propeptide segment (PIIINP). This results in Tsk2 mutation phenotypes including accumulation of COL3A1 and induction of COL1A1(38). A major function of the Col3a1 gene is promoting blood vessel development. Vasculopathy in Tsk2/+ mouse have not yet been reported and need to be investigated. Tsk2/+ mouse displays histopathological and biochemical abnormalities including mononuclear cell infiltration in the dermis and adipose tissues, increased collagen accumulation (37). Interestingly, these mice present anti-Scl70 and anti-centromere antibodies, major frequently presented autoantibodies in patients with SSc (39). Furthermore, an important interspecies comparative genomic data analysis revealed that Tsk2/+ mice skin tissue showed similar gene expression to the fibroproliferative subset of SSc patients skin. Bleomycin and Tsk2/+ mice shared enrichment of TGFβ signaling pathway activation (9). Therefore, Tsk2/+ mouse model is a helpful tool to study inflammation, fibrosis, vasculopathy and autoimmunity in pathogenesis of SSc.

Sclerodermatous graft-versus-host disease (SclGVHD) mouse model of systemic sclerosis

The Murine Scl GVHD model is produced by transplanting B10.D2 spleen cells to lethally irradiated Rag-2 KO Balb/c mice (lacking mature T and B cells). After 21 days of the transplantation, these animals demonstrated the fibrotic effects of dermal thickening and progressive fibrosis in internal organs including lung and kidney, as well as vascular damage with immune system of T cell and macrophage activation/infiltration around perivascular area in organs, presence of anti-Scl-70 auto-antibodies (40, 41). All of these disease characteristics are seen in a high proportion of patients with diffuse SSc. Furthermore, SSc inflammatory skin and its associated IFN signaling molecules such as Stat1, Irf1, Ccl5, IFfi30, AIf1, Ccl2 and Jak3 are highly upregulated in early scl GVHD animal model (9). Taken together, the murine Scl GVHD model may provide a great tool for understanding the immunological responses such as immune reconstitution, present of IFN signaling and autoantibodies involved in SSc pathogenesis.

Fra-2 (Fos-related antigen-2) transgenic mouse model of systemic sclerosis

Microvascular injury is involved in the pathogenesis of fibrosis in SSc. The Fra-2 transgenic mouse model displays both fibrosis and vascular characteristics with immune dysregulation of human SSc (42). Fra-2 tg mouse phenotype starts from an age of 9 weeks with increased perivascular inflammatory cell infiltration followed by increased accumulation of collagen and extracellular matrix proteins in the skin. Furthermore, these mice developed a progressive vascular feature of SSc pulmonary arterial hypertension (SSc-PAH) including vascular inflammation and lumen obliteration. IFN signature and other cytokine/chemokines of Igf-1, Il-6, Il-1β, Ifn-γ, Ccl1–4, Cxcl2, Cxcl10, Cxcl11 are presented in these mice (42). Nintedanib, an anti-fibrotic medicine approved for the treatment of SSc-ILD (43, 44), ameliorates vascular remodeling, fibrosis, and endothelial cell apoptosis in the Fra-2 tg model. Another interesting study with both of Fra-2 tg mice and intratracheal bleomycin injected mice model study demonstrated that the antifibrotic drug Pirfenidone treatment aggravated pulmonary inflammation, fibrosis, and vascular remodeling in Fra-2 tg mice whereas attenuated bleomycin induced pulmonary remodeling (45). Pirfenidone is an approved treatment option for IPF, but not for SSc-ILD. The initial clinical trials demonstrated good tolerance of pirfenidone among patients with SSc-ILD but its efficacy for fibrotic manifestations of SSc has not been established (46). However, more recent trials have revealed limited or no significant benefits (47). In the other hands, pirfenidone aggravated pulmonary inflammation, fibrosis, and vascular remodeling in Fra-2 tg mice. Increased IL-4 levels and eosinophil numbers of pronounced immune response and disrupted endothelial integrity was observed in pirfenidone-treated Fra-2 tg mice (45). It remains to be seen whether these findings on the lack of efficacy of pirfenidone translate to human studies.

TNF-Tg mouse model of Connect Tissue Disorders-Pulmonary Arterial Hypertension (CTD-PAH) or (SSc-PAH)

Pulmonary arterial hypertension (PAH) is one of the complication features of SSc with the highest mortality.

A recent study demonstrated that TNF-Tg mouse represents a novel animal model of connective tissue disease PAH (CTD-PAH) including SSc-PAH (48). TNF-Tg mice exhibit a gradual development of pulmonary arterial thickening, vascular fibrosis, and luminal occlusion. These changes correlate with ventricular myocyte hypertrophy and fibrosis. The pathology closely resembles human CTD-PAH, specifically SSc-PAH. In patients with SSc-PAH, lung tissues showed elevated TNF signaling and significant similarities in immune pathway dysregulation. Additionally, analysis of the 100 most differentially expressed genes between diseased and healthy lungs derived from five different GEO data sets in the RNA-seq data from the TNF-Tg and WT mice, the gene expression pattern in TNF-Tg mice closely resembled that observed in SSc-ILD, while it was distincting with idiopathic PAH (IPAH) or idiopathic pulmonary fibrosis (IPF) (48). In summary, TNF-Tg mouse model exhibited dysregulation of angiogenesis, endothelin, apoptosis, and B-cell activation pathways, making it a beneficial tool for investigating the pathogenesis of SSc-PAH.

Fli1 & Klf5-deficient mouse model of systemic sclerosis

Friend leukemia integration 1 (Fli1), a member of the E-twenty-six (ETS) transcription factor family, was first identified as a proto-oncogene in Friend virus-induced erythroleukemia in mice (49). In skin, Fli1 is expressed in several types of cells including endothelial, hematopoietic cells, dermal fibroblasts, and keratinocytes, and plays an important role in vascular development and angiogenesis, ECM remodeling, and keratinocytes function. Fli was epigenetically downregulated in the lesional skin of SSc patients. Interestingly, mice with double heterozygous deficiency of Klf5 and Fli1 replicate the epigenetic phenotype of SSc skin and spontaneously manifest several SSc features, including fibrosis and vasculopathy of the skin and lung, B cell activation, and autoantibody production (50). These findings suggest that the epigenetic downregulation of Fli1 and KLF5 plays a pivotal role in triggering the pathogenic triad of SSc. Notably, mice with targeted deletion of the Fli1 carboxy-terminal activation (CTA) domain (Fli1^ΔCTA/ΔCTA) revealed a significant upregulation of fibrillar collagen genes, and abnormally thin and unmatured collagen fibrils similar to those observed in the SSc skin without developing skin fibrosis (51). Interestingly, mice with Fli1 deficiency specifically in Keratin 14-expressing epithelial cells drive systemic autoimmunity and skin and esophagus fibrosis (52). Taken together, these findings highlight the important role of Fli1 in skin fibrosis and Fli1 deficient mice as a useful vasculopathy and fibrosis model of SSc.

Xenotransplant mouse model of scleroderma

Plasmacytoid dendritic cells (pDCs), specialized innate immune cells with anti-viral functions, play a pivotal role in the pathogenesis of autoimmune disorders including SSc (53). A recent study developed a novel SSc model by xenotransplanted human pDCs into immunocompromised Nonobese diabetic/severe combined immunodeficiency (NOD SCID) mice treated with Aldara (inflammatory model), or bleomycin (fibrotic model) (54). Xenotransplantation of human pDCs to animals markedly amplified the in vivo skin response to TLR-9 agonist-induced type I IFNs, while also significantly enhancing both the fibrotic and immune response to bleomycin. This induction can be repressed in mice receiving the antibodies against BDCA2, a novel plasmacytoid dendritic cell–specific type II C-type lectin (55). Overall, this animal model has a great potential to study human pDCs and immune cells in SSc. Additionally, transplanting inflammatory cells offers a promising approach to create new SSc animal models.

Anti–DNA topoisomerase I (anti–topo I) antibody induced mouse model of systemic sclerosis

Presence of anti–topo I antibody is a hall mark of diffuse SSc (dcSSc) subset and is strongly associated with interstitial lung disease, it predicts more severe disease course. Immunization of C57BL/6 wildtype mice with subcutaneous injection of recombinant human topo I (Topo GEN) and Freund’s complete adjuvant (CFA) H37Ra in back skin 4 times of 2 weeks, induced skin and lung fibrosis and autoimmune abnormalities similar in dcSSc patients. Skin and lung fibrosis and inflammatory cell infiltration developed during the first 8 weeks and began to resolve 6 weeks later. Topo- I IgM and IgG autoantibodies were elevated in these animals. IL-6, IL-17, TGFβ cytokine levels were upregulated and IL-10 level was downregulated with fibrosis in this animal model (56). Indeed, the anti–topo I antibody-induced mouse model could serve as an excellent tool for investigating autoimmunity and the role of B cells in the pathogenesis of SSc. This model has the potential to provide valuable insights into the development of novel therapeutic strategies targeting B cells in this autoimmune disease.

Conclusions

SSc is a complex autoimmune disease involving different pathological aspects of immunity, fibrosis, and vasculopathy. To better comprehend the pathogenesis of SSc, it is essential to explore the molecular mechanisms using experimental animal models. While there is no animal model that perfectly replicates SSc, the use of single or combined animal models remains a valuable tool for investigating and understanding the signaling pathways involved in SSc pathogenesis. Such research is crucial for developing targeted treatment interventions and advancing our knowledge of this complex autoimmune disorder.

Key points.

• This review offers an updated overview of various animal model studies, providing valuable insights into the signaling pathways and cellular processes implicated in the development and progression of SSc. It serves as a resource for understanding the selection of a suitable animal model to investigate specific signaling pathways involved in SSc pathogenesis.

• Animal models play a vital role in biomedical research as they enable the investigation of the intricate interactions between multiple cell types involved in fibrosis development, a complexity that cannot be fully recapitulated in tissue & cell culture systems.

• Although no single animal model can completely mimic all aspects of human SSc, researchers frequently employ a combination and best choice of different animal models to gain a more comprehensive understanding of the disease relevant molecular mechanisms.