Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Feb 16;113(7):1865-70.
doi: 10.1073/pnas.1519906113. Epub 2016 Feb 1.

Enhanced stability of tristetraprolin mRNA protects mice against immune-mediated inflammatory pathologies

Sonika Patial  1 , Alan D Curtis 2nd  2 , Wi S Lai  1 , Deborah J Stumpo  1 , Georgette D Hill  3 , Gordon P Flake  4 , Mark D Mannie  2 , Perry J Blackshear  5
Affiliations

Enhanced stability of tristetraprolin mRNA protects mice against immune-mediated inflammatory pathologies

Sonika Patial et al. Proc Natl Acad Sci U S A. .

Abstract

Tristetraprolin (TTP) is an inducible, tandem zinc-finger mRNA binding protein that binds to adenylate-uridylate-rich elements (AREs) in the 3'-untranslated regions (3'UTRs) of specific mRNAs, such as that encoding TNF, and increases their rates of deadenylation and turnover. Stabilization of Tnf mRNA and other cytokine transcripts in TTP-deficient mice results in the development of a profound, chronic inflammatory syndrome characterized by polyarticular arthritis, dermatitis, myeloid hyperplasia, and autoimmunity. To address the hypothesis that increasing endogenous levels of TTP in an intact animal might be beneficial in the treatment of inflammatory diseases, we generated a mouse model (TTPΔARE) in which a 136-base instability motif in the 3'UTR of TTP mRNA was deleted in the endogenous genetic locus. These mice appeared normal, but cultured fibroblasts and macrophages derived from them exhibited increased stability of the otherwise highly labile TTP mRNA. This resulted in increased TTP protein expression in LPS-stimulated macrophages and increased levels of TTP protein in mouse tissues. TTPΔARE mice were protected from collagen antibody-induced arthritis, exhibited significantly reduced inflammation in imiquimod-induced dermatitis, and were resistant to induction of experimental autoimmune encephalomyelitis, presumably by dampening the excessive production of proinflammatory mediators in all cases. These data suggest that increased systemic levels of TTP, secondary to increased stability of its mRNA throughout the body, can be protective against inflammatory disease in certain models and might be viewed as an attractive therapeutic target for the treatment of human inflammatory diseases.

Keywords: AU-rich elements; deadenylation; inflammation; mRNA stability.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. S1.
Fig. S1.
Generation and characterization of TTPΔARE mice. (A) Shown is the Zfp36 (TTP) mRNA 3′UTR sequence from the stop codon to the end of the transcript; the polyadenylation signal is underlined. The segment deleted in the TTPΔARE allele is highlighted in red. (B) Schematic diagrams of the WT Zfp36 allele, the design of the targeting construct, and the targeted allele after Cre-based excision. The targeting construct contained the mutated exon 2 with a 136-base deletion in the 3′UTR. (C) PCR analysis of genomic DNA from WT, heterozygous, and homozygous TTPΔARE mice. The locations of the two primers (P1 and P2) used for genotyping are shown in B. (D) Body weights of male (n = 6) and female (n = 4) mice of the indicated genotypes at 8–12 wk of age. (E) Serum levels of triglycerides (Tgl), cholesterol (Chl), HDL, LDL, and (F) alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) (n = 4), all measured at 8–12 wk of age. Data are represented as mean ± SEM.
Fig. S2.
Fig. S2.
Histopathological analysis (H&E) of tissues of TTP∆ARE and WT mice. Representative images from each tissue are shown (n = 4).
Fig. S3.
Fig. S3.
Immunohistochemical analysis of liver, spleen, and thymus of TTP∆ARE and WT mice. The following primary antibodies were used to show the presence of various populations of immune cells: Ly6G: Neutrophils, CD3: T cells, CD45: Leucocytes, Pax5: B cells, and F4/80: Macrophages. Representative images from each tissue are shown (n = 4).
Fig. 1.
Fig. 1.
TTP mRNA expression and stability, and TTP protein expression in primary cells and tissues derived from homozygous TTPΔARE mice. (A) Relative levels of TTP mRNA under basal (i.e., unstimulated) conditions in serum-deprived BMDMs (n = 3–4). (B) Time course of expression of TTP mRNA before and after stimulation (LPS; 1 µg/mL) in BMDMs. Data are expressed as a percentage of WT at 1 h (n = 3–4). (C) TTP mRNA decay in BMDMs and (D) MEFs. BMDMs or MEFs were stimulated with LPS (1 µg/mL) or 10% FBS (vol/vol), respectively, for 1 h, followed by treatment with actinomycin D. Percent remaining mRNA was measured by real-time RT-PCR (n = 4). The insets show semilogarithmic decay plots of the same data, analyzed by nonlinear regression. The approximate half-lives were: WT (BMDMs), ∼32 min; TTPΔARE (BMDMs), ∼80 min; WT (MEFs), ∼28 min; TTPΔARE (MEFs), ∼54 min. (E) TTP protein levels in BMDMs under basal and LPS- (1 µg/mL) stimulated conditions. Tubulin was used as a loading control. (F) TTP protein expression in mouse tissues. The lane labeled TTP KO contains an equal amount of protein from the respective TTP KO mouse tissue, included as a negative control. Actin was used as a loading control. Statistical analysis was performed by two-tailed Student’s t test for A, C, and D and by two-way ANOVA for B. Error bars represent SEM; *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. S4.
Fig. S4.
Expression of known or suspected TTP targets in LPS-stimulated BMDMs. (A) Cells were either left unstimulated or were stimulated with LPS (1 µg/mL), RNA was extracted, and real-time RT-PCR was performed using transcript-specific primers for Tnf, Il1b, Cxcl2, Il10, Il6, and Il23a mRNAs. Values were normalized to Gapdh mRNA, and the fold changes were calculated relative to basal levels (n = 4). Statistical analysis was performed by two-way ANOVA (**P < 0.01, ***P < 0.001). (B) Cells were either left unstimulated or were stimulated with LPS (1 µg/mL); cell culture supernatants were collected and assayed for the indicated cytokines using 5-plex Milliplex MAP mouse cytokine/chemokine magnetic bead panel (EMD Millipore). Data were normalized to the total RNA (n = 4). Statistical analysis was performed by an unpaired Student’s t test for each time point (*P < 0.05, **P < 0.01, ***P < 0.001).
Fig. S5.
Fig. S5.
Expression of TNF, IL-10, IL-1B, and CXCL2 in mouse serum in response to LPS injections. WT and TTPΔARE mice were injected i.p. with LPS at 0.5, 3, or 20 mg/kg body weight, as indicated, and then blood was collected at the indicated times and serum was assayed for cytokines (n = 5 or 6). Closed circles with solid lines represent WT and open circles with dotted lines represent TTPΔARE. Statistical analysis was performed by two-tailed unpaired Student’s t test. Error bars represent SEM; *P < 0.05, **P < 0.01, ****P < 0.0001.
Fig. 2.
Fig. 2.
Effect of the homozygous TTPΔARE mutation on CAIA. (A) Shown are the percent body weight changes and (B) clinical arthritis scores over a period of 9 d of CAIA (n = 14, WT; n = 13,TTPΔARE). (C) Representative H&E-stained sections of tarsal joints from WT and TTPΔARE mice. Arrows indicate the presence of intense inflammatory synovial cellular infiltrates in and around the joint spaces in the WT mice, but not in the TTPΔARE mice. (D) Histopathology scores were determined for three lesions (i.e., subacute periarticular inflammation, synovial cell hyperplasia, and fibrosis). Severity was graded on a four-point scale. Mean severity scores are shown (n = 7). (E) Serum levels of G-CSF and (F) IL6 from days 0, 7, and 9 of CAIA induction (n = 7). Statistical analysis was performed by two-tailed Student’s t test at each time point for A, B, E, and F and each lesion in D. Error bars represent SEM; *P < 0.05, **P < 0.01.
Fig. 3.
Fig. 3.
Inflammation in IMQ-induced dermatitis. (A) Increase in skinfold thickness, measured as the difference between skin fold thickness on day 0 and day 6 (n = 7–8). (B) Representative H&E staining of untreated (Left, control) or IMQ-treated (Middle and Right, IMQ) skin from mice of the indicated genotypes. a, acanthosis; d, dermal infiltration; pu, pustule. The right panel shows higher-power magnifications of the inset in the middle panel, demonstrating more infiltrating cells in the WT compared with the TTPΔARE section. (C) Shown are Ly6G immunostained sections from control (Left) and IMQ-treated (Right) mice of the indicated genotypes (n = 4–5). (D) Ly6G positive cells in Fig. 4C were quantitated as the number of cells in one high-power field (400×) in an area of maximal infiltration from each animal (n = 4–5). (E) Histopathology scores were determined for five lesions, namely, epidermal hyperplasia, hyperkeratosis, parakeratosis, parakeratotic inflammation, and dermal inflammation. Severity was graded on a four-point scale. Each mean score is depicted by a black line and each point represents data from one animal (n = 7–8). (F) NanoString gene expression analysis was performed on RNA isolated from affected skin. Shown are the normalized counts for the 11 transcripts that were significantly down-regulated in the TTPΔARE group compared with the WT group (n = 6). Statistical analysis was performed by two-tailed unpaired Student’s t test for A, D, E, and F. Error bars represent SEM; *P < 0.05, **P < 0.01.
Fig. 4.
Fig. 4.
Effect of the TTPΔARE mutation on EAE. (A) Shown are means ± SEM of the percent body weight loss and (B) clinical disease scores observed over a period of 30 d during the course of EAE (n = 11,WT; n = 10,TTPΔARE). (C) Shown are data on the prevalence, the cumulative disease scores (mean and median), and the maximal disease scores (mean and median) for the WT and TTPΔARE groups. Cumulative EAE scores were calculated by summing daily scores for each mouse across the designated time course of disease. Maximal scores were calculated as the most severe EAE score for each mouse. Mice that did not exhibit EAE had a score of zero for the cumulative and maximal scores, and these scores were included in the group average. Pairwise comparisons were analyzed by two-tailed t tests. Cumulative scores, P = 0.003; maximal scores, P = 0.0018.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Blackshear PJ. Tristetraprolin and other CCCH tandem zinc-finger proteins in the regulation of mRNA turnover. Biochem Soc Trans. 2002;30(Pt 6):945–952. - PubMed
    1. Brooks SA, Blackshear PJ. Tristetraprolin (TTP): Interactions with mRNA and proteins, and current thoughts on mechanisms of action. Biochim Biophys Acta. 2013;1829(6-7):666–679. - PMC - PubMed
    1. Taylor GA, et al. A pathogenetic role for TNF alpha in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency. Immunity. 1996;4(5):445–454. - PubMed
    1. Carballo E, Lai WS, Blackshear PJ. Feedback inhibition of macrophage tumor necrosis factor-alpha production by tristetraprolin. Science. 1998;281(5379):1001–1005. - PubMed
    1. Carballo E, Blackshear PJ. Roles of tumor necrosis factor-alpha receptor subtypes in the pathogenesis of the tristetraprolin-deficiency syndrome. Blood. 2001;98(8):2389–2395. - PubMed

Publication types

LinkOut - more resources