Limits...
Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity.

Tun-Kyi A, Finn G, Greenwood A, Nowak M, Lee TH, Asara JM, Tsokos GC, Fitzgerald K, Israel E, Li X, Exley M, Nicholson LK, Lu KP - Nat. Immunol. (2011)

Bottom Line: Toll-like receptors (TLRs) shape innate and adaptive immunity to microorganisms.The enzyme IRAK1 transduces signals from TLRs, but mechanisms for its activation and regulation remain unknown.We found here that TLR7 and TLR9 activated the isomerase Pin1, which then bound to IRAK1; this resulted in activation of IRAK1 and facilitated its release from the receptor complex to activate the transcription factor IRF7 and induce type I interferons.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Toll-like receptors (TLRs) shape innate and adaptive immunity to microorganisms. The enzyme IRAK1 transduces signals from TLRs, but mechanisms for its activation and regulation remain unknown. We found here that TLR7 and TLR9 activated the isomerase Pin1, which then bound to IRAK1; this resulted in activation of IRAK1 and facilitated its release from the receptor complex to activate the transcription factor IRF7 and induce type I interferons. Consequently, Pin1-deficient cells and mice failed to mount TLR-mediated, interferon-dependent innate and adaptive immune responses. Given the critical role of aberrant activation of IRAK1 and type I interferons in various immune diseases, controlling IRAK1 activation via inhibition of Pin1 may represent a useful therapeutic approach.

Show MeSH

Related in: MedlinePlus

Pin1 is required for IRF7 activation and IFN-α production upon TLR ligation in vitro(a, b) Pin1 is required for IRF7 activation in response to TLR7 or TLR9 activation. Pin1 WT and KO cells transiently co-expressing a UAS(GAL)-reporter plasmid, Gal4-IRF7 and TLR7 (a) or TLR9 (b) were stimulated with R-848 or CpG, respectively, followed by luciferase assay 12 h later using renilla luciferase to normalize for transfection efficiency.(c, d) Re-expression of Pin1, but not its mutants, fully rescues impaired IRF7 activation and IFN-α production in Pin1 KO cells. Pin1 WT and KO MEFs stably expressing IRAK1 were transiently co-transfected with UAS(GAL) and Gal4-IRF7 and empty vector (EV), Pin1, WW domain mutant (W34A) or PPIase domain mutant (K63A), followed by luciferase assay (c) and IFN-α ELISA (d), with Pin1 WT MEFs stably expressing IRAK1 transfected with EV as a control. Expression levels of WT, W34A and K63A Pin1 proteins are shown below graphs in (c) and (d).(e) Overexpression of KD IRAK1 inhibits IRF7 activity in Pin1 WT, but does not affect basal IRF7 activity in Pin1 KO MEFs. Pin1 WT and KO MEFs were transiently transfected with Gal4-IRF7, UAS(Gal), MyD88 (20 ng) and various amounts of KD Irak1 or control vector, as indicated, followed by assaying IRF7 activity using Renilla as a control for normalization.(f, g) Pin1 KO or IRAK1 mutations that prevent IRAK1 from being a Pin1 substrate abolish IRF7 activation and IFN-α production. Pin1 WT and KO cells stably expressing empty vector (EV), IRAK1 or IRAK1 mutants S110A, S131, S144, S173A, 3A (S131+S144+S173A) or KD were co-transfected with UAS(GAL) and Gal4-IRF7 to assess IRF7 reporter activity (f) or with IRF7 to measure IFN-α production (g). Expression levels of IRAK1 and its various mutants are shown below the graph (f).(h, i) Pin1 KO or Irak1 mutations that prevent IRAK1 from being a Pin1 substrate abolish antiviral activity. VSV production in plaque-forming units (PFU) per ml 24 h after infection of monolayer L cells (0.1 PFU/cell) previously treated with supernatants from Pin1 WT and KO cells stably expressing EV, IRAK1 or IRAK1 mutants S110A, S131, S144, S173A, 3A or KD (h), with representative pictures of VSV plaques shown in (i). ND, not detectable. Results shown are means ± s.d. of triplicates.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3298750&req=5

Figure 6: Pin1 is required for IRF7 activation and IFN-α production upon TLR ligation in vitro(a, b) Pin1 is required for IRF7 activation in response to TLR7 or TLR9 activation. Pin1 WT and KO cells transiently co-expressing a UAS(GAL)-reporter plasmid, Gal4-IRF7 and TLR7 (a) or TLR9 (b) were stimulated with R-848 or CpG, respectively, followed by luciferase assay 12 h later using renilla luciferase to normalize for transfection efficiency.(c, d) Re-expression of Pin1, but not its mutants, fully rescues impaired IRF7 activation and IFN-α production in Pin1 KO cells. Pin1 WT and KO MEFs stably expressing IRAK1 were transiently co-transfected with UAS(GAL) and Gal4-IRF7 and empty vector (EV), Pin1, WW domain mutant (W34A) or PPIase domain mutant (K63A), followed by luciferase assay (c) and IFN-α ELISA (d), with Pin1 WT MEFs stably expressing IRAK1 transfected with EV as a control. Expression levels of WT, W34A and K63A Pin1 proteins are shown below graphs in (c) and (d).(e) Overexpression of KD IRAK1 inhibits IRF7 activity in Pin1 WT, but does not affect basal IRF7 activity in Pin1 KO MEFs. Pin1 WT and KO MEFs were transiently transfected with Gal4-IRF7, UAS(Gal), MyD88 (20 ng) and various amounts of KD Irak1 or control vector, as indicated, followed by assaying IRF7 activity using Renilla as a control for normalization.(f, g) Pin1 KO or IRAK1 mutations that prevent IRAK1 from being a Pin1 substrate abolish IRF7 activation and IFN-α production. Pin1 WT and KO cells stably expressing empty vector (EV), IRAK1 or IRAK1 mutants S110A, S131, S144, S173A, 3A (S131+S144+S173A) or KD were co-transfected with UAS(GAL) and Gal4-IRF7 to assess IRF7 reporter activity (f) or with IRF7 to measure IFN-α production (g). Expression levels of IRAK1 and its various mutants are shown below the graph (f).(h, i) Pin1 KO or Irak1 mutations that prevent IRAK1 from being a Pin1 substrate abolish antiviral activity. VSV production in plaque-forming units (PFU) per ml 24 h after infection of monolayer L cells (0.1 PFU/cell) previously treated with supernatants from Pin1 WT and KO cells stably expressing EV, IRAK1 or IRAK1 mutants S110A, S131, S144, S173A, 3A or KD (h), with representative pictures of VSV plaques shown in (i). ND, not detectable. Results shown are means ± s.d. of triplicates.

Mentions: This suggestion was further supported by our findings from IRAK1-mediated IRF7 functional assays. Specifically, Pin1 KO abolished IRF7 reporter activity following TLR7 or TLR9 stimulation (Fig. 6a, b), and these defects were fully rescued by Pin1, but not its binding-inactive- or isomerase-defective mutant, as measured by IRF7 reporter activity and IFN-α production (Fig. 6c, d). To further investigate the role of Pin1 and IRAK1 kinase activity in IRF7 activation, we co-expressed MyD88, a Gal4-IRF7 reporter construct and various amounts of KD IRAK1 in Pin1 WT and KO MEFs. IRF7 activation in WT cells decreased as the amount of transfected KD IRAK1 was increased. In contrast, IRF7 activation was consistently lower in Pin1 KO cells and unaffected by the amount of KD IRAK1 transfected (Fig. 6e). These results demonstrate that both Pin1 and IRAK1 kinase activity are necessary for activation of IRF7. These findings are consistent with the previous findings 1) that IRAK1, but not its KD mutant, phosphorylates IRF714, 2) that IRAK1 kinase activity is necessary for the transcriptional activity of IRF7, but not NF-κB14, 3) that KD IRAK1 inhibits MyD88-induced IRF7 activation in a dominant-negative manner14, and 4) that inhibition of IRAK kinase activity with a synthetic inhibitor prevents IRF7 phosphorylation, but not NF-κB phosphorylation in CpG stimulated pDCs33.


Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity.

Tun-Kyi A, Finn G, Greenwood A, Nowak M, Lee TH, Asara JM, Tsokos GC, Fitzgerald K, Israel E, Li X, Exley M, Nicholson LK, Lu KP - Nat. Immunol. (2011)

Pin1 is required for IRF7 activation and IFN-α production upon TLR ligation in vitro(a, b) Pin1 is required for IRF7 activation in response to TLR7 or TLR9 activation. Pin1 WT and KO cells transiently co-expressing a UAS(GAL)-reporter plasmid, Gal4-IRF7 and TLR7 (a) or TLR9 (b) were stimulated with R-848 or CpG, respectively, followed by luciferase assay 12 h later using renilla luciferase to normalize for transfection efficiency.(c, d) Re-expression of Pin1, but not its mutants, fully rescues impaired IRF7 activation and IFN-α production in Pin1 KO cells. Pin1 WT and KO MEFs stably expressing IRAK1 were transiently co-transfected with UAS(GAL) and Gal4-IRF7 and empty vector (EV), Pin1, WW domain mutant (W34A) or PPIase domain mutant (K63A), followed by luciferase assay (c) and IFN-α ELISA (d), with Pin1 WT MEFs stably expressing IRAK1 transfected with EV as a control. Expression levels of WT, W34A and K63A Pin1 proteins are shown below graphs in (c) and (d).(e) Overexpression of KD IRAK1 inhibits IRF7 activity in Pin1 WT, but does not affect basal IRF7 activity in Pin1 KO MEFs. Pin1 WT and KO MEFs were transiently transfected with Gal4-IRF7, UAS(Gal), MyD88 (20 ng) and various amounts of KD Irak1 or control vector, as indicated, followed by assaying IRF7 activity using Renilla as a control for normalization.(f, g) Pin1 KO or IRAK1 mutations that prevent IRAK1 from being a Pin1 substrate abolish IRF7 activation and IFN-α production. Pin1 WT and KO cells stably expressing empty vector (EV), IRAK1 or IRAK1 mutants S110A, S131, S144, S173A, 3A (S131+S144+S173A) or KD were co-transfected with UAS(GAL) and Gal4-IRF7 to assess IRF7 reporter activity (f) or with IRF7 to measure IFN-α production (g). Expression levels of IRAK1 and its various mutants are shown below the graph (f).(h, i) Pin1 KO or Irak1 mutations that prevent IRAK1 from being a Pin1 substrate abolish antiviral activity. VSV production in plaque-forming units (PFU) per ml 24 h after infection of monolayer L cells (0.1 PFU/cell) previously treated with supernatants from Pin1 WT and KO cells stably expressing EV, IRAK1 or IRAK1 mutants S110A, S131, S144, S173A, 3A or KD (h), with representative pictures of VSV plaques shown in (i). ND, not detectable. Results shown are means ± s.d. of triplicates.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3298750&req=5

Figure 6: Pin1 is required for IRF7 activation and IFN-α production upon TLR ligation in vitro(a, b) Pin1 is required for IRF7 activation in response to TLR7 or TLR9 activation. Pin1 WT and KO cells transiently co-expressing a UAS(GAL)-reporter plasmid, Gal4-IRF7 and TLR7 (a) or TLR9 (b) were stimulated with R-848 or CpG, respectively, followed by luciferase assay 12 h later using renilla luciferase to normalize for transfection efficiency.(c, d) Re-expression of Pin1, but not its mutants, fully rescues impaired IRF7 activation and IFN-α production in Pin1 KO cells. Pin1 WT and KO MEFs stably expressing IRAK1 were transiently co-transfected with UAS(GAL) and Gal4-IRF7 and empty vector (EV), Pin1, WW domain mutant (W34A) or PPIase domain mutant (K63A), followed by luciferase assay (c) and IFN-α ELISA (d), with Pin1 WT MEFs stably expressing IRAK1 transfected with EV as a control. Expression levels of WT, W34A and K63A Pin1 proteins are shown below graphs in (c) and (d).(e) Overexpression of KD IRAK1 inhibits IRF7 activity in Pin1 WT, but does not affect basal IRF7 activity in Pin1 KO MEFs. Pin1 WT and KO MEFs were transiently transfected with Gal4-IRF7, UAS(Gal), MyD88 (20 ng) and various amounts of KD Irak1 or control vector, as indicated, followed by assaying IRF7 activity using Renilla as a control for normalization.(f, g) Pin1 KO or IRAK1 mutations that prevent IRAK1 from being a Pin1 substrate abolish IRF7 activation and IFN-α production. Pin1 WT and KO cells stably expressing empty vector (EV), IRAK1 or IRAK1 mutants S110A, S131, S144, S173A, 3A (S131+S144+S173A) or KD were co-transfected with UAS(GAL) and Gal4-IRF7 to assess IRF7 reporter activity (f) or with IRF7 to measure IFN-α production (g). Expression levels of IRAK1 and its various mutants are shown below the graph (f).(h, i) Pin1 KO or Irak1 mutations that prevent IRAK1 from being a Pin1 substrate abolish antiviral activity. VSV production in plaque-forming units (PFU) per ml 24 h after infection of monolayer L cells (0.1 PFU/cell) previously treated with supernatants from Pin1 WT and KO cells stably expressing EV, IRAK1 or IRAK1 mutants S110A, S131, S144, S173A, 3A or KD (h), with representative pictures of VSV plaques shown in (i). ND, not detectable. Results shown are means ± s.d. of triplicates.
Mentions: This suggestion was further supported by our findings from IRAK1-mediated IRF7 functional assays. Specifically, Pin1 KO abolished IRF7 reporter activity following TLR7 or TLR9 stimulation (Fig. 6a, b), and these defects were fully rescued by Pin1, but not its binding-inactive- or isomerase-defective mutant, as measured by IRF7 reporter activity and IFN-α production (Fig. 6c, d). To further investigate the role of Pin1 and IRAK1 kinase activity in IRF7 activation, we co-expressed MyD88, a Gal4-IRF7 reporter construct and various amounts of KD IRAK1 in Pin1 WT and KO MEFs. IRF7 activation in WT cells decreased as the amount of transfected KD IRAK1 was increased. In contrast, IRF7 activation was consistently lower in Pin1 KO cells and unaffected by the amount of KD IRAK1 transfected (Fig. 6e). These results demonstrate that both Pin1 and IRAK1 kinase activity are necessary for activation of IRF7. These findings are consistent with the previous findings 1) that IRAK1, but not its KD mutant, phosphorylates IRF714, 2) that IRAK1 kinase activity is necessary for the transcriptional activity of IRF7, but not NF-κB14, 3) that KD IRAK1 inhibits MyD88-induced IRF7 activation in a dominant-negative manner14, and 4) that inhibition of IRAK kinase activity with a synthetic inhibitor prevents IRF7 phosphorylation, but not NF-κB phosphorylation in CpG stimulated pDCs33.

Bottom Line: Toll-like receptors (TLRs) shape innate and adaptive immunity to microorganisms.The enzyme IRAK1 transduces signals from TLRs, but mechanisms for its activation and regulation remain unknown.We found here that TLR7 and TLR9 activated the isomerase Pin1, which then bound to IRAK1; this resulted in activation of IRAK1 and facilitated its release from the receptor complex to activate the transcription factor IRF7 and induce type I interferons.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Toll-like receptors (TLRs) shape innate and adaptive immunity to microorganisms. The enzyme IRAK1 transduces signals from TLRs, but mechanisms for its activation and regulation remain unknown. We found here that TLR7 and TLR9 activated the isomerase Pin1, which then bound to IRAK1; this resulted in activation of IRAK1 and facilitated its release from the receptor complex to activate the transcription factor IRF7 and induce type I interferons. Consequently, Pin1-deficient cells and mice failed to mount TLR-mediated, interferon-dependent innate and adaptive immune responses. Given the critical role of aberrant activation of IRAK1 and type I interferons in various immune diseases, controlling IRAK1 activation via inhibition of Pin1 may represent a useful therapeutic approach.

Show MeSH
Related in: MedlinePlus