Serum amyloid A 1 protein isoforms induce Rheumatoid Arthritis

Background : SAA1 in RA pathogenesis and its complications remains unknown, making early diagnosis and risk prevention difficult. This study is to determine the pathogenetic mechanisms of three different SAA1 protein isoforms in RA progression. Methods: We modified an experimental adenovirus infection protocol in order to successfully introduce SAA1.2, SAA1.3, SAA1.5 gene alleles into the rear knee joints of C57BL/6 mice. Micro-computed tomography (micro-CT) analysis was applied to determine changes in bone morphology and density. Immunohistochemistry (IHC), flow cytometry, ELISA and real-time PCR were used to investigate disease progression and cytokine alterations in the course of adenoviral SAA-induced knee joint inflammation and bone destruction. Results: The pathogenetic functions of SAA1.2, SAA1.3 and SAA1.5 protein isoforms in promoting the initiation and progression of RA were determined. We established that SAA1.2 was the most aggressive factor in RA induction and progression. Mechanistically, we found that the arthritis-inducing effect of SAA1.2 transcription in the knee joints and mutant SAA1 protein secretion in blood results in stimulation of immune responses, leading to CD8 + T cell and pro-inflammatory cytokine elevation, with subsequent synovial inflammation and bone destruction. Conclusions: These findings indicate that SAA1 protein isoforms, particularly SAA1.2, play a significant role in the induction and progression of RA and may have potential value in the early diagnosis and severity prediction for RA.

inflammation due to lower secreting ability of pro-inflammatory cytokines [15]. To our knowledge, the biological roles of these SAA1 isoforms including our newly identified SAA1.2 mutation have not yet been comprehensively investigated.
Here, we aim to clarify the role of the SAA 1.2, SAA1.3 and SAA 1.5 alleles as a pathogenesis factor or an inhibitory factor in the development and outcome of RA. We C57BL/6 mice (10-14 weeks old) was used in our studies, after one weeks adaptive feeding, all mice were randomly and evenly divided into 4 groups (10 mice each), including SAA1.2, SAA1.3, SAA1.5 adenovirus injection groups and the EGFP control group.
The rear right knee joints were injected laterally with 1 × 10 8 pfu of adenovirus containing either EGFP and or one of the three SAA1 isoforms. The animals were sacrificed at 1, 2, 4, or 6 weeks after injection. The study protocol was approved by the institutional committee The concentrations of the SAA, and the inflammatory factors IL-6, IL-22, MMP-3, MMP-9, IL-17, IL-23, and G-CSF were measured in mouse serum using commercially available ELISA kits (R&D, USA). According to the manufacturer's instructions, the blood samples were allowed to clot for 2 h at room temperature before being centrifuged for 20 min at 2000 x g. The serum was removed and assayed immediately or after storage at ≤-20°C. Both samples and standards were run in triplicate.

Western blot analysis
Liver, kidney and lung tissues were lysed with ice-cold RIPA lysis buffer. The protein samples were electrophoretically separated and transferred to nitrocellulose (NC) membranes. The membranes were incubated with primary antibodies at 4°C overnight and incubated with a secondary fluorescent antibody for 1 hour at room temperature. Bands were visualized on a LI-COR Odessy scanner (Belfast, USA). Primary antibodies human SAA1 was purchased from Abcam (Abcam, UK) and GAPDH from Cell Signaling Technology (Danvers, USA).

Statistical analysis
All the data are presented as the mean ± SD or SEM (indicated in each figure) of 3 individual experiments. Differences were analyzed by one-way ANOVA using Graph Pad Prism 5.

1.
Adenoviral expression of three SAA1 isoforms induced bone destruction of knee joints in C57BL/6 mice To identify the roles of these three SAA1 isoforms in RA, we created a timeline for animal experiments (Fig 1a). Adenoviruses carrying SAA1 alleles and an EGFP control were injected in a single dose into the articular cavity of the right knee in mice; computed tomography CT scores were monitored, and tissues were collected at 4 time points (the 1 st , 2 nd , 4 th and 6 th weeks). We observed no differences in body weight among the groups during this 6-week period, suggesting that adenovirus infection did not induce toxicity ( Fig   S1a). RT-PCR confirmed successful gene integration and transcription in the bone cartilage throughout the experimental period; SAA1 mRNA levels were elevated in week 1 and gradually decreased from weeks 2 to 6, but the levels remained significantly higher than those of EGFP-treated animals (Fig 1b). An increase in secreted-SAA levels in serum was also observed (fig S6a).
Then, we analyzed changes in including trabecular bone mineral density (BMD), tissue mineral density (TMD), trabecular number (Tb.N), porosity percent (TOT) and the trabecular volume rate (BV/TV) using axial micro-CT in both legs. In the right leg, bone destruction occurred from weeks 2 to 4 and gradually recovered by week 6; the CT scores of the SAA1.2, SAA1.3, and SAA1.5 groups decreased by 27.8%, 26.7% and 10.9% in week 2 and were restored to decreases of 8.7%, 15.8% and 5.8%, respectively, in week 6, compared to those of the EGFP group. For BMD, SAA1.2 showed an 8.7% decrease in week 2, and SAA1.2 and SAA1.3 decreased by 12.3% and 11.3%, respectively, in week 4 and were restored to normal values in week 6. Other parameters also showed the most significant change in week 2, except for TMD (Fig 1c-d, Fig S1b). To visualize bone destruction, we reconstructed a 3D model of the mouse tibia and observed remarkable attenuation of the trabecular bone in the SAA1 groups, especially in the SAA1.2 group in week 4 (Fig 1e); the bone destruction recovered by week 6, which was consistent with a decrease in the SAA1 level, indicating that stress reaction recovery had occurred.
Surprisingly, bone destruction also occurred in the left leg, although no injection procedures were performed. BMD showed significant decreases of 13.2% and 14.7% in the SAA1.2 and SAA1.3 groups, respectively, in week 4, and these values were restored to normal in week 6, suggesting that blood circulation of the SAA1 protein induces systematic inflammatory responses, as reported for human RA (Fig 5a & S7).

Three SAA 1 isoforms induced synovial and systemic inflammation
Histological scoring [19] and qualitative scoring of the degree of synovitis were used to assess synovial inflammation (Table 1) On the left knee joints biopsy, we did not observe significant bone erosion (Fig S9, 10).
IHC analysis confirmed SAA1 protein expression from week 1 until week 4 in the synovium, growth plate, and even deep into the bone (Fig 2e-h, Fig S2-4). Interestingly, all three SAA1 isoforms protein expression were also found in left knee joints on 1 st week, and the protein expression remained high until the 4 th week (Fig S9, 10).
3. Three SAA1 isoforms induced T cell mediated immune response and infiltration into articular joints and release of pro-inflammatory cytokines RA is characterized by T cell infiltration in the synovial membranes [20]. Flow cytometric analysis of mouse spleens in week 2 showed that SAA1.2 induced a 10.9% decrease in CD8 + T cells and an 11.3% increase in CD4 + T cells (Fig 3a, b), resulting in an increase in the CD4 + /CD8 + ratio. No changes in CD8 + or CD4 + T cells were observed in the spleen in other groups throughout the 6-week study period. On the other hand, compared to the EGFP-treated mice, all SAA1 isoforms induced changes in CD4 + and CD8 + T cells in the blood in week 4 (Fig 3c, d).
RT-PCR and ELISA results showed that interleukin (IL)-6 and IL-22 expression was remarkably stimulated in week 1 at right leg and blood circulation of mice with SAA1 isoform transfected (Fig 4a, b), consistent with the recent report that robust production of IL-22 induces SAA1 production and inflammation [21], further explaining why SAA1.2 caused the most serious bone damage (Fig 1c & d). Previous reports demonstrated that adenovirus injection (Fig 4g, h). At the same time, the MMP inhibitor TIMP-1 (tissue inhibitor of metalloproteinases 1) exhibited trends opposite to those of the MMPs level ( Fig   S5e). Interestingly, we observed SAA1 protein accumulation in key organs, including liver, lung and kidney (Fig 5c, d). In addition, similar cytokines elevation was also observed at the left leg (Fig 5b, S8 & S9), further confirming that SAA1 is circulating in blood to left leg after expression at the right knee and induce arthritis condition.

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