Cyclin-dependent kinase inhibitor p21, via its C-terminal domain, is essential for resolution of murine inflammatory arthritis

Abstract

Objective: The mechanism responsible for persistent synovial inflammation in rheumatoid arthritis (RA) is unknown. Previously, we demonstrated that expression of the cyclin-dependent kinase inhibitor p21 is reduced in synovial tissue from RA patients compared to osteoarthritis patients and that p21 is a novel suppressor of the inflammatory response in macrophages. The present study was undertaken to investigate the role and mechanism of p21-mediated suppression of experimental inflammatory arthritis.

Methods: Experimental arthritis was induced in wild-type or p21-/- (C57BL/6) mice, using the K/BxN serum-transfer model. Mice were administered p21 peptide mimetics as a prophylactic for arthritis development. Lipopolysaccharide-induced cytokine and signal transduction pathways in macrophages that were treated with p21 peptide mimetics were examined by Luminex-based assay, flow cytometry, or enzyme-linked immunosorbent assay.

Results: Enhanced and sustained development of experimental inflammatory arthritis, associated with markedly increased numbers of macrophages and severe articular destruction, was observed in p21-/- mice. Administration of a p21 peptide mimetic suppressed activation of macrophages and reduced the severity of experimental arthritis in p21-intact mice only. Mechanistically, treatment with the p21 peptide mimetic led to activation of the serine/threonine kinase Akt and subsequent reduction of the activated isoform of p38 MAPK in macrophages.

Conclusion: These are the first reported data to reveal that p21 has a key role in limiting the activation response of macrophages in an inflammatory disease such as RA. Thus, targeting p21 in macrophages may be crucial for suppressing the development and persistence of RA.

Figure 1. Increased and prolonged inflammatory arthritis and elevated cytokine in p21^−/− mice

(A) Shown is the change in ankle circumference and clinical score of arthritic mice (n=15/group at day 7, 10/group at day 14, and n=5/group at day 25). Data shown are representative of at least two independent experiments. (B) Representative day 7 ankle section stained with H & E. (C) H&E stained ankle sections were scored by a pathologist blinded to the study. Inflam.=inflammation, Synov.=synovial, Cartil.=cartilage, Lymph=lymphocytes, PMN=polymorphonuclear cells, Extra-articular=extra-articular inflammation. (D) Representative day 25 ankle sections stained for TRAP. (E) TRAP stained ankle sections were scored for TRAP-positive cells per 40× field. (F) microCT imaging of p21−/− ankles. (G, H) Serum levels of IL-6 (G) and IL-1α (H) were measured using Luminex-based assay on serum collected at day 7from arthritic ankles. Values represent mean ± SE as compared by Student's t-test (*p<0.05).

Figure 2. p21^−/− mice display increased inflammatory cell infiltrate

Arthritis was induced in Wt and p21^−/− mice as in Figure 1 and ankles (n=10/group) were prepared for histological analysis. (A–C) Representative antigen-retrieved paraffin-embedded ankle sections (Day 7) were stained with antibody to (A) CD45, (B) F4/80 or (C) PCNA. SL=synovial lining, P=pannus, B=bone, BM=bone marrow, C=cartilage. (D) The number of positive cells in the indicated region per high powered field was counted by a pathologist blinded to the study. Values represent the mean of at least 3 sections/ankle and 3 fields/section ± SE, compared by Student's t-test (*p<0.05). Macs=macrophages. Prolif.=proliferating cells.

Figure 3. aa 141–160 reduces the severity of arthritis in vivo

Arthritis was induced in Wt mice as in Figure 1. Mice were IP injected with peptide (10mg/kg) 30 minutes prior to injection of K/BxN serum and daily throughout the experiment (n=8/group). Shown is (A, C) the change in ankle circumference and (B, D) clinical score for p21^+/+ (Wt) and p21^−/− mice, respectively. Values represent mean ± SE as compared by Student's t-test (*p<0.05 for aa 141–160 as compared to Tat-Ctrl, † p<0.05 for aa 141–160 as compared to Tat, data not significant for aa 15–40, aa 46–65, or aa 63–77 as compared to Tat-Ctrl or Tat at any time point). Data shown are representative of at least two independent experiments.

Figure 4. Treatment with aa 141–160 suppresses articular destruction

Wt mice were treated with peptide, arthritis was induced as in Figure 3 and ankles (n=16/group) were prepared for histological analysis. (A) Representative ankle sections were stained with H & E. SL=synovial lining, P=pannus, B=bone, BM=bone marrow, C=cartilage. (B) H&E stained ankle sections were scored on a 0–5 scale by a pathologist blinded to the study as previously described (11, 21, 45, 46). Values represent the mean of at least 3 sections/ankle and 3 fields/section ± SE, compared by Student's t-test (*p<0.05 as compared to Tat-Ctrl, †p<0.05 as compared to Tat). Data shown are representative of at least two independent experiments.

Figure 5. Reduced proliferation and inflammatory cell infiltrate is observed in ankles from aa 141–160 treated mice

Wt mice were treated with peptide and arthritis was induced as in Figure 5 and ankles (n=16/group) were prepared for histological analysis. (A) Representative antigen-retrieved paraffin-embedded ankle sections were stained with antibody to CD45, F4/80 or PCNA. (B) The number of positive cells in the indicated region per high powered field was counted by a pathologist blinded to the study. Values represent the mean of at least 3 sections/ankle and 3 fields/section ± SE, compared by Student's t-test (*p<0.05 as compared to Tat-Ctrl, †p<0.05 as compared to Tat). Macs=macrophages. Prolif.=proliferating cells. Data shown are representative of at least two independent experiments.

Figure 6. aa 141–160 enters into macrophages and reduces production of inflammatory cytokines following TLR stimulation in vitro

(A) Peritoneal macrophages were incubated with no peptide, Tat-Ctrl, or aa 141–160 peptide conjugated to FITC for 2 hours and images were taken. (B, C) Peritoneal macrophages were incubated with no peptide or with a Tat-peptide corresponding to various domains of p21 conjugated to FITC for 2 hours at (B) 37°C or (C) 4°C or 37°C. Shown in (B) is the overlay of all the cells treated with the TAT-conjugated, FITC labeled peptides at 37°C. (D–F) Supernatants from LPS-stimulated peritoneal macrophages incubated aa 141–160 or control peptide were analyzed by ELISA for (D) TNFα, (E) IL-6, and (F) IL-1β. (G-1) Cell lysates from (D–F) were analyzed for expression of (G) phosphorylated AKT, (H) phosphorylated p38, (I) and total IκB by Luminex-based assay. Data were normalized to cell number. Values represent mean ± SE of fold change relative to untreated cells as compared by Student's t-test (*p<0.05). Data shown are representative of two independent experiments.