Pim1 promotes IFN-β production by interacting with IRF3

www.nature.com/emm ARTICLE OPEN Pim1 promotes IFN-β production by interacting with IRF3 Ryeojin Ko1,2, Jeongin Seo1, Hana Park1, Nawon Lee1 and Soo Young Lee 1,2 ✉ 1234567890();,: © The Author(s) 2022 The Pim (proviral integration site for Moloney murine leukemia virus) proteins compose a serine threonine kinase family whose members regulate cell proliferation, migration and cell survival. However, whether Pim kinases participate in innate immune responses is unclear. Here, we show for the first time that Pim1 plays an essential role in the production of interferon (IFN)-β by macrophages after their Toll-like receptor (TLR) pathway is activated by pathogen-associated molecular patterns (PAMPs). Specifically, Pim1 was quickly upregulated in an NF-κB-dependent manner after TLR stimulation with PAMPs. Pim1 deficiency reduced TLR3- or TLR4-stimulated IFN-β and IFN-stimulated gene (ISG) expression but not proinflammatory cytokine expression in macrophages. Mechanistically, Pim1 specifically upregulates IRF3 phosphorylation and nuclear translocation. However, this role is not dependent on Pim1 kinase activity. Rather, Pim1 appears to promote IRF3 phosphorylation by enhancing the formation of IFN-β signaling complexes composed of TRIF, TRAF3, TBK1, and IRF3. Poly (I:C)-treated Pim1−/− mice produced less serum IFN-β and were less likely to survive than wild-type mice. These findings show for the first time that Pim1 participates in TLR-mediated IFN-β production, thus revealing a novel target for controlling antiviral innate immune responses. Experimental & Molecular Medicine (2022) 54:2092–2103; https://doi.org/10.1038/s12276-022-00893-y INTRODUCTION Innate immune cells form the first line of host defense against pathogens because they express molecules called pattern recognition receptors (PRRs) on their surface or endosomes. PPRs recognize viral or bacterial molecules called pathogen-associated molecular patterns (PAMPs). Upon binding of PAMPs, PRRs activate multiple signaling cascades that cause innate immune cells to produce inflammatory cytokines1–3 and two key subtypes of the type I interferon (IFN) family, namely, IFN-α and IFN-β; the latter is a highly conserved cytokine that plays critical roles in antiviral innate immune responses4,5. Classical examples of PRRPAMP interactions involve Toll-like receptor 3 (TLR3) and TLR4, which are highly conserved PRRs that recognize double-stranded RNA (dsRNA) and lipopolysaccharide (LPS), respectively6,7. These PRR-PAMP interactions induce the selective recruitment of an immune adaptor protein known as toll-interleukin 1 (IL-1) receptor homology (TIR) domain-containing adaptor inducing IFN-β (TRIF), which binds to the TLR and then recruits downstream signaling molecules that ultimately induce the production of IFN-β8–10. Specifically, TRIF forms a signaling complex with TNF receptorassociated factor (TRAF) that activates tank-binding kinase 1 (TBK1)11,12, which in turn phosphorylates the master transcription factor called interferon regulatory factor 3 (IRF3)13,14. This phosphorylation event causes IRF3 to dimerize, translocate into the nucleus, and induce the expression of IFN-β. IFN-β then induces the expression of IFN-stimulated genes (ISGs) through the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway15,16. Pim is a highly conserved serine/threonine kinase with three isoforms, namely, Pim117–19, Pim220 and Pim321. The Pim1 gene contains the upstream CUG start codon, and its Pim1L and Pim1S isoforms are produced by alternative translation18. Pim kinases, which have extensive amino acid homology, are constitutively active and play critical roles in multiple cellular functions22,23, including cell cycle control24, growth25, proliferation26, migration27, apoptosis28,29 and survival30. Indeed, numerous studies have shown that all Pim kinases are oncogenic proteins that promote tumorigenesis via diverse signaling pathways31–33. Pim kinases also participate in adaptive immune responses, again via disparate mechanisms34. For example, Pim1 promotes lymphocyte proliferation and survival by suppressing apoptosis35 and promoting NFATc1 activity36. Moreover, it enhances CD8 + T-cell survival, promotes CD8 + T-cell memory, and fosters B-cell proliferation37–39. While Pim2 also promotes B-cell survival, it inhibits T-cell immune responses; this is achieved by downregulating T-cell production of pro-inflammatory cytokines40. In contrast, while Pim3 also inhibits T-cell responses, it accomplishes this by inhibiting CD4 + and CD8 + T-cell proliferation and activation41. In addition, while all Pim kinases are expressed in both Th1 and Th2 cells, their expression is higher in Th1 cells, and they promote Th1-cell differentiation from precursor Th cells42. In contrast, very little is known about the potential role of the Pim kinase family in innate immune responses. Some very limited recent evidence suggests that these kinases can also participate in this arm of the immune system, especially during viral infection. In particular, the results of two studies suggest that Pim1 may be able to modulate virus-induced type I IFN signaling, which is an important mediator of innate immunity43,44. Here, we expand this finding by conducting exploratory analyses with Pim1-knockout mice and RNA-seq analyses. We show for the first time that Pim1 facilitates the innate immune responses that are driven by TLR- 1 Department of Life Science, Ewha Womans University, Seoul, Korea. 2The Research Center for Cellular Homeostasis, Ewha Womans University, Seoul, Korea. ✉email: Received: 15 April 2022 Revised: 26 September 2022 Accepted: 3 October 2022 Published online: 29 November 2022 R. Ko et al. 2093 mediated IFN-β production. Specifically, we observed that Pim1 expression was elevated soon after TLR stimulation and that Pim1 deficiency significantly reduced the phosphorylation and nuclear translocation of IRF3. Our other findings suggest that Pim1 promotes IFN-β expression by enhancing the formation of a cell surface signaling complex composed of TRIF, TRAF3, TBK1, and IRF3; this complex then induces IRF3 phosphorylation. Importantly, the kinase activity of Pim1 was not needed for this function. Our findings thus show that Pim1 can positively regulate the TLR signaling pathway. These observations may provide insights into potential approaches to controlling antiviral responses. MATERIALS AND METHODS Mice Pim1−/− mice were generated by Macrogen via CRISPR/Cas9-mediated genome editing. All mice were on the C57BL/6 genetic background and were bred in the animal facility under specific-pathogen-free conditions. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Ewha Womans University (No. 19-006). Cells Murine BMDMs were generated by flushing bone marrow cells from the femurs and tibias of 8- to 10-week-old male C57BL/6 mice, suspending them in Dulbecco’s modified Eagle’s medium (DME (...truncated)