Decreased collagen-induced arthritis severity and adaptive immunity in MKK-6-deficient mice

University of California San Diego at La Jolla, CA 92093, USA.
Arthritis & Rheumatology (Impact Factor: 7.76). 03/2012; 64(3):678-87. DOI: 10.1002/art.33359
Source: PubMed


The MAPK kinases MKK-3 and MKK-6 regulate p38 MAPK activation in inflammatory diseases such as rheumatoid arthritis (RA). Previous studies demonstrated that MKK-3 or MKK-6 deficiency inhibits K/BxN serum-induced arthritis. However, the role of these kinases in adaptive immunity-dependent models of chronic arthritis is not known. The goal of this study was to evaluate MKK-3 and MKK-6 deficiency in the collagen-induced arthritis (CIA) model.
Wild-type (WT), MKK-3(-/-) , and MKK-6(-/-) mice were immunized with bovine type II collagen. Disease activity was evaluated by semiquantitative scoring, histologic assessment, and micro-computed tomography. Serum anticollagen antibody levels were quantified by enzyme-linked immunosorbent assay. In vitro T cell cytokine response was measured by flow cytometry and multiplex analysis. Expression of joint cytokines and matrix metalloproteinases (MMPs) was determined by quantitative polymerase chain reaction.
MKK-6 deficiency markedly reduced arthritis severity compared with that in WT mice, while the absence of MKK-3 had an intermediate effect. Joint damage was minimal in arthritic MKK-6(-/-) mice and intermediate in MKK-3(-/-) mice compared with WT mice. MKK-6(-/-) mice had modestly lower levels of pathogenic anticollagen antibodies than did WT or MKK-3(-/-) mice. In vitro T cell assays showed reduced proliferation and interleukin-17 (IL-17) production by lymph node cells from MKK-6(-/-) mice in response to type II collagen. Gene expression of synovial IL-6, MMP-3, and MMP-13 was significantly inhibited in MKK-6-deficient mice.
Reduced disease severity in MKK-6(-/-) mice correlated with decreased anticollagen antibody responses, indicating that MKK-6 is a crucial regulator of inflammatory joint destruction in CIA. MKK-6 is a potential therapeutic target in complex diseases involving adaptive immune responses, such as RA.

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Available from: Esther Cory, Jul 11, 2014
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    • "We recently showed that p38αlysM mice, which lack p38α expression in macrophages, have increased arthritis severity in passive serum transfer and antigen-induced arthritis models [12]. In contrast, MKK3- or MKK6-deficiency reduces arthritis severity and joint destruction [13,14]. In addition, MKK3, but not MKK6, is required for optimal p38 activation in synovitis, whereas MKK6-deficiency is associated with lower IL-6, IL-17 and anti-collagen antibody production [14,15]. "
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    ABSTRACT: Background Conventional p38α inhibitors have limited efficacy in rheumatoid arthritis, possibly because p38 blockade suppresses the counter-regulatory mechanisms that limit inflammation. In contrast, targeting the upstream MAP kinase kinases, MKK3 and MKK6, partially maintains p38-mediated anti-inflammatory responses in bone marrow-derived macrophages (BMDM). In this study, we explored the mechanisms that preserve anti-inflammatory gene expression by evaluating differential regulation of IL-10 and p38-dependent anti-inflammatory genes in MKK3−/−, MKK6−/−, and p38 inhibitor-treated wildtype cells. Methods BMDM from wild type (WT), MKK3−/−, and MKK6−/− mice were pre-treated with p38 inhibitor SB203580 (SB), JNK inhibitor SP600125 (SP), and/or ERK inhibitor PD98059 (PD) and stimulated with LPS. Supernatant protein levels were measured by multiplex bead immunoassay. mRNA expression was determined by qPCR and protein expression by Western blot analysis. De novo IL-10 mRNA synthesis was quantified in cells treated with ethynyl-uridine and LPS followed by reverse transcription and qPCR. mRNA half-life was measured in LPS-treated cells that were then incubated with actinomycin D ± SB203580. Results Pre-treatment of WT BMDM with p38 inhibitor significantly reduced IL-10 production in the three groups, while ERK and JNK inhibitors had minimal effects. IL-10 production was significantly decreased in MKK3−/− BMDM compared with either WT or MKK6−/− cells. IL-10 mRNA expression was modestly reduced in MKK3−/− BMDM but was preserved in MKK6−/− cells compared with WT. De novo IL-10 mRNA synthesis was inhibited in MKK3−/− and p38 inhibitor pre-treated cells, but not MKK6−/− cells compared with WT. IL-10 mRNA half-life was markedly reduced in p38 inhibitor-treated WT cells while MKK-deficiency had minimal effect. DUSP1 mRNA levels were preserved in MKK-deficient cells but not in p38 inhibitor-treated WT cells. Tristetraprolin mRNA and protein levels were reduced in p38 inhibitor-treated WT cells compared with MKK6−/− cells. Conclusion Unlike p38-inhibition, the absence of MKK6 mostly preserves IL-10 and TTP protein expression in BMDM. MKK6-deficiency also spares DUSP1 and IL-1RA, which are key negative regulators of the inflammatory response. Together, these data suggest that MKK6 is a potential therapeutic target in RA.
    Full-text · Article · May 2014 · Journal of Inflammation
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    • "The decrease in osteoclast numbers in MKK6-deficient mice could be due to decreased p38-regulated cytokine production, such as IL-6, since a direct effect on osteoclasts was not observed. The bone protection is consistent with our previous studies showing reduced inflammation and joint destruction in MKK3−/− and MKK6−/− mice, where a direct effect on osteoclasts for MKK3 and an indirect effect by blocking cytokines for MKK6 were found [22]. "
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    ABSTRACT: Bone mass is maintained by osteoclasts that resorb bone and osteoblasts that promote matrix deposition and mineralization. Bone homeostasis is altered in chronic inflammation as well as in post-menopausal loss of estrogen, which favors osteoclast activity that leads to osteoporosis. The MAPK p38α is a key regulator of bone loss and p38 inhibitors preserve bone mass by inhibiting osteoclastogenesis. p38 function is regulated by two upstream MAPK kinases, namely MKK3 and MKK6. The goal of this study was to assess the effect of MKK3- or MKK6-deficiency on osteoclastogenesis in vitro and on bone loss in ovariectomy-induced osteoporosis in mice. We demonstrated that MKK3 but not MKK6, regulates osteoclast differentiation from bone marrow cells in vitro. Expression of NFATc1, a master transcription factor in osteoclastogenesis, is decreased in cells lacking MKK3 but not MKK6. Expression of osteoclast-specific genes Cathepsin K, osteoclast-associated receptor and MMP9, was inhibited in MKK3-/- cells. The effect of MKK-deficiency on ovariectomy-induced bone loss was then evaluated in female WT, MKK3-/- and MKK6-/- mice by micro-CT analysis. Bone loss was partially inhibited in MKK3-/- as well as MKK6-/- mice, despite normal osteoclastogenesis in MKK6-/- cells. This correlated with the lower osteoclast numbers in the MKK-deficient ovariectomized mice. These studies suggest that MKK3 and MKK6 differentially regulate bone loss due to estrogen withdrawal. MKK3 directly mediates osteoclastogenesis while MKK6 likely contributes to pro-inflammatory cytokine production that promotes osteoclast formation.
    Full-text · Article · Jan 2014 · PLoS ONE
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    ABSTRACT: TNFα inhibitor therapy has greatly improved the treatment of patients with rheumatoid arthritis, however at least 30% do not respond. We aimed to investigate insertions and deletions (INDELS) associated with response to TNFα inhibitors in patients with rheumatoid arthritis (RA). In the DANBIO Registry we identified 237 TNFα inhibitor naïve patients with RA (81% women; median age 56 years; disease duration 6 years) who initiated treatment with infliximab (n = 160), adalimumab (n = 56) or etanercept (n = 21) between 1999 and 2008 according to national treatment guidelines. Clinical response was assessed at week 26 using EULAR response criteria. Based on literature, we selected 213 INDELS potentially related to RA and treatment response using the GeneVa® (Compugen) in silico database of 350,000 genetic variations in the human genome. Genomic segments were amplified by polymerase chain reaction (PCR), and genotyped by Sanger sequencing or fragment analysis. We tested the association between genotypes and EULAR good response versus no response, and EULAR good response versus moderate/no response using Fisher's exact test. At baseline the median DAS28 was 5.1. At week 26, 68 (29%) patients were EULAR good responders, while 81 (34%) and 88 (37%) patients were moderate and non-responders, respectively. A 19 base pair insertion within the CD6 gene was associated with EULAR good response vs. no response (OR = 4.43, 95% CI: 1.99-10.09, p = 7.211×10(-5)) and with EULAR good response vs. moderate/no response (OR = 4.54, 95% CI: 2.29-8.99, p = 3.336×10(-6)). A microsatellite within the syntaxin binding protein 6 (STXBP6) was associated with EULAR good response vs. no response (OR = 4.01, 95% CI: 1.92-8.49, p = 5.067×10(-5)). Genetic variations within CD6 and STXBP6 may influence response to TNFα inhibitors in patients with RA.
    Full-text · Article · Jun 2012 · PLoS ONE
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