Physical examination
The cynomolgus monkeys with spontaneous joint abnormalities generally develop symptoms such as hunchback, muscle atrophy, lameness, tail stiffness, restricted spinal activity and higher spinal rigidity. Both spine and the peripheral joints of the diseased monkeys were found to be swollen and immobile(Figure 1A). Most of the diseased monkeys have enlarged, stiff knees with limited mobility and are unable to stretch properly. All diseased monkeys’ tails are stiff and have a bamboo-like exterior.
After monitoring closely in the animals’ postures and movements, we further measured the mobility of their joints as depicted in the images (Fig. 1B). The curvatures of the spine of diseased monkeys are significantly higher than that of the normal. When compared to the control group, the curvatures of bilateral knee joints were significantly reduced, while the stretches were significantly increased; the curvatures of the right hip joint were significantly increased, the bilateral hip extensions were significantly reduced. There were no significant differences in the curvature and extension of bilateral elbow joints. It is shown that the lesion significantly changes the mobility of these joints.
Incidence And Family Aggregation
Through physical examination, we screened out nearly 100 animals with abnormal joints and abnormal movements from about 20,000 cynomolgus monkeys, further confirmed that those animals possessed spinal lesions though X-ray examination and biochemical testing. Refined identification allowed 57 cynomolgus monkeys to be diagnosed with AS, and the incidence rate was about 0.275% in the farm, which is similar to the likelihood as in human beings. Among these AS cynomolgus monkeys, 19 were males (33.33%) and 38 were females (66.67%) (Fig. 1C). Those AS monkeys were clustered between 4 and 10 years old (38 monkeys), which is effectively converted to human beings from 16 to 40 years old. There are 6 AS monkeys (10.53%) which are less than 4 years old, which is considered the pre-adolescent period of cynomolgus. The youngest diseased animal we diagnosed was 2 years old, converted to about 8 years old as in human beings. There were 13 AS cynomolgus monkeys older than 10 years old (22.81%) with generally more severe symptoms.
We further checked the animal archives to link up for the husbandry information and found that some AS monkeys were in fact close relatives (Fig. 1D). It was found that 5 AS cynomolgus monkeys (5/57, 8.77%) had family aggregation distributed in 2 separated bloodlines. The family tree of family 1 shown in Fig. 1 clearly indicated that two granddaughters of a single male cynomolgus monkey have ankylosing spondylitis. In family 2, two grandchildren of a female cynomolgus monkey were diagnosed with ankylosing spondylitis, while the uncle of this female AS cynomolgus monkey also developed ankylosing spondylitis.
X-ray Examination And CT Scan
Radiographs reveal erosive changes at the corners of the vertebral bodies in the early stages of disease together with outgrowth of bony spurs known as syndesmophytes in the later stages. When these syndesmophytes and their adjacent vertebral bodies are fused together, the spine shall appear as a single piece and is aptly described as bamboo spine. Similar lesions can also be found for knee joints, wrist joints and at the vertebral of the tails. CT scan had indicated a general rough surface for sacroiliac joints which are fused in severe cases ( Fig. 2A). X-ray examination showed that 57 cynomolgus monkeys had different degrees of joint abnormalities in the central joint, tail joint and limb joints ( Fig. 2B). To characterize the disease progression in AS monkeys, the lesions frequencies of each joint across 2 years disease time were deduced by X-ray examination. In the first physical and X-ray examination, 46 abnormal animals were found. The lesions frequencies of each joint at the first examination were summarized in Table 1. However, during the fourth examination, there was an obvious aggravation in disease symptoms. All 57 AS animals were found to have abnormal axial and peripheral joints, especially in the spine and sacroiliac joint. The order of joint lesions frequency is: sacroiliac joint(57/57) > knee joint(55/57) > lumbar vertebra(53/57) > elbow joint(8/57) > wrist and ankle joint(6/57). The increase in lesions is suggestive to a debilitating nature of AS at these diseased joints, with a higher tendency of developing lesions at joints which would be subjected to more mechanical stress, while small joints such as fingers and toes were rarely affected. This is an overt distinguishing feature different from rheumatoid arthritis (RA).
Table 1
Statistics of lesion involvement sites in AS cynomolgus monkeys
Involvement site | Before AS(n = 46) | After AS(n = 57) |
Axial joint | | |
Sacroiliac joint | 29(63.0) | 57(100.0) |
Caudal vertebrae | 26(56.5) | 48(84.2) |
Lumbar vertebra | 23(50.0) | 53(93.0) |
Thoracic vertebra | 18(39.1) | 36(63.2) |
Cervical vertebra | 13(28.3) | 24(42.1) |
Peripheral joint | | |
Knee joint | 41(89.1) | 55(96.5) |
Elbow joint | 3(6.5) | 8(14.0) |
Wrist joint | 6(13.0) | 6(10.5) |
Ankle joint | 1(2.2) | 6(10.5) |
Toe joint | 0 | 3(5.3) |
Finger joint | 0 | 3(5.3) |
Note.Data presented as n (%). |
Comparison of the diseased progressing in AS cynomolgus monkeys by radiographic examination and the time required
We performed X-ray examination of disease animals once every six months. We found that disease progression in these animals is largely consistent with that of AS patients. Following a period of bone loss, new bone forms as spurs, which bridges the vertebrae to be fused into a single unit (Fig. 2C). By comparing the X-ray images of two-years follow-up examination, we further calculated how long it takes for these AS cynomolgus monkeys to change from bone erosion period to bone formation period (Table 2). We found that it takes about 7 months to change from bone erosion to osteophyte formation, about 10 months to change from osteophyte formation to bony bridge, while the transition from bony bridge to bamboo-like structures shall take around 12 months. It is suggested that the time from bone erosion to osteophyte formation is shorter, and it takes much longer for the osteophyte formation to develop into the final bamboo-like change. This also explains the small number of animal samples in the bone erosion period when we evaluate the bone metabolic indices of AS animals in the later study. In addition, we also counted the time required for the development of different parts of the spine in the AS monkeys. It was found that it takes around 13 months for the AS monkeys with abnormal peripheral joints to develop abnormalities in axial joints. Furthermore, it takes around 8 months to develop from having abnormal lumbar vertebras to then affect the thoracic vertebras. Thereafter, it takes around another 12 months for the affected thoracic vertebras to develop into diseased cervical vertebras.
Table 2
Statistics of disease progresses in AS cynomolgus monkeys
Disease progresses | AS(n = 57) | Length of time (Month) |
Uncertain(There were abnormalities in both mid-axis and peripheral joints when first examination) | 40(70.2) | / |
Peripheral joints to mid-axis joints | 17(29.8) | 13.07 ± 6.63 |
Lumbar vertebra to thoracic vertebra | 9 | 8.44 ± 1.33 |
Thoracic vertebra to Cervical vertebra | 3 | 12.54 ± 1.08 |
Thoracic vertebra to lumbar vertebra | 2 | 9.00 ± 1.41 |
Erosion to osteophyte in the Vertebra | 17(29.8) | 7.63 ± 1.67 |
Osteophyte to bone bridge in the Vertebra | 10(17.5) | 10.40 ± 4.67 |
Bone bridge to bamboo spine | 6(10.5) | 12.50 ± 3.99 |
Note.Data presented as n (%) |
Cytokines Of Inflammation
To further distinguish AS from other similar diseases, we checked the expression of serum cytokines in diseased animals (Fig. 3A). When compared to normal monkeys, AS monkeys showed a significant increase in ESR and CRP levels, which represents two important indicators of inflammatory activity. However, there were no significant change in MCP-1, IgM-RF, Anti-CCP antibody, PCT and ASO levels. These results suggested that animals may be excluded from rheumatoid arthritis and infectious arthritis. More importantly, serum cytokine examination revealed that IL-17 and TNF-α levels were significantly elevated in AS monkeys when compared to normal monkeys ( Fig. 3B ), while IL-1β, IL-2, IL-8, IL-6, IL-10, IL-12 and IFN-ɣ levels did not deviate significantly. Importantly, IL-17 and TNF-α elevation are deemed characteristic features of AS clinical patients. The above results suggest that the diseased animals demonstrated similar characteristics as in the AS patients in clinical settings.
Hematological Examination
Hematological examination showed that the PLT significantly increased, while HGB and MCHC significantly decreased in AS monkeys when compared to those in the control group, which indicated that the AS animal might have mild anemia symptoms ( Fig. 4 ). WBC increased significantly compared to the control group, indicating that the AS monkeys suffer inflammatory responses. Further cytology analysis in identifying individual leukocyte cell types revealed that neither leukocytes, eosinophils, basophils, nor monocytes were significantly expanded in population between AS group and control group, while lymphocytes were decreased and neutrophils were increased significantly in the AS group. It is suggested that the increase of WBC in AS cynomolgus monkeys was mainly due to the increase of neutrophils. Serum biochemical indicators showed that the levels of ALP, GLOB and TP in AS cynomolgus monkeys increased significantly when compared to the control group, while serum ALB, Ca and P decreased significantly. Changes in ALP, Ca, and P may be associated with changes in bone metabolismwhile changes in GLOB, TP, and ALB may be associated with nutritional malabsorption and chronic inflammation in AS animals.
Pathological changes of lumbar and caudal vertebrae in AS animals
The pathological changes of the caudal spine were illustrated in Fig. 5. The surface of the normal cynomolgus monkey caudal cartilage is intact and smooth, while inside the cartilage plate, chondrocytes are arranged tightly and orderly. However, the intervertebral space of the AS monkey caudal joints were much narrower when compared to the control ( Fig. 5B ); heterotopic ossification were observed in the cartilage. The cartilage was ossified from the intervertebral space side and inner layer. In severe cases, the cartilage was completely ossified. The remaining chondrocytes were irregularly arranged. Histological examination also demonstrated that the annulus fibrosus of normal disc were regularly arranged and filled with diffuse extracellular matrix, with spindlelike fibroblasts running parallel to these fibrils ( Fig. 5B ). By contrast, the annulus fibrosus of animals with AS were irregularly organized and did not contain any extracellular matrix. Furthermore, fewer fibrils were observed in these samples. Sporadically, the direct transformation of fibrous cells into woven bone was apparent ( Fig. 5B ).
In the AS cynomolgus lumbar vertebral sections, we found the following features, including cartilage destruction, chondroid metaplasia, bony spur formation, synovial hyperplasia, intra-articular fibrous stands, and vascular proliferation. These are all pathological features of AS patients observed on the clinical samples. The normal cynomolgus monkey intervertebral annulus fibrosus were arranged neatly, the chondrocytes in the cartilage plate were arranged in longitudinal rows and the cartilage plate was connected with the subchondral bone without erosion. The intervertebral disc space of the AS cynomolgus vertebral body gradually became narrow as the disease progressed, and the intervertebral space disappeared completely in the final stage when cartilage fusion.
In the early stage, thickening of the anterior ligament and synovial hyperplasia, can be observed, with inflammatory cell infiltration and obvious fibrous tissue hyperplasia( Fig. 5C ). More importantly, there are a large amount of small blood vessels and osteoclasts in these fibrous tissues ( Fig. 5Ab and Fig. 5Ca ). The fibrous tissue was infiltrated by large numbers of monocytes ( Fig. 5Cc ). Small blood vessels also occurred in the bone marrow of AS monkeys, but rarely ( Fig. 5Cb ). We compared the vessels numbers of the bone marrow and ligaments of normal and monkey HE stained sections. The result is shown in Fig. 5C. The number of anterior ligament vessels of AS monkeys was significantly increased in comparison with normal monkeys, which indicated vascular invasion in the diseased disc. The destruction of the intervertebral disc cartilage plate can be observed at the early stage, with disc destruction combined with heterotopic bone formation. As a result, bony nodules can be observed in the deep-zone of the cartilage plate near the subchondral plate( Fig. 5A ).
The spur formation area is frequently situated around the anterior vertebral ligament during the syndesmophyte formation stage, where they extend from the vertebral body edge of the upper and lower sides of the disc, surrounding the proliferating fibrous tissue. Inside the spur formation area heterotopic ossification and chondroid metaplastic foci could be found ( Fig. 5Aa ). Cells inside the chondroid metaplastic foci, also known as calcified fibrocartilage, were not densely packed. Also, cells were rarely arranged in columns or hypertrophy like normal cartilage plates. The internal structure of the spur was similar to cancellous bone, and osteoblasts were attached to the bone surface. Finally, both the original and the ectopic cartilaginous tissues were replaced by bone at the cartilage fusion stage. Here, the original cartilages were fused, and chondral fusion was the predominant mode of ankyloses ( Fig. 5A ).
Bone Formation / Bone Resorption Markers
We divided AS animals into two subgroups of bone formation group and bone erosion group according to their X-ray results. After biochemical analysis in determining the serum bio-marker levels of bone turnover, we found that three bone formation indicators (BAP, OC and PINP) and one bone resorption indicator (β-CTx) were significantly reduced in AS animals when compared to the control ( Fig. 6 ). Moreover, there were no significantly changes of the above bone turnover markers between the bone erosion subgroup and bone formation subgroup. The 25-(OH)VD3 content was upregulated with trends similar to the bone turnover serum bio-markers. In contrast, PTH and calcitonin—two important hormones in bone metabolism—had no significant change not only between the AS group and control group, but also demonstrated no change between two subgroups.
SNaPshot Sequencing
We performed genetic analysis for the 20 AS-related SNPs that have been reported in literature [14–16]. The first step is to determine whether there existed nucleotide changes between the reference genome sequences of human and cynomolgus monkeys in these reported SNPs. It was found that among these reported AS-related SNPs, 10 SNPs (rs30187, rs11209026, rs1004819, rs11465804, rs10889677, rs1495965, rs6556416, rs2297909, rs11249215, rs11616188)are identical to the reference gene sequence of human and cynomolgus monkeys. We further confirmed whether these SNPs are located on the exons of the gene. As shown in the Table 3, only 4 SNPs are located in the exon region. However, the two HLA-related SNPs, rs4349859 and rs13202464, which have the highest OR, are not in the exon region of the HLA-B gene. Since HLA-B27 was recognized as important effector of AS, we chose rs4349859 (corresponding loci: Macaca_fascicularis_5.0:4:139402704) for SNP shot sequencing though rs4349859 is located on the introns. Among the 4 SNPs located in exons, rs30187 and rs10889677 are non-synonymous coding SNPs, we performed SNP shot sequencing on rs30187 and rs10889677(corresponding loci: Macaca_fascicularis_5.0: chr6:95192910 and chr1:1600952276) in the genome of 55 monkeys (AS = 34, control = 21).
Table 3
Consistency between Homo sapiens and Macaca fascicularis for sNPs within previously confirmed risk loci
Homo sapiens SNP | Gene | Ref | OR | P | Exon variant | Consist-ency | Nonsyno-nymous | Reference |
rs27434 | ERAP1 | A | 1.19 | 5.3 × 10− 12 | √ | × | √ | [15] |
rs30187 | ERAP1 | T | 1.35 | 1.8 × 10− 27 | √ | √ | √ | [15] |
rs11209026 | IL23R | G | 1.48 | 2.3 × 10− 17 | √ | √ | √ | [15] |
rs1004819 | IL23R | G | 1.20 | 1.1 × 10− 5 | × | √ | × | [13] |
rs10489629 | IL23R | T | 0.83 | 0.00011 | × | × | × | [13] |
rs11465804 | IL23R | T | 0.68 | 0.0002 | × | √ | × | [13] |
rs1343151 | IL23R | G | 0.80 | 1.0 × 10− 5 | × | × | × | [13] |
rs10889677 | IL23R | C | 1.30 | 1.3 × 10− 6 | √ | √ | √ | [13] |
rs11209032 | IL23R | G | 1.30 | 7.5 × 10− 9 | × | × | × | [13] |
rs1495965 | IL23R | C | 1.20 | 3.1 × 10− 6 | × | √ | × | [13] |
rs4349859 | HLA-B | G | 40.80 | < 10− 200 | × | √ | × | [14] |
rs13202464 | HLA-B | G | 27.60 | < 10− 200 | × | × | × | [15] |
rs4389526 | ANTXR2 | A | 1.15 | 9.4 × 10− 8 | × | × | × | [15] |
rs6556416 | IL12B | C | 1.18 | 1.9 × 10− 8 | × | √ | × | [15] |
rs2297909 | KIF21B | G | 1.14 | 5.2 × 10− 12 | × | √ | × | [15] |
rs11249215 | RUNX3 | A | 1.19 | 9.2 × 10− 11 | × | √ | × | [15] |
rs11616188 | LTBR | A | 1.21 | 4.1 × 10− 12 | × | √ | × | [15] |
rs8070463 | TBKBP1 | C | 1.13 | 5.3 × 10− 8 | × | × | × | [15] |
rs10440635 | PTGER4 | A | 1.10 | 0.017 | × | × | × | [14] |
rs10781500 | CARD9 | T | 1.08 | 0.041 | × | × | × | [14] |
Ref., reference allele; OR, odds ratio calculated using the discovery sample unless otherwise stated; Consistency, Identity between Homo sapiens and Macaca fascicularis. |
The minor allele in AS group was considered as a risk factor. According to the results of SNP shot sequencing, there was no minor alleles detected in SNP genotyping of these loci in AS or control animals(Table 4). The genotypes of these sites are consistent with the reference genome, and also consistent in between AS and control groups. These results suggested that these 3 AS-related SNPs found in clinical studies may not be involved in the pathogenesis of AS cynomolgus.
Table 4
Association results for SNPs within previously confirmed risk loci
Gene | SNP | Ref. | AS group (n = 34) | Control group (n = 21) |
ERAP1 | rs30187 | A | 34(100%) | 21(100%) |
IL23R | rs10889677 | C | 34(100%) | 21(100%) |
HLA-B | rs4349859 | C | 34(100%) | 21(100%) |