TY - JOUR
T1 - Myeloid-specific gene deletion of protein phosphatase 2A magnifies MyD88- and TRIF-dependent inflammation following endotoxin challenge
AU - Sun, Lei
AU - Pham, Tiffany T.
AU - Cornell, Timothy T.
AU - McDonough, Kelli L.
AU - McHugh, Walker M.
AU - Blatt, Neal B.
AU - Dahmer, Mary K.
AU - Shanley, Thomas P.
N1 - Funding Information:
This work was supported by National Institutes of Health Grant R01 GM66839-07A2 and a Jeanette Ferrantino Professorship (to T.P.S.). We thank Dr. Thomas L. Saunders at the Transgenic Animal Model Core of the University of Michigan's Biomedical Research Core Facilities for helping us to accomplish this work. The gene-knockout vector ploxPFlpneo was provided by Dr. Thom Saunders. We also acknowledge the assistance of Elizabeth Hughes, Keith Childs, Galina Gavrilina, Helena Mueller, and Debora VanHeyningen at the Transgenic Animal Core for providing the following services: electroporation injection of PP2ACα conditional-knockout gene-targeting vector into embryonic stem cells JM.8.F6 (C57BL/6N) and G418-resistant embryonic stem cell clone selection, expansion of embryonic stem cells containing PP2ACα-neo-fl allele and injection into blastocysts obtained from the mating of C57BL/6-BrdCrHsd-Tyrc females with B6 (Cg)-Tyr〈c2J〉/J males, chimera production, and subsequent breeding with Albino B6 females to achieve germline transmission of the targeted allele.
Publisher Copyright:
Copyright © 2016 by The American Association of Immunologists, Inc.
PY - 2017/1/1
Y1 - 2017/1/1
N2 - Protein phosphatase 2A (PP2A) is a member of the intracellular serine/threonine phosphatases. Innate immune cell activation triggered by pathogen-associated molecular patterns is mediated by various protein kinases, and PP2A plays a counter-regulatory role by deactivating these kinases. In this study, we generated a conditional knockout of the a isoform of the catalytic subunit of PP2A (PP2ACα). After crossing with myeloid-specific cre-expressing mice, effective gene knockout was achieved in various myeloid cells. The myeloid-specific knockout mice (lyM-PP2Afl/fl) showed higher mortality in response to endotoxin challenge and bacterial infection. Upon LPS challenge, serum levels of TNF-α, KC, IL-6, and IL-10 were significantly increased in lyM-PP2Afl/fl mice, and increased phosphorylation was observed in MAPK pathways (p38, ERK, JNK) and the NF-κB pathway (IKKα/β, NF-κB p65) in bone marrow-derived macrophages (BMDMs) from knockout mice. Heightened NF-κB activation was not associated with degradation of IκBα; instead, enhanced phosphorylation of the NF-κB p65 subunit and p38 phosphorylation-mediated TNF-α mRNA stabilization appear to contribute to the increased TNF-α expression. In addition, increased IL-10 expression appears to be due to PP2ACα-knockout-induced IKKα/β hyperactivation. Microarray experiments indicated that the Toll/IL-1R domain-containing adaptor inducing IFN-β/ TNFR-associated factor 3 pathway was highly upregulated in LPS-treated PP2ACα-knockout BMDMs, and knockout BMDMs had elevated IFN-α/β production compared with control BMDMs. Serum IFN-β levels from PP2ACα-knockout mice treated with LPS were also greater than those in controls. Thus, we demonstrate that PP2A plays an important role in regulating inflammation and survival in the setting of septic insult by targeting MyD88- and Toll/IL-1R domain-containing adaptor inducing IFN-β-dependent pathways.
AB - Protein phosphatase 2A (PP2A) is a member of the intracellular serine/threonine phosphatases. Innate immune cell activation triggered by pathogen-associated molecular patterns is mediated by various protein kinases, and PP2A plays a counter-regulatory role by deactivating these kinases. In this study, we generated a conditional knockout of the a isoform of the catalytic subunit of PP2A (PP2ACα). After crossing with myeloid-specific cre-expressing mice, effective gene knockout was achieved in various myeloid cells. The myeloid-specific knockout mice (lyM-PP2Afl/fl) showed higher mortality in response to endotoxin challenge and bacterial infection. Upon LPS challenge, serum levels of TNF-α, KC, IL-6, and IL-10 were significantly increased in lyM-PP2Afl/fl mice, and increased phosphorylation was observed in MAPK pathways (p38, ERK, JNK) and the NF-κB pathway (IKKα/β, NF-κB p65) in bone marrow-derived macrophages (BMDMs) from knockout mice. Heightened NF-κB activation was not associated with degradation of IκBα; instead, enhanced phosphorylation of the NF-κB p65 subunit and p38 phosphorylation-mediated TNF-α mRNA stabilization appear to contribute to the increased TNF-α expression. In addition, increased IL-10 expression appears to be due to PP2ACα-knockout-induced IKKα/β hyperactivation. Microarray experiments indicated that the Toll/IL-1R domain-containing adaptor inducing IFN-β/ TNFR-associated factor 3 pathway was highly upregulated in LPS-treated PP2ACα-knockout BMDMs, and knockout BMDMs had elevated IFN-α/β production compared with control BMDMs. Serum IFN-β levels from PP2ACα-knockout mice treated with LPS were also greater than those in controls. Thus, we demonstrate that PP2A plays an important role in regulating inflammation and survival in the setting of septic insult by targeting MyD88- and Toll/IL-1R domain-containing adaptor inducing IFN-β-dependent pathways.
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U2 - 10.4049/jimmunol.1600221
DO - 10.4049/jimmunol.1600221
M3 - Article
C2 - 27872207
AN - SCOPUS:85006978080
SN - 0022-1767
VL - 198
SP - 404
EP - 416
JO - Journal of Immunology
JF - Journal of Immunology
IS - 1
ER -