Arthritis severity locus Cia4 is an early regulator of IL-6, IL-1β, and NF-κB activators' expression in pristane-induced arthritis

Abstract

Cia4 is a locus on rat chromosome 7 that regulates disease severity and joint damage in models of rheumatoid arthritis, including pristane-induced arthritis (PIA). To identify molecular processes regulated by Cia4, synovial tissues from MHC-identical DA (severe erosive) and DA.F344(Cia4) congenics (mild nonerosive) rats were collected at preclinical and recent onset stages following the induction of PIA and analyzed for gene expression levels. Il6 levels were significantly higher in DA compared with congenics on day 10 (135-fold) after PIA induction (preclinical stage) and remained increased on days 14 (47.7-fold) and 18 (29.41-fold). Il6 increased before Il1b suggesting that Il6 could be driving Il1b expression and early synovial inflammation; 187 genes had significantly different expression levels and included inflammatory mediators increased in DA such Slpi (10.94-fold), Ccl7 (5.17-fold), and Litaf (2.09-fold). Syk or NF-κB activating and interacting genes, including Cd74 Ccl21, were increased in DA; 59 genes implicated in cancer-related phenotypes were increased in DA. Genes involved in cell metabolism, transport across membranes, and tissue protection such as Dgat1, Dhcr7, and Slc1a1 were increased in DA.F344(Cia4) congenics; 21 genes differentially expressed or expressed in only one of the strains were located within the Cia4 interval and could be the gene accounting for the arthritis effect. In conclusion, the Cia4 interval contains at least one new arthritis gene that regulates early Il6, Il1b expression, and other inflammatory mediators. This gene regulates the expression of cancer genes that could mediate the development of synovial hyperplasia and invasion, and cartilage and bone destruction.

Keywords: autoimmunity; genetic prognosis; positional; rheumatoid.

Fig. 1.

Map of the Cia4 congenic interval on rat chromosome 7. Left column shows simple sequence length polymorphism (SSLP) markers and their position in the physical map of rat chromosome 7. Black bar (45.5 Mb) identifies the region homozygous for F344 alleles.

Fig. 2.

DA.F344(Cia4) congenics develop a significantly milder form of disease in pristane- (PIA) and collagen-induced arthritis (CIA). Male and female DA.F344(Cia4) congenic rats (○) had significantly lower arthritis severity scores both in PIA (A and B) and CIA (D and E) compared with DA rats (●). E: median arthritis severity index (ASI), which provides information of cumulative arthritis severity over time, was also significantly reduced in congenic males and females, both in PIA (C) and CIA (F). *P ≤ 0.01, Mann-Whitney test for difference between arthritis severity scores at each time point and between ASI.

Fig. 3.

Histology section of joints from DA and DA.F344(Cia4) congenics. A: histologic section of a midfoot collected 18 days after the induction of pristane-induced arthritis shows pronounced synovial inflammatory infiltration in DA (×10 magnification), but not in DA.F344(Cia4) congenics (×20 magnification; hematoxylin and eosin staining).

Fig. 4.

Cia4 regulates the expression of key proinflammatory cytokines. Expression of IL-6, IL-1β, and TNF-α was measured by quantitative (q)PCR using cDNA from synovial tissue collected 10 (DA n = 8, Cia4 n = 6), 14 (DA n = 9, Cia4 n = 7), or 18 days (DA n = 5, Cia4 n = 8) postinduction of PIA. *P ≤ 0.05, #P ≤ 0.008 (t-test).

Fig. 5.

Network interactions among the differentially expressed genes. A: genes expressed in reduced levels in DA.F344(Cia4) congenics and reciprocally increased in DA and implicated in the activation or interaction with the Spleen tyrosine kinase (Syk) and NF-κB pathways. B: genes expressed in increased levels in DA.F344(Cia4) congenics and implicated in metabolism and transport across membranes and centered around the PDGF and insulin gene and pathways. Red, gene expression in increased levels in the respective strain; numbers below gene symbol: top, P value; bottom, fold-difference compared with DA. Red boxes highlight central pathways.

Fig. 6.

Validation of microarray results with qPCR. qPCR and microarray fold differences of genes expressed in increased levels in DA (n = 6, red circles) or in DA.F4344(Cia4) congenics' (n = 6, green circles) synovial tissues were significantly correlated (Pearson's correlation coefficient =0.916, P = 0.003). Exceptions were Cysltr1, Ptgs2, and Rrp7a. The microarray fold for Rpl30 was 92.76, and the qPCR fold-difference was 1.57 (qPCR P = 0.038, t-test).