We have previously shown that Mkp-1deficient mice produce elevated TNF-a, IL-6, and IL-10 following systemic Escherichia coli infection, and they exhibited increased mortality, elevated bacterial burden, and profound metabolic alterations. To understand the function of Mkp-1 during bacterial infection, we performed RNA-sequencing analysis to compare the global gene expression between E. coliinfected wild-type and Mkp-1 2/2 mice. A large number of IFN-stimulated genes were more robustly expressed in E. coliinfected Mkp-1 2/2 mice than in wild-type mice. Multiplex analysis of the serum cytokine levels revealed profound increases in IFN-b, IFN-g, TNF-a, IL-1a and b, IL-6, IL-10, IL-17A, IL-27, and GMSF levels in E. coliinfected Mkp-1 2/2 mice relative to wildtype mice. Administration of a neutralizing Ab against the receptor for type I IFN to Mkp-1 2/2 mice prior to E. coli infection augmented mortality and disease severity. Mkp-1 2/2 bone marrowderived macrophages (BMDM) produced higher levels of IFN-b mRNA and protein than did wild-type BMDM upon treatment with LPS, E. coli, polyinosinic:polycytidylic acid, and herring sperm DNA. Augmented IFN-b induction in Mkp-1 2/2 BMDM was blocked by a p38 inhibitor but not by an JNK inhibitor. Enhanced Mkp-1 expression abolished IFN-b induction by both LPS and E. coli but had little effect on the IFN-b promoter activity in LPSstimulated RAW264.7 cells. Mkp-1 deficiency did not have an overt effect on IRF3/7 phosphorylation or IKK activation but modestly enhanced IFN-b mRNA stability in LPS-stimulated BMDM. Our results suggest that Mkp-1 regulates IFN-b production primarily through a p38-mediated mechanism and that IFN-b plays a beneficial role in E. coliinduced sepsis.

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Mkp-1 is a critical negative regulator of the inflammatory response and plays a crucial role for bacterial clearance in an animal model of sepsis. Mkp-1−/− mice exhibit augmented inflammation, increased bacterial burden, elevated organ damage, and increased mortality relative to Mkp-1+/+ mice following systemic E. coli infection. To understand the immunological functions of Mkp-1, we performed RNA-seq analysis with mouse livers. Under normal conditions, 357 genes were differentially expressed between Mkp-1+/+ and Mkp-1−/− mice. However, following E. coli infection > 5,400 genes displayed differential expression between the two groups. Among genes upregulated by E. coli infection and exacerbated by Mkp-1 knockout are IL-6, IL-10, as well as a large number of IFN-inducible genes including Mx1/2, Gbp2/3, Isg15, Usp18, Ifitm1-3, STAT3, and immune checkpoint protein PD-L1. qRT-PCR and Western blot analyses verified E. coli infection-induced PD-L1 expression and further exacerbation in Mkp-1−/− mice. Examination of the RNA-seq dataset also revealed a clear interferon signature. ELISA analyses on the serum revealed low IFN-β levels in uninfected Mkp-1+/+ and Mkp-1−/− mice, increased IFN-β levels in E. coli-infected Mkp-1+/+ mice, and further augmented IFN-β levels in E. coli-infected Mkp-1−/− mice. Similarly, Mkp-1−/− bone marrow-derived macrophages also produced more IFN-β than Mkp-1+/+ macrophages following stimulation with either LPS or heat-killed E. coli. Analysis of the signaling pathways critical for IFN-β expression indicates a marked increase in TBK1 activity. Our studies suggest that exaggerated expression of type I interferons and PD-L1 contribute to the phenotypes of Mkp-1−/− mice in the model of sepsis.

Pde3B hydrolyzes both cAMP and cGMP, important second messengers in a variety of cellular processes. Pde3B has been shown to regulate NLRP3 inflammasome activity in adipose tissue. To understand whether Pde3B plays a role in the macrophage response to bacterial infection, we derived macrophages from the bone marrow of wildtype and Pde3B knockout mice. Macrophages were then stimulated with LPS for 6 h or left untreated, and total RNA was prepared for RNA-seq analyses. Our data indicate that Pde3B expression was inhibited by LPS in macrophages. Knockout of Pde3B resulted in significant changes in the expression of a large group of genes: 427 genes were up-regulated by at least 2-fold while 428 genes were down-regulated by more than 50% in LPS stimulated macrophages as the result of Pde3B deficiency. Gene profiling indicates that a number of biological processes are affected by Pde3B knockout, including cellular ion/cation homeostasis, immune system processes, and chemotaxis. Interestingly, Pde3B deficiency disturbed the expression of several chemokines: Cxcl-1, -2, and -3 expression were decreased by 96, 60, and 75%, respectively. In contrast, Ccl-4, -7, -12, -22, Cxcl-9 and -l11 expression were enhanced 1.8, 1.5, 1.9, 2, 3, and 3-fold, respectively. Differential expression of dual specificity phosphatase (Dusp)-1, -2, -3, -4, -5, -6, -10, -11, and -16 were also seen between LPS-stimulated wildtype and Pde3B-deficient macrophages. Since the Dusps are important regulators of MAP kinases that control the expression of chemokines, alterations in the expression of Dusps provides a plausible explanation for the changes in chemokine expression. Our studies suggest that Pde3B may regulate chemokine expression via modulating MAP kinases.