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Zinc Finger Protein ZBTB20 Promotes Toll-Like Receptor-Triggered Innate

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Zinc Finger Protein ZBTB20 Promotes Toll-Like Receptor-Triggered Innate Zinc finger protein ZBTB20 promotes toll-like receptor-triggered innate immune responses by repressing IκBα gene transcription Xingguang Liua,1, Peng Zhanga,1, Yan Baoa,1, Yanmei Hana, Yan Wangb, Qian Zhangc, Zhenzhen Zhana, Jun Mengd, Yingke Lia, Nan Lia, Weiping J. Zhangb,2, and Xuetao Caoa,c,d,2 aNational Key Laboratory of Medical Immunology and Institute of Immunology and bDepartment of Pathophysiology, Second Military Medical University, Shanghai 200433, China; cNational Key Laboratory of Molecular Biology and Department of Immunology, Chinese Academy of Medical Sciences, Beijing 100730, China; and dInstitute of Immunology, Zhejiang University School of Medicine, Hangzhou 310058, China Edited by Genhong Cheng, University of California, Los Angeles, CA, and accepted by the Editorial Board May 21, 2013 (received for review January 24, 2013) Toll-like receptor (TLR) signaling is critical in innate response signaling is tightly regulated to maintain the immunological against invading pathogens. However, the molecular mechanisms homeostasis. Previous studies have shown that the TLR pathway for full activation of TLR-triggered innate immunity need to be is positively or negatively regulated by various signaling mole- fully elucidated. The broad complex tramtrack bric-a-brac/poxvirus cules (7–9). However, the underlying molecular mechanism and zinc finger (BTB/POZ) family is a class of transcription factors through which TLR response is properly initiated and fully ac- involved in many biological processes. However, few BTB/POZ tivated still needs to be further clarified. proteins were reported to function in innate immune response. Zinc The broad complex tramtrack bric-a-brac/poxvirus and zinc fi finger and BTB domain-containing 20 (ZBTB20), a member of BTB/POZ nger (BTB/POZ) family is a class of nuclear DNA binding family, functions in neurogenesis and represses α-fetoprotein transcription factors that is involved in a variety of biological gene transcription in liver. However, the immunological functions processes, including development, differentiation, tumorigenesis, of ZBTB20 remain unknown. Here, we found that myeloid cell- and chromatin remodeling (10, 11). However, up to now, few fi specific ZBTB20 KO mice were resistant to endotoxin shock and BTB/POZ zinc nger proteins were reported to function in in- Escherichia coli fi nate immune response. Zinc finger and BTB domain-containing -caused sepsis. ZBTB20 de ciency attenuated TLR- fi triggered production of proinflammatory cytokines and type I IFN 20 (ZBTB20), also known as HOF and zinc nger protein 288 κ α (12, 13), is identified in human dendritic cells (DCs) and named in macrophages, which attributed to higher abundance of I B pro- fi tein and impaired activity of NF-κB. Furthermore, ChIP and next as dendritic cell-derived BTB/POZ zinc nger (14). ZBTB20 is generation high-throughput DNA sequencing assay showed that a unique member of BTB/POZ family and functions primarily as ZBTB20 specifically bound to IκBα gene promoter (+1to+60 region) a transcriptional repressor by the N-terminal BTB/POZ domain and conserved C-terminal C H Krüppel-type zinc finger. By after TLR activation. ZBTB20 could inhibit IκBα gene transcription, 2 2 κ α κ generating ZBTB20 KO mice, we show that ZBTB20 is a unique govern I B protein expression, and then promote NF- B activation. IMMUNOLOGY regulator that directly binds to the α-fetoprotein (Afp) promoter Therefore, transcriptional repressor ZBTB20 is needed to promote and controls postnatal AFP repression (15). Moreover, it can full activation of TLR signaling and TLR-triggered innate immune β IκBα regulate -cell function through transcriptional suppression of response by selectively suppressing the suppressor gene fructose-1,6-bisphosphatase 1 and thus, becomes crucial for transcription. postnatal survival and glucose homeostasis in mice (16, 17). In addition, ZBTB20 is essential for the specification of CA1 field oll-like receptors (TLRs) are important pattern recognition identity in the developing hippocampus (18). Treceptors that can detect the conserved structures in pathogens Our previous studies show that ZBTB20 is widely expressed in and initiate protective innate immune responses (1). After the human hematopoietic tissues, including DCs, monocytes, B cells, recognition of pathogen-associated molecule patterns, TLRs recruit and T cells (14). However, the precise role of ZBTB20 in im- adaptor molecules, such as myeloid differentiation protein 88 and mune response, especially in TLR-triggered innate response, has toll-interleukin 1 receptor domain-containing adaptor protein- not been established yet. Here, we found that myeloid cell- inducing IFN-β (TRIF), to activate subsequent signaling pathways specific ZBTB20 KO mice were resistant to endotoxin shock and and induce the production of proinflammatory cytokines and type I Escherichia coli-caused sepsis, which was caused by the attenu- IFN (2). On stimulation, most TLRs recruit myeloid differentiation ated production of proinflammatory cytokines and type I IFN in protein 88 and use the downstream molecules, such as IL-1 TLR-triggered or bacterial-challenged macrophages. Further- receptor-associated kinases, TNF receptor-associated factor 6, more, ZBTB20 specifically bound to IκBα gene promoter, re- and TGF-β–activated kinase 1, to activate inhibitor of κB kinases pressed IκBα gene transcription, and then inhibited IκBα protein (IKKs). The IKK complex can phosphorylate IκBα and lead to expression, thus promoting NF-κB activation and leading to the degradation of IκBα by ubiquitination. Then, NF-κB, which the enhanced TLR-triggered cytokine production. Therefore, has been inhibited by IκBα, is released to translocate into nu- ZBTB20 can act as a transcriptional repressor of the inhibitory cleus and initiate the transcription of genes encoding various proinflammatory mediators (3). Apart from that, TLR3 and TLR4 can signal through a TRIF-dependent pathway, which activates Author contributions: X.L. and X.C. designed research; X.L., P.Z., Y.B., Y.H., Y.W., Q.Z., Z.Z., TNF receptor-associated factor family member-associated NF-κB J.M., Y.L., and N.L. performed research; X.L., W.J.Z., and X.C. analyzed data; and X.L., W.J.Z., activator binding kinase 1 to phosphorylate IFN regulatory factor 3 and X.C. wrote the paper. (IRF3), leading to the production of type I IFN. In addition, TRIF The authors declare no conflict of interest. canalsointeractwithTNFreceptor-associatedfactor6andmediate This article is a PNAS Direct Submission. G.C. is a guest editor invited by the Editorial NF-κB activation, leading to the proinflammatory cytokine pro- Board. duction and enhanced type I IFN production (4). 1X.L., P.Z., and Y.B. contributed equally to this work. Although full activation of TLR signaling is critical for host 2To whom correspondence may be addressed. E-mail: [email protected] or defense to eliminate the invading microbial pathogens, excessive [email protected]. activation of TLRs pathways may contribute to the pathogenesis This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of inflammatory and autoimmune diseases (5, 6). Thus, TLR 1073/pnas.1301257110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1301257110 PNAS | July 2, 2013 | vol. 110 | no. 27 | 11097–11102 Downloaded by guest on September 26, 2021 gene IκBα, promoting full activation of TLR-triggered innate mice exhibit impaired TLR-triggered inflammatory responses immune responses. and are more resistant to endotoxin shock, indicating that ZBTB20 plays an important role in the full activation of TLR-triggered Results innate immune responses. Generation of Myeloid Cell-Specific ZBTB20 KO Mice. ZBTB20 null To assess the role of ZBTB20 in the host innate response to mice, generated by homologous recombination, displayed a stark pathogen infection, MZB20KO mice were challenged with intact phenotype characterized by postnatal growth retardation, met- Gram-negative E. coli or Gram-positive Listeria monocytogenes. abolic dysfunction, and lethality (16). To specifically investigate After infection with E. coli, the production of TNF and IL-6 in the function of ZBTB20 in immune cell, especially in macro- serum of MZB20KO mice was significantly less than the pro- phages, we created myeloid cell-specific ZBTB20 KO mice duction in serum of their control littermates (Fig. 2A). Accord- (MZB20KO mice) using the LysM-Cre/loxP recombination sys- ingly, MZB20KO mice had a smaller load of E. coli bacteria fi tem, which allowed for specific and highly efficient Cre-mediated in the blood (Fig. 2B), consistent with published nding that fl deletion of loxP-flanked target genes in myeloid cells, mainly proin ammatory cytokines promote the dissemination of E. coli including mature macrophages and granulocytes. MZB20KO (20). After infection with L. monocytogenes, MZB20KO mice also fl fl fl mice were generated by repeated mating of ZBTB20 ox/ ox mice produced fewer proin ammatory cytokines and had a greater fl fl (15) with LysM-cre mice (19). MZB20KO (ZBTB20 ox/ ox/LysM-Cre) bacterial load in the spleen and liver (Fig. 2 C and D). These data fl fl fi fl and control mice (ZBTB20 ox/ ox) were viable, fertile, and nor- indicate that ZBTB20 de ciency attenuates the in ammatory in- mal in size, and they displayed no gross physical or behavioral nate response of host and protects mice from lethal challenge by abnormalities. Efficient deletion of Zbtb20 gene in MZB20KO endotoxin shock and bacteria-caused sepsis. fi peritoneal macrophages was con rmed at the mRNA and pro- fi tein levels (Fig. S1). No deletion was detected in T cells, and slight Impaired Cytokine Production in TLR-Triggered ZBTB20-De cient deletion was observed in splenic DCs from MZB20KO mice (Fig. Macrophages. Macrophages are the main mediator of TLR- triggered inflammatory responses in vivo (1). Next, we assessed S1). MZB20KO and control mice also had similar numbers of fi splenic or bone marrow-derived macrophages, DCs, and perito- whether ZBTB20 de ciency attenuated the production of pro- inflammatory cytokines and type I IFN in TLR-triggered mac- neal macrophages (Fig.
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