Poster 206: TIPE2 Mitigates the Histopathology of Osteoarthritis in An Accelerate Aging Mouse Model
Authors: Huard Matthieu , Gao Xueqin , Huard Johnny , Huard Johnny , Guo Ping 
Published: July 2022

Objectives:

It has been reported that pain occurs after the onset of OA and is often associated with inflammatory synovial expression of tumor necrotic factor (TNFα), suggesting that TNFα is one of the main factors causing inflammation, pain and OA development in the joints (1). Inhibition of TNFα could be a potential approach to reduce inflammation in patients with OA. However, most anti-TNFα treatments in clinical trials with antibodies or inhibitors to reduce inflammation in OA have yielded conflicting results (2). It is essential to explore novel and more efficient approaches to modulate the expression of TNFα and inflammation in patients with painful OA. TNFα-induced protein 8-like 2 (TIPE2) was found to regulate the immune system’s homeostasis, and thus regulate inflammation. Zmpste24 deficient mouse (Z24-/-) is a reliable model of human Hutchinson-Gilford progeria. They are incapable of producing lamin A, an essential component of the nuclear envelope, and exhibit profound nuclear architecture abnormalities and multiple histopathological defects that phenocopy an accelerated aging process (3). Z24-/- mice are spontaneously and progressively developed OA around three months old. To investigate the role of TIPE2 on OA in this accelerated aging model, we have performed intra-articular injections of adeno-associated virus(AAV) carrying the TIPE2 gene in the knee of Z24-/- mice. Our results indicate that the overexpression of TIPE2 ameliorates osteoarthritis phenotype through the reduction of inflammation and senescent cells, suggesting that TIPE2 gene delivery may provide a novel anti-inflammatory therapy to alleviate the aging related OA in the clinic.

Methods:

Construction of the AAV-TIPE2 gene expression vector. Construction was performed as previously described (4). AAV production and titration. Viral production and purification of AAV2 were carried out as previously described (5). The concentration was determined using an AAV titration kit (Takara). The titer for AAV2-TIPE2 was 4.7 × 10⁁12 GCP/ml. Animals. Both male and female Z24-/- (B6.129SZmpste24tm1Sgy/Mmucd) mice at 12 weeks old were used for this study. The animal protocol was approved by the Institutional Animal Welfare Committee at the Colorado State University. TIPE2 treatment. AAV2-TIPE2 (20ul) was intra-articular injected into the right knee joint of Z24-/- mice and the same volume of PBS was injected into their left knee joint, which was used as control. At four weeks post-injection, mice were euthanized, and the knee joints were excised for decalcification, paraffin embedding, sectioning and histology analysis. Histology. H&E and Safranin O staining were performed following the manufacture protocol. The AC damage shown in the Safranin O staining images will be further evaluated using the Mankin scoring system(6). Immune staining. TNF-α and p16 immune staining was performed as the previous description(7). Immunofluorescence images were taken using a Nikon upright microscope. β-Gal staining. β-Gal staining was performed using the senescence staining kit (Cell Signaling). Statistical analysis. All values are expressed as the means ± SD. Statistical analyses were performed using Microsoft Excel software. Data were analyzed by the independent samples t-test compared between 2 groups at each time point using 2 tails. A value of P < 0.05 was considered statistically significant.

Results:

TIPE2 alleviates OA progression by regulating cell inflammatory responses. Since the inflammatory factor TNFα plays a key role in OA progression we first confirmed that TNFα is highly expressed in OA chondrocytes in the knee joint of Z24-/- mice at three months old (Fig.1). As we expected, TIPE2 treatment significantly decreased TNFα expression in chondrocytes of the OA knee (Fig. 1). TIPE2 treatment prevents cartilage degradation. We saw the difference in Safranin O–positive staining intensity in the AC (articular cartilage) of TIPE2 treated mice and PBS control mice. The glycosaminoglycan content (red) is degraded in the Z24-/- cartilage (PBS control), but cartilage degradation was reduced in Z24-/- mice treated with TIPE2 (Fig.2). TIPE2 treatment decreased β-Gal + senescent cells in the OA knee. OA as a whole joint disease, we observed the Z24 -/- mice chondrocytes exhibiting a variety of senescent-associated phenotypes. β-gal staining was observed in a subset of chondrocytes and subchondral bone close to the lesion sites of the OA. TIPE2 alleviates OA progression by targeting senescent cells. We observed that a significant decrease of TNFα positive cells (Fig. 1) and β-Gal positive cells (yellow arrow indicated in Fig.3, # p<0.05) by intra-articular injection of AAV TIPE2. This decrease is further confirmed by the determination of p16 expression in the Z24 -/- mice by TIPE2 treatment (Fig.3, *p<0.03). These findings suggest that TIPE2 can suppress OA progression via targeting inflammation factor TNFα in vivo.

Conclusions:

OA has become an emerging health challenge that occurs in older people. TNF-α is one of the main factors causing inflammation, pain and OA development in the joints. Here we first report that OA spontaneously occurred in aging joints of Z24-/- mice, and using a TNF-α antagonist, TIPE2, could attenuate OA development by decreasing inflammation through a reduction in the number of senescent cells. It has been reported that cellular senescence and the senescence-associated secretory phenotype (SASP) that drive and promote chronic inflammation in multiple age-related chronic diseases(8). Our results provide evidence that the downregulation of the inflammation factor TNF-α is capable to reduce cell senescence in this OA animal model. In conclusion, our research demonstrated a connection between TNF-α and senescence in aging caused OA. Our results indicate that TIPE2, as a TNF-α regulator, modulates inflammation can be used as a treatment for OA. [Figure: see text][Figure: see text][Figure: see text]