- Email: [email protected]
8-mediated signaling
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Differential regulation of IL-23 production in M1 macrophages by TIR8/ SIGIRR through TLR4- or TLR7/8-mediated signaling Rui Yamaguchia,b, Arisa Sakamotoa, Takatoshi Yamamotoa, Shinji Naraharaa, Hiroyuki Sugiuchia, ⁎ Yasuo Yamaguchia, a b
Graduate School of Medical Science, Kumamoto Health Science University, Kitaku Izumi-machi 325, Kumamoto 861-5598, Japan Graduate School of Medical Science, Kumamoto University School of Medicine, Chuo-ku Honjo 1-1-1, Kumamoto 860-8556, Japan
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A B S T R A C T
Keywords: Granulocyte–macrophage colony-stimulating factor IL-12p40 IL-23 Interferon regulatory factor 5 Single immunoglobulin interleukin-1 receptorrelated molecule
Cross-talks between toll-like receptors (TLRs) including various negative regulatory mechanisms are many unknown. We investigated the differential mechanism of IL-23 production in M1 macrophages by single immunoglobulin interleukin-1 receptor-related (SIGIRR) molecule through TLR4 or TLR7/8. IL-12p40 production by M1 macrophages pretreated with human neutrophil elastase (HNE) was synergistically enhanced IL-12p40, but not IL-23 production, after exposure to lipopolysaccharide (LPS). LPS (a TLR4 agonist) induced a slight increase of IL-23 production, while Resiquimod (a TLR7/8 agonist) significantly enhanced IL-23 production. Expression of SIGIRR protein, a negative regulator of TLR4, was higher in M1 macrophages than in monocytes. Interestingly, SIGIRR siRNA induced a slight increment of IL-23 production after exposure of macrophages to LPS, while IL-23 production in response to Resiquimod was significantly upregulated by SIGIRR siRNA. Silencing SIGIRR enhanced IRF4 protein level determined by western blotting or ELISA. IRF4 siRNA dramatically restored IL-23 production after exposure to Resiquimod in macrophages transfected with SIGIRR siRNA. In conclusion, production of IL-23 is differentially regulated in M1 macrophages by SIGIRR through TLR4- or TLR7/8-mediated signaling. SIGIRR is both a negative regulator of TLR4 and a positive regulator of TLR7/8.
1. Introduction The interleukin 12 (IL-12) family includes IL-12, IL-23, IL-27, and IL-35. Among the members of this family, IL-12 and IL-23 induce Th1 cells or Th17 cells, respectively [1-3]. IL-23 is composed of two subunits, which are IL-12p40 and IL-23p19. Toll-like receptors (TLRs) influence the balance between production of different IL-12 family cytokines [1]. TLRs stimulated with various pathogens induce the production of different IL-12 family members [4]. Inflammatory cytokines are produced by stimulation with all TLRs through myeloid differentiation primary response gene 88 (MyD88) signaling. TRAF6 exerts TLR2- and TLR4-mediated proinflammatory cytokine production. Activation of TRAF6-dependent signaling is associated with induction of IL-12 [5]. In addition, the interferon regulatory factor (IRF) family is activated by MyD88 -dependent and/or -independent TLR signaling pathways. IRF5 is elevated in M1
macrophages and activates IL-23p19 transcription [6]. One of critical downstream mediators of TLR7/8 signaling is IRF5. Therefore, we investigated the mechanisms regulating IL-23 production by M1 macrophages after exposure to TLR7/8 agonists. Both innate and adaptive immunity are regulated by the TLR family. A balance between stimulatory and inhibitory immune response needs to maintain homeostatic immunity. Thus, there are many negative regulatory immune systems for TLRs [7]. Transmembrane protein regulators include TRAILR, SIGIRR, ST2, and RP105. Among them, SIGIRR is known to negatively regulate TLR4-mediated signaling. The specificity protein 1 (SP1) binds to SIGIRR proximal promoter and enhances its transcription [8]. GM-CSF also induces the activation of transcription factor SP1 [9]. IRFs are master regulators of TLR signaling. IRF4 interacts with MyD88 to negatively regulate TLR signaling and GM-CSF upregulates IRF4 expression [10]. Transcription factor Kruppel-like factor 2 (KLF2)
Abbreviations: ELISA, enzyme-linked immunosorbent assay; GM−CSF, granulocytemacrophage colony-stimulating factor; HNE, human neutrophil elastase; IL, interleukin; IRF, interferon regulatory factor; KLF2, kruppel-like factor 2; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; PAR, protease-activated receptor; SIGIRR, single immunoglobulin interleukin-1 receptor-related molecule; siRNA, small interfering RNA; SP1, specificity protein 1; TIR, toll/interleukin-1 receptor; TLR, toll-like receptor; TRAF6, tumor necrosis factor receptor-associated factor 6 ⁎ Corresponding author. E-mail address: [email protected] (Y. Yamaguchi). http://dx.doi.org/10.1016/j.cyto.2017.08.014 Received 7 May 2017; Received in revised form 14 August 2017; Accepted 19 August 2017 1043-4666/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Yamaguchi, R., Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2017.08.014
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(10 μM) for 6 h. The protein levels of IRF5 in whole-cell lysates was determined by ELISA. In addition, HNE (50 μM)- or AC264613 (10 μM)treated M1 macrophages with or without GB83 (4 μM) were stimulated with Resiquimod (5 μM) for 6hr and IL-23 levels were determined by ELISA. Moreover, SIGIRR protein levels in whole-cell lysates of monocytes or M1 macrophages were measured by ELISA (RayBiotech, Norcross, GA) with an anti-SIGIRR monoclonal antibody.
is a negative regulator of pro-inflammatory activity [11]. It has been reported that IRF4 is activated by KLF2 and that KLF2 is reciprocally upregulated by IRF4 [12]. Activation of TLR4 also induces IRF4 expression in macrophages [13], and proinflammatory cytokine production after TLR stimulation is negatively regulated by IRF4 [14]. It was reported that IRF4 interferes with MyD88/IRF5 signaling [15]. TLR7/8 signaling is mediated by IRF5 [16], and IL-12 is down-regulated by TLR7/8 while IL-23 is upregulating [17]. Cross-talk between TLRs is still not well understood. The pattern of cytokine production is dependent on engagement of different TLR with various pathogens [18]. Cellular signaling by various combinations of TLR2-9 agonists show inert, stimulatory, synergistic, or inhibitory activities. Among them, the interaction of TLR4-meadiated with TLR7/8-mediated signaling [19], which may be caused by crosstalk with these TLRs. We reported synergistic elevation of IL-12p40 by M1 macrophages pretreated with human neutrophil elastase after lipopolysaccharide (LPS) exposure. Neutrophil elastase activated AR-2 and then transactivated EGFR/TLT4 signaling [20]. This time, we examined the influence of inter-relationships among TLR4 and TLR7/8 agonists on IL-23 production by M1 macrophages.
2.5. RNA interferences with SP1, SIGIRR, or IRF4 siRNA Transfection of M1 macrophages with siRNAs for SP1 (50 nM), SIGIRR (50 nM), IRF4 (50 nM) or control siRNA-A (Santa Cruz Biotechnology, Santa Cruz, CA) was performed day 7–8 of cell culture using Lipofectamine (Life Technologies, Carlsbad, CA). IL-23 protein levels in whole-cell lysates were measured by ELISA. 2.6. Effect of siRNA for SIGIRR, SP1, or IRF4 on IL-23 production by M1 macrophages stimulated with Resiquimod
2. Materials and methods
After transfection of M1 macrophages with siRNA for SIGIRR, SP1, or IRF4, the cells were stimulated with LPS (10 ng) and/or Resiquimod (5 μM) for 6 h and IL-23 protein was measured by ELISA.
2.1. Ethics statement
2.7. Western blotting or ELISA for IRF4 and SIGIRR
The Board of Ethics in Kumamoto Health Science University approved to obtain blood from volunteers in conformity with the declaration of Helsinki after obtaining their informed consent (No. 26–20).
M1 macrophages were transfected with siRNA for SIGIRR, KLF2 or SP1. Then IRF4 or SIGIRR protein production by the transfected cells was detected by western blotting of whole-cell lysates with an antimouse monoclonal antibody for IRF4 or SIGIRR, respectively (Santa Cruz Biotechnology, Santa Cruz, CA). Whole-cell lysates of M1 macrophages stimulated with LPS (10 ng) for 6 h were utilized as the positive control. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was also detected by western blotting with an anti-GAPDH antibody (Santa Cruz Biotechnology). Furthermore, IRF4 and SIGIRR protein levels in wholecell lysates were measured by ELISA (RayBiotech or MyBioSource, respectively).
2.2. Chemicals and reagents Recombinant human GM-CSF, human neutrophil elastase (HNE) and Escherichia coli 0111:B4 lipopolysaccharide (LPS) were purchased from SERVA Electrophoresis (Heidelberg, Germany), Tocris Bioscience (Bristol, UK), or Sigma-Aldrich (St. Louis, MO) respectively. Proteaseactivated receptor (PAR)-2 agonists (AC264613), Tocris Bioscience and PAR-2 antagonist (GB83), Axon Medchem, Reston, VA were purchased to study the induction of IRF5. TLR7/8 agonist, Resiquimod, was obtained from ChemScene Chemicals, Monmouth Junction, NJ.
3. Results Stimulation of M1 macrophages with LPS enhanced IL-12p40 levels, whereas HNE (50 μM) alone did not. Synergistic elevation of IL-12p40 was observed after exposure to HNE and LPS (Fig. 1a). LPS (a TLR4 agonist) induced a slight increment of IL-23 levels. Unexpectedly, synergistic upregulation of IL-23 levels was not observed by treatment with HNE and LPS, unlike production of IL-12p40 (Fig. 1b). Resiquimod significantly upregulated IL-23 production (Fig. 2). Next, we elvaluated the roles of HNE and LPS on IL-23 production by M1 macrophages. A PAR-2 agonists, HNE or AC264613 attenuated IRF5 production by M1 macrophages, while GB83 (a PAR-2 antagonist) restored it (Fig. 3). Pretreatment of M1 macrophages with HNE or AC264613 reduced IL-23 production by these cells after stimulation with Resiquimod, whereas GB83 restored it (Fig. 4). The level of SIGIRR protein was significantly higher in M1 macrophages than in monocytes (Fig. 5). Interestingly, transfection of macrophages with SIGIRR siRNA led to a slight increment of IL-23 production after stimulation with LPS. Unexpectedly, SIGIRR siRNA dramatically blunted IL-23 production by M1 macrophages in response to Resiquimod. Importantly, silencing of IRF4 restored Resiquimod-stimulated IL-23 production by macrophages transfected with SIGIRR siRNA. Surprisingly, IL-23 levels in M1 macrophages in response to Resiquimod stimulation was diminished by adding LPS. SP1 siRNA also significantly attenuated IL-23 levels in response to Resiquimod (Fig. 6). Western blotting and ELISA showed that transfection of macrophages with SIGIRR siRNA or SP1 siRNA led to increased expression of IRF4 compared to that in untreated M1 macrophages, while transfection with KLF2 siRNA reduced IRF4 expression. Transfection with SIGIRR or SP1 siRNA reduced the expression of
2.3. Induction of M1 macrophages Peripheral blood mononuclear cells (PBMCs) were obtained from heparinized blood samples [21]. PBMCs collected using Lymphoprep gradients (Axis-Shield PoC As, Norway) were suspended with Lymphocyte medium for thawing (BBLYMPH1, Zen-Bio, Inc. Research Triangle Park, NC). The monocytes were stained with CD14-phycoerythrin (PE) mouse anti-human monoclonal antibody (Life technologies, Staley Road Grand Island, NY). The purity of monocytes was determined by Fluorescence Activated Cell Sorting (FACS), showing 86.28 ± 0.14% (mean ± SE, n=64, 83.6–89.1). M1 macrophages were obtained after monocytes stimulated with recombinant human GM-CSF on days 1, 3, and 6 of culture [22]. Macrophages (on day 9 of culture) were utilized as M1 macrophages in this study. 2.4. ELISA for IL-12p40, IL-23, IRF5, and SIGIRR M1 macrophages were incubated with HNE (50 μM) for 6 h and then treated with LPS (10 ng) for 6 h. The IL-12p40 and IL-23 protein levels in whole-cell lysates were determined by ELISA with an anti-IL12p40 antibody or anti-IL23 antibody, respectively (Abcam, Cambridge, UK). Additionally, after M1 macrophages treated with Resiquimod (5 μM) or LPS (10 ng) for 6hr, IL-23 levels were determined by ELISA. M1 macrophages were treated with HNE (50 μM) or AC264613 2
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Fig. 1. Effect of HNE and LPS on IL-12p40 or IL-23 production by macrophages M1 macrophages (on day 9 of culture) were pretreated with HNE (50 μM) for 6 h and then stimulated with LPS (10 ng) for 6 h. IL-12p40 or IL-23 protein was measured in whole-cell lysates by ELISA. (a) IL-12p40 (b) IL-23 Data were obtained from cells of three donors and represent the mean ± SE. P < 0.05; **P < 0.01 (with Bonferroni’s correction); N.S., not significant.
Fig. 2. IL-23 production by macrophages after stimulation with LPS or Resiquimod M1 macrophages (day 9) were stimulated with LPS (10 ng) or Resiquimod (5 μM) for 6 h and IL-23 protein was measured by ELISA. Data were obtained from cells of three donors and represent the mean ± SE. *P < 0.05; **P < 0.01 (with Bonferroni’s correction); N.S., not significant. Fig. 3. Effect of HNE or AC-264613 on IRF5 production by macrophages M1 macrophages (day 9) were treated with HNE (50 μM) or AC-264613 (10 μM) and IRF5 protein was measured by ELISA. After pretreatment of macrophages with GB83 (4 μM), the cells were stimulated with HNE (50 μM) or AC-264613 (10 μM) for 6 h and IRF5 protein was measured in whole-cell lysates by ELISA. Data were obtained from cells of three donors and represent the mean ± SE. *P < 0.05; **P < 0.01 (with Bonferroni’s correction); N.S., not significant.
SIGIRR protein (Fig. 7). 4. Discussion The present study demonstrated that stimulation of M1 macrophages (on day 9 of culture) with HNE and LPS led to synergistic upregulation of IL-12p40 production. IL-12 and IL-23 share the IL-12p40 subunit, but only IL-23 targets the p19 subunit. Unexpectedly, there was no synergistic increment of IL-23 production after stimulation of macrophages with both HNE and LPS, so we investigated the detailed influence of HNE and LPS on IL-23 production by M1 macrophages. While LPS (a TLR4 agonist) induced a slight increase of IL-23 production by macrophages, we found that Resiquimod (a TLR7/8 agonist) significantly upregulated IL-23 production. IRF5 is a downstream mediator of TLR7/8 signaling [23]. We found that HNE or AC264613 (a PAR-2 agonist) attenuated the IRF5 protein level in macrophages, while
GB83 (a PAR-2 antagonist) restored IRF5 after stimulation with HNE or AC264613. Pretreatment of macrophages with HNE or AC264613 diminished IL-23 production in response to stimulation with Resiquimod, whereas GB83 restored the response of IL-23 to Resiquimod. We also found that the level of SIGIRR protein was much higher in M1 macrophages than in monocytes. GM-CSF has been reported to activate transcription factor SP1 [9]. The proximal promoter region of SIGIRR has a binding site for the transcription factor SP1, a zinc finger protein that binds directly to DNA and enhances SIGIRR gene transcription [8]. This study showed that transfection of macrophages with 3
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Fig. 6. Effect of SIGIRR siRNA, IRF4 siRNA, or SP1 siRNA on IL-23 production by macrophages after stimulation with LPS and/or Resiquimod M1 macrophages (day 9) transfected with SIGIRR siRNA, IRF4 siRNA, or SP1 siRNA were stimulated with LPS (10 ng) and/or Resiquimod (5 μM) and IL-23 protein was measured by ELISA. Data were obtained from cells of three donors and represent the mean ± SE. **P < 0.01 (with Bonferroni’s correction); N.S., not significant.
Fig. 4. Effect of HNE or AC-264613 on IL-23 production by macrophages after stimulation with Resiquimod M1 macrophages (day 9) pretreated with HNE (50 μM) or AC264613 (10 μM) for 6 h in presence or absence of GB83 (4 μM) were stimulated with Resiquimod (5 μM) for 6 h and IL-23 protein was measured by ELISA. Data were obtained from cells of three donors and represent the mean ± SE. **P < 0.01 (with Bonferroni’s correction; N.S., not significant.
SIGIRR deficiency has been shown to significantly enhance the expression of IRF4 [22], which represses IRF5 [25]. In the present study, transfection of macrophages with SIGIRR siRNA or SP1 siRNA led to upregulation of IRF4 protein expression as determined by western blotting or ELISA. LPS reduced SIGIRR protein expression compared to that in untreated M1 macrophages. This finding is in agreement with the observation that stimulation by LPS reduces binding of SP1 to the SIGIRR promoter [26]. IRF4 is known to serve as an inhibitor of TLR signaling via binding to MyD88, which impairs its interaction with IRF5 and other downstream signaling elements [15]. Interestingly, we found that small interfering RNA for IRF4 dramatically restored IL-23 production by SIGIRR siRNA-transfected macrophages stimulated with Resiquimod. LPS signaling has also been reported to counteract SP1dependent promoter activation of SIGIRR via the TLR4-p38MAPK pathway [26], while GM-CSF induces the activation of SP1 [9]. Interestingly, we found that IL-23 production was significantly enhanced in response to stimulation of macrophages with Resiquimod alone, while it was blunted after stimulation with both LPS and Resiquimod. GM-CSF has been reported to induce IRF4 [27], and IRF4 also is induced by LPS through activation of TLR4 [13]. Indeed, LPS has been shown to upregulate IRF4 mRNA expression [15]. We also found that LPS enhanced the level of IRF4 protein, as determined by western blotting or ELISA. In this study, we demonstrated that small interfering RNA for IRF4 dramatically restored IL-23 production after exposure of macrophages to LPS and Resiquimod, while silencing of SP1 dramatically attenuated the response of IL-23 to Resiquimod. SIGIRR deficiency has been shown to significantly enhance IRF4 expression [25]. This study also demonstrated that transfection of macrophages with SIGIRR siRNA led to upregulation of IRF4 protein, as determined by western blotting or ELISA. Kruppel-like Factor 2 (KLF2) is a member of a large family of SP1-related transcription factors. It was reported that the SP1binding site is the functional responsive element of KLF2 [28]. KLF2 is involved in direct transactivation of the IRF4 promoter, so KLF2 is a direct target of IRF4 [12]. In the present study, transfection of macrophages with KLF2 siRNA led to a decrease of IRF4 expression, as
Fig. 5. SIGIRR protein levels in monocytes and M1 macrophages SIGIRR protein levels were measured by ELISA on day 9 of culture. Data were obtained from cells of three donors and represent the mean ± SE. **P < 0.01 (with Bonferroni’s correction).
SP1 siRNA reduced SIGIRR protein expression compared to that in untreated M1 macrophages. SIGIRR is a membrane protein with a critical role in the negative regulation of TLR4-mediated signaling [24]. Thus, stimulation of macrophages with LPS alone induced a slight increment of IL-23 production, whereas SIGIRR siRNA further increased the response of IL-23 to LPS. Unexpectedly, we found that SIGIRR siRNA significantly attenuated IL-23 production after stimulation of macrophages with Resiquimod, unlike the response to LPS. Thus, SIGIRR may be a positive regulator of TLR7/8-mediated signaling.
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Fig. 7. Western blotting and ELISA for detection of IRF4 or SIGIRR After transfection of M1 macrophages (day 9) with siRNA for SIGIRR, KLF2 or SP1, IRF4 or SIGIRR protein was detected by western blotting or ELISA of whole-cell lysates. Whole-cell lysates of M1 macrophages stimulated with LPS (10 ng) for 6 h were utilized as the positive control. The density of each IRF4 or SIGIRR band was normalized to that of GAPDH. Samples were tested in triplicate and three separate experiments were performed. Data were obtained from cells of three donors and represent the mean ± SE. *P < 0.05; **P < 0.01 (with Bonferroni’s correction); N.S., not significant (a) Representative western blot. (b) Densitometry data. (c) ELISA for IRF4. (d) ELISA for SIGIRR.
transactivation of the IRF4 promoter by KLF2 in the absence of an SP1binding site. Accordingly, IL-23 production was suppressed by IRF4.
determined by western blotting or ELISA. We also found that SIGIRR siRNA significantly attenuated IL-23 production by macrophages exposed to Resiquimod, whereas silencing of IRF4 dramatically restored IL-23 production by SIGIRR siRNA-transfected macrophages stimulated with Resiquimod. In addition, we found that silencing of SP1 significantly attenuated the response of IL-23 to stimulation with Resiquimod. These data suggests that SP1 deficiency results in increased
5. Conclusion We examined the influence of LPS and Resiquimod (TLR4 and TLR7/8 agonists) on IL-23 production by M1 macrophages. GM-CSF 5
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axis maintains myeloma cell survival, Nat. Commun. 7 (2016) 10258–10273. [13] J. Eguchi, X. Kong, M. Tenta, X. Wang, S. Kang, E.D. Rosen, Interferon regulatory factor 4 regulates obesity-induced inflammation through regulation of adipose tissue macrophage polarization, Diabetes 62 (2013) 3394–3403. [14] K. Honma, H. Udono, T. Kohno, K. Yamamoto, A. Ogawa, T. Takemori, A. Kumatori, S. Suzuki, T. Matsuyama, K. Yui, Interferon regulatory factor 4 negatively regulates the production of proinflammatory cytokines by macrophages in response to LPS, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 16001–16006. [15] H. Negishi, Y. Ohba, H. Yanai, A. Takaoka, K. Honma, K. Yui, T. Matsuyama, T. Taniguchi, K. Honda, Negative regulation of Toll-like-receptor signaling by IRF-4, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 15989–15994. [16] A. Schoenemeyer, B.J. Barnes, M.E. Mancl, E. Latz, N. Goutagny, P.M. Pitha, K.A. Fitzgerald, D.T. Golenbock, The interferon regulatory factor, IRF5, is a central mediator of toll-like receptor 7 signaling, J. Biol. Chem. 280 (2005) 17005–17012. [17] F. Gerosa, B. Baldani-Guerra, L.A. Lyakh, G. Batoni, S. Esin, R.T. Winkler-Pickett, M.R. Consolaro, M. De Marchi, D. Giachino, A. Robbiano, M. Astegiano, A. Sambataro, R.A. Kastelein, G. Carra, G. Trinchieri, Differential regulation of interleukin 12 and interleukin 23 production in human dendritic cells, J. Exp. Med. 205 (2008) 1447–1461. [18] R.S. Tan, B. Ho, B.P. Leung, J.L. Ding, TLR cross-talk confers specificity to innate immunity, Int. Rev. Immunol. 33 (2014) 443–453. [19] T.K. Ghosh, D.J. Mickelson, J.C. Solberg, K.E. Lipson, J.R. Inglefield, S.S. Alkan, TLR-TLR cross talk in human PBMC resulting in synergistic and antagonistic regulation of type-1 and 2 interferons, IL-12 and TNF-alpha, Int. Immunopharmacol. 7 (2007) 1111–1121. [20] R. Yamaguchi, T. Yamamoto, A. Sakamoto, S. Narahara, H. Sugiuchi, Y. Yamaguchi, Neutrophil elastase enhances IL-12p40 production by lipopolysaccharide-stimulated macrophages via transactivation of the PAR-2/EGFR/TLR4 signaling pathway, Blood Cells Mol. Dis. 59 (2016) 1–7. [21] R.M. Strieter, D.G. Remick, J.P.3rd Lynch, M. Genord, C. Raiford, R. Spengler, S.L. Kunkel, Differential regulation of tumor necrosis factor-alpha in human alveolar macrophages and peripheral blood monocytes: a cellular and molecular analysis, Am. J. Respir. Cell. Mol. Biol. 1 (1989) 57–63. [22] R. Yamaguchi, T. Yamamoto, A. Sakamoto, Y. Ishimaru, S. Narahara, H, E. Hirose, Y. Yamaguchi, Mechanism of interleukin-13 production by granulocyte-macrophage colony-stimulating factor-dependent macrophages via protease-activated receptor-2, Blood Cells Mol. Dis. 55 (2015) 21–26. [23] H.J. Martin, J.M. Lee, D. Walls, S.D. Hayward, Manipulation of the toll-like receptor 7 signaling pathway by Epstein-Barr virus, J. Virol. 81 (2007) 9748–9758. [24] M.F. Gulen, Z. Kang, K. Bulek, W. Youzhong, T.W. Kim, Y. Chen, C.Z. Altuntas, K. Sass Bak-Jensen, M.J. McGeachy, J.S. Do, H. Xiao, G.M. Delgoffe, B. Min, J.D. Powell, V.K. Tuohy, D.J. Cua, X. Li, The receptor SIGIRR suppresses Th17 cell proliferation via inhibition of the interleukin-1 receptor pathway and mTOR kinase activation, Immunity 32 (2010) 54–66. [25] D. Xu, F. Meyer, E. Ehlers, L. Blasnitz, L. Zhang, Interferon regulatory factor 4 (IRF4) targets IRF-5 to regulate Epstein-Barr virus transformation, J. Biol. Chem. 286 (2011) 18261–18267. [26] K. Ueno-Shuto, K. Kato, Y. Tasaki, M. Sato, K. Sato, Y. Uchida, H. Sakai, T. Ono, M.A. Suico, K. Mitsutake, N. Tokutomi, H. Kai, T. Shuto, Lipopolysaccharide decreases single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR) expression by suppressing specificity protein 1 (Sp1) via the Toll-like receptor 4 (TLR4)-p38 pathway in monocytes and neutrophils, J. Biol. Chem. 289 (2014) 18097–18109. [27] D.C. Lacey, A. Achuthan, A.J. Fleetwood, H. Dinh, J. Roiniotis, G.M. Scholz, M.W. Chang, S.K. Beckman, A.D. Cook, J.A. Hamilton, Defining GM-CSF- and macrophage-CSF-dependent macrophage responses by in vitro models, J. Immunol. 188 (2012) 5752–5765. [28] J. Wu, J.B. Lingrel, KLF2 inhibits Jurkat T leukemia cell growth via upregulation of cyclin-dependent kinase inhibitor p21WAF1/CIP1, Oncogene 23 (2004) 8088–8096.
upregulated SIGIRR expression by macrophages. The influence of crosstalk between TLR4 and TLR7/8 on IL-23 production was differentially regulated by SIGIRR. SIGIRR is both a negative regulator of TLR4 and a positive regulator of TLR7/8. Acknowledgement This study was partly supported by a Kumamoto Health Science University special fellowship grant (No. 27-A-1). Conflict of interest declaration The authors declare that there are no conflicts of interest. References [1] C.L. Langrish, B.S. McKenzie, N.J. Wilson, R. de Waal Malefyt, R.A. Kastelein, D.J. Cua, IL-12 and IL-23: master regulators of innate and adaptive immunity, Immunol. Rev. 202 (2004) 96–105. [2] C.A. Hunter, New IL-12-family members: IL-23 and IL-27, cytokines with divergent functions, Nat. Rev. Immunol. 5 (2005) 521–531. [3] R.A. Kastelein, C.A. Hunter, D.J. Cua, Discovery and biology of IL-23 and IL-27: related but functionally distinct regulators of inflammation, Annu. Rev. Immunol. 25 (2007) 221–242. [4] S. Goriely, M.F. Neurath, M. Goldman, How microorganisms tip the balance between interleukin-12 family members, Nat. Rev. Immunol. 8 (2008) 81–86. [5] N.J. Mason, J. Fiore, T. Kobayashi, K.S. Masek, Y. Choi, C.A. Hunter, TRAF6-dependent mitogen-activated protein kinase activation differentially regulates the production of interleukin-12 by macrophages in response to Toxoplasma gondii, Infect. Immun. 72 (2004) 5662–5667. [6] T. Krausgruber, K. Blazek, T. Smallie, S. Alzabin, H. Lockstone, N. Sahgal, T. Hussell, M. Feldmann, I.A. Udalova, IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses, Nat. Immunol. 12 (2011) 231–238. [7] F.Y. Liew, D. Xu, E.K. Brint, L.A. O'Neill, Negative regulation of toll-like receptormediated immune responses, Nat. Rev. Immunol. 5 (2005) 446–458. [8] C. Kadota, S. Ishihara, M.M. Aziz, M.A. Rumi, N. Oshima, Y. Mishima, I. Moriyama, T. Yuki, Y. Amano, Y. Kinoshita, Down-regulation of single immunoglobulin interleukin-1R-related molecule (SIGIRR)/TIR8 expression in intestinal epithelial cells during inflammation, Clin. Exp. Immunol. 162 (2010) 348–361. [9] Y. Ohoka, A. Yokota-Nakatsuma, N. Maeda, H. Takeuchi, M. Iwata, Retinoic acid and GM-CSF coordinately induce retinal dehydrogenase 2 (RALDH2) expression through cooperation between the RAR/RXR complex and Sp1 in dendritic cells, PLoS One. 9 (2014) e96512. [10] A. Achuthan, A.D. Cook, M.C. Lee, R. Saleh, H.W. Khiew, M.W. Chang, C. Louis, A.J. Fleetwood, D.C. Lacey, A.D. Christensen, A.T. Frye, P.Y. Lam, H. Kusano, K. Nomura, N. Steiner, I. Förster, S.L. Nutt, M. Olshansky, S.J. Turner, J.A. Hamilton, Granulocyte macrophage colony-stimulating factor induces CCL17 production via IRF4 to mediate inflammation, J. Clin. Invest. 126 (2016) 3453–3466. [11] H. Das, A. Kumar, Z. Lin, W.D. Patino, P.M. Hwang, M.W. Feinberg, P.K. Majumder, M.K. Jain, Kruppel-like factor 2 (KLF2) regulates proinflammatory activation of monocytes, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 6653–6658. [12] H. Ohguchi, T. Hideshima, M.K. Bhasin, G.T. Gorgun, L. Santo, M. Cea, M.K. Samur, N. Mimura, R. Suzuki, Y.T. Tai, R.D. Carrasco, N. Raje, P.G. Richardson, N.C. Munshi, H. Harigae, T. Sanda, J. Sakai, K.C. Anderson, The KDM3A-KLF2-IRF4
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Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/cytokine
Differential regulation of IL-23 production in M1 macrophages by TIR8/ SIGIRR through TLR4- or TLR7/8-mediated signaling Rui Yamaguchia,b, Arisa Sakamotoa, Takatoshi Yamamotoa, Shinji Naraharaa, Hiroyuki Sugiuchia, ⁎ Yasuo Yamaguchia, a b
Graduate School of Medical Science, Kumamoto Health Science University, Kitaku Izumi-machi 325, Kumamoto 861-5598, Japan Graduate School of Medical Science, Kumamoto University School of Medicine, Chuo-ku Honjo 1-1-1, Kumamoto 860-8556, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords: Granulocyte–macrophage colony-stimulating factor IL-12p40 IL-23 Interferon regulatory factor 5 Single immunoglobulin interleukin-1 receptorrelated molecule
Cross-talks between toll-like receptors (TLRs) including various negative regulatory mechanisms are many unknown. We investigated the differential mechanism of IL-23 production in M1 macrophages by single immunoglobulin interleukin-1 receptor-related (SIGIRR) molecule through TLR4 or TLR7/8. IL-12p40 production by M1 macrophages pretreated with human neutrophil elastase (HNE) was synergistically enhanced IL-12p40, but not IL-23 production, after exposure to lipopolysaccharide (LPS). LPS (a TLR4 agonist) induced a slight increase of IL-23 production, while Resiquimod (a TLR7/8 agonist) significantly enhanced IL-23 production. Expression of SIGIRR protein, a negative regulator of TLR4, was higher in M1 macrophages than in monocytes. Interestingly, SIGIRR siRNA induced a slight increment of IL-23 production after exposure of macrophages to LPS, while IL-23 production in response to Resiquimod was significantly upregulated by SIGIRR siRNA. Silencing SIGIRR enhanced IRF4 protein level determined by western blotting or ELISA. IRF4 siRNA dramatically restored IL-23 production after exposure to Resiquimod in macrophages transfected with SIGIRR siRNA. In conclusion, production of IL-23 is differentially regulated in M1 macrophages by SIGIRR through TLR4- or TLR7/8-mediated signaling. SIGIRR is both a negative regulator of TLR4 and a positive regulator of TLR7/8.
1. Introduction The interleukin 12 (IL-12) family includes IL-12, IL-23, IL-27, and IL-35. Among the members of this family, IL-12 and IL-23 induce Th1 cells or Th17 cells, respectively [1-3]. IL-23 is composed of two subunits, which are IL-12p40 and IL-23p19. Toll-like receptors (TLRs) influence the balance between production of different IL-12 family cytokines [1]. TLRs stimulated with various pathogens induce the production of different IL-12 family members [4]. Inflammatory cytokines are produced by stimulation with all TLRs through myeloid differentiation primary response gene 88 (MyD88) signaling. TRAF6 exerts TLR2- and TLR4-mediated proinflammatory cytokine production. Activation of TRAF6-dependent signaling is associated with induction of IL-12 [5]. In addition, the interferon regulatory factor (IRF) family is activated by MyD88 -dependent and/or -independent TLR signaling pathways. IRF5 is elevated in M1
macrophages and activates IL-23p19 transcription [6]. One of critical downstream mediators of TLR7/8 signaling is IRF5. Therefore, we investigated the mechanisms regulating IL-23 production by M1 macrophages after exposure to TLR7/8 agonists. Both innate and adaptive immunity are regulated by the TLR family. A balance between stimulatory and inhibitory immune response needs to maintain homeostatic immunity. Thus, there are many negative regulatory immune systems for TLRs [7]. Transmembrane protein regulators include TRAILR, SIGIRR, ST2, and RP105. Among them, SIGIRR is known to negatively regulate TLR4-mediated signaling. The specificity protein 1 (SP1) binds to SIGIRR proximal promoter and enhances its transcription [8]. GM-CSF also induces the activation of transcription factor SP1 [9]. IRFs are master regulators of TLR signaling. IRF4 interacts with MyD88 to negatively regulate TLR signaling and GM-CSF upregulates IRF4 expression [10]. Transcription factor Kruppel-like factor 2 (KLF2)
Abbreviations: ELISA, enzyme-linked immunosorbent assay; GM−CSF, granulocytemacrophage colony-stimulating factor; HNE, human neutrophil elastase; IL, interleukin; IRF, interferon regulatory factor; KLF2, kruppel-like factor 2; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; PAR, protease-activated receptor; SIGIRR, single immunoglobulin interleukin-1 receptor-related molecule; siRNA, small interfering RNA; SP1, specificity protein 1; TIR, toll/interleukin-1 receptor; TLR, toll-like receptor; TRAF6, tumor necrosis factor receptor-associated factor 6 ⁎ Corresponding author. E-mail address: [email protected] (Y. Yamaguchi). http://dx.doi.org/10.1016/j.cyto.2017.08.014 Received 7 May 2017; Received in revised form 14 August 2017; Accepted 19 August 2017 1043-4666/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Yamaguchi, R., Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2017.08.014
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(10 μM) for 6 h. The protein levels of IRF5 in whole-cell lysates was determined by ELISA. In addition, HNE (50 μM)- or AC264613 (10 μM)treated M1 macrophages with or without GB83 (4 μM) were stimulated with Resiquimod (5 μM) for 6hr and IL-23 levels were determined by ELISA. Moreover, SIGIRR protein levels in whole-cell lysates of monocytes or M1 macrophages were measured by ELISA (RayBiotech, Norcross, GA) with an anti-SIGIRR monoclonal antibody.
is a negative regulator of pro-inflammatory activity [11]. It has been reported that IRF4 is activated by KLF2 and that KLF2 is reciprocally upregulated by IRF4 [12]. Activation of TLR4 also induces IRF4 expression in macrophages [13], and proinflammatory cytokine production after TLR stimulation is negatively regulated by IRF4 [14]. It was reported that IRF4 interferes with MyD88/IRF5 signaling [15]. TLR7/8 signaling is mediated by IRF5 [16], and IL-12 is down-regulated by TLR7/8 while IL-23 is upregulating [17]. Cross-talk between TLRs is still not well understood. The pattern of cytokine production is dependent on engagement of different TLR with various pathogens [18]. Cellular signaling by various combinations of TLR2-9 agonists show inert, stimulatory, synergistic, or inhibitory activities. Among them, the interaction of TLR4-meadiated with TLR7/8-mediated signaling [19], which may be caused by crosstalk with these TLRs. We reported synergistic elevation of IL-12p40 by M1 macrophages pretreated with human neutrophil elastase after lipopolysaccharide (LPS) exposure. Neutrophil elastase activated AR-2 and then transactivated EGFR/TLT4 signaling [20]. This time, we examined the influence of inter-relationships among TLR4 and TLR7/8 agonists on IL-23 production by M1 macrophages.
2.5. RNA interferences with SP1, SIGIRR, or IRF4 siRNA Transfection of M1 macrophages with siRNAs for SP1 (50 nM), SIGIRR (50 nM), IRF4 (50 nM) or control siRNA-A (Santa Cruz Biotechnology, Santa Cruz, CA) was performed day 7–8 of cell culture using Lipofectamine (Life Technologies, Carlsbad, CA). IL-23 protein levels in whole-cell lysates were measured by ELISA. 2.6. Effect of siRNA for SIGIRR, SP1, or IRF4 on IL-23 production by M1 macrophages stimulated with Resiquimod
2. Materials and methods
After transfection of M1 macrophages with siRNA for SIGIRR, SP1, or IRF4, the cells were stimulated with LPS (10 ng) and/or Resiquimod (5 μM) for 6 h and IL-23 protein was measured by ELISA.
2.1. Ethics statement
2.7. Western blotting or ELISA for IRF4 and SIGIRR
The Board of Ethics in Kumamoto Health Science University approved to obtain blood from volunteers in conformity with the declaration of Helsinki after obtaining their informed consent (No. 26–20).
M1 macrophages were transfected with siRNA for SIGIRR, KLF2 or SP1. Then IRF4 or SIGIRR protein production by the transfected cells was detected by western blotting of whole-cell lysates with an antimouse monoclonal antibody for IRF4 or SIGIRR, respectively (Santa Cruz Biotechnology, Santa Cruz, CA). Whole-cell lysates of M1 macrophages stimulated with LPS (10 ng) for 6 h were utilized as the positive control. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was also detected by western blotting with an anti-GAPDH antibody (Santa Cruz Biotechnology). Furthermore, IRF4 and SIGIRR protein levels in wholecell lysates were measured by ELISA (RayBiotech or MyBioSource, respectively).
2.2. Chemicals and reagents Recombinant human GM-CSF, human neutrophil elastase (HNE) and Escherichia coli 0111:B4 lipopolysaccharide (LPS) were purchased from SERVA Electrophoresis (Heidelberg, Germany), Tocris Bioscience (Bristol, UK), or Sigma-Aldrich (St. Louis, MO) respectively. Proteaseactivated receptor (PAR)-2 agonists (AC264613), Tocris Bioscience and PAR-2 antagonist (GB83), Axon Medchem, Reston, VA were purchased to study the induction of IRF5. TLR7/8 agonist, Resiquimod, was obtained from ChemScene Chemicals, Monmouth Junction, NJ.
3. Results Stimulation of M1 macrophages with LPS enhanced IL-12p40 levels, whereas HNE (50 μM) alone did not. Synergistic elevation of IL-12p40 was observed after exposure to HNE and LPS (Fig. 1a). LPS (a TLR4 agonist) induced a slight increment of IL-23 levels. Unexpectedly, synergistic upregulation of IL-23 levels was not observed by treatment with HNE and LPS, unlike production of IL-12p40 (Fig. 1b). Resiquimod significantly upregulated IL-23 production (Fig. 2). Next, we elvaluated the roles of HNE and LPS on IL-23 production by M1 macrophages. A PAR-2 agonists, HNE or AC264613 attenuated IRF5 production by M1 macrophages, while GB83 (a PAR-2 antagonist) restored it (Fig. 3). Pretreatment of M1 macrophages with HNE or AC264613 reduced IL-23 production by these cells after stimulation with Resiquimod, whereas GB83 restored it (Fig. 4). The level of SIGIRR protein was significantly higher in M1 macrophages than in monocytes (Fig. 5). Interestingly, transfection of macrophages with SIGIRR siRNA led to a slight increment of IL-23 production after stimulation with LPS. Unexpectedly, SIGIRR siRNA dramatically blunted IL-23 production by M1 macrophages in response to Resiquimod. Importantly, silencing of IRF4 restored Resiquimod-stimulated IL-23 production by macrophages transfected with SIGIRR siRNA. Surprisingly, IL-23 levels in M1 macrophages in response to Resiquimod stimulation was diminished by adding LPS. SP1 siRNA also significantly attenuated IL-23 levels in response to Resiquimod (Fig. 6). Western blotting and ELISA showed that transfection of macrophages with SIGIRR siRNA or SP1 siRNA led to increased expression of IRF4 compared to that in untreated M1 macrophages, while transfection with KLF2 siRNA reduced IRF4 expression. Transfection with SIGIRR or SP1 siRNA reduced the expression of
2.3. Induction of M1 macrophages Peripheral blood mononuclear cells (PBMCs) were obtained from heparinized blood samples [21]. PBMCs collected using Lymphoprep gradients (Axis-Shield PoC As, Norway) were suspended with Lymphocyte medium for thawing (BBLYMPH1, Zen-Bio, Inc. Research Triangle Park, NC). The monocytes were stained with CD14-phycoerythrin (PE) mouse anti-human monoclonal antibody (Life technologies, Staley Road Grand Island, NY). The purity of monocytes was determined by Fluorescence Activated Cell Sorting (FACS), showing 86.28 ± 0.14% (mean ± SE, n=64, 83.6–89.1). M1 macrophages were obtained after monocytes stimulated with recombinant human GM-CSF on days 1, 3, and 6 of culture [22]. Macrophages (on day 9 of culture) were utilized as M1 macrophages in this study. 2.4. ELISA for IL-12p40, IL-23, IRF5, and SIGIRR M1 macrophages were incubated with HNE (50 μM) for 6 h and then treated with LPS (10 ng) for 6 h. The IL-12p40 and IL-23 protein levels in whole-cell lysates were determined by ELISA with an anti-IL12p40 antibody or anti-IL23 antibody, respectively (Abcam, Cambridge, UK). Additionally, after M1 macrophages treated with Resiquimod (5 μM) or LPS (10 ng) for 6hr, IL-23 levels were determined by ELISA. M1 macrophages were treated with HNE (50 μM) or AC264613 2
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Fig. 1. Effect of HNE and LPS on IL-12p40 or IL-23 production by macrophages M1 macrophages (on day 9 of culture) were pretreated with HNE (50 μM) for 6 h and then stimulated with LPS (10 ng) for 6 h. IL-12p40 or IL-23 protein was measured in whole-cell lysates by ELISA. (a) IL-12p40 (b) IL-23 Data were obtained from cells of three donors and represent the mean ± SE. P < 0.05; **P < 0.01 (with Bonferroni’s correction); N.S., not significant.
Fig. 2. IL-23 production by macrophages after stimulation with LPS or Resiquimod M1 macrophages (day 9) were stimulated with LPS (10 ng) or Resiquimod (5 μM) for 6 h and IL-23 protein was measured by ELISA. Data were obtained from cells of three donors and represent the mean ± SE. *P < 0.05; **P < 0.01 (with Bonferroni’s correction); N.S., not significant. Fig. 3. Effect of HNE or AC-264613 on IRF5 production by macrophages M1 macrophages (day 9) were treated with HNE (50 μM) or AC-264613 (10 μM) and IRF5 protein was measured by ELISA. After pretreatment of macrophages with GB83 (4 μM), the cells were stimulated with HNE (50 μM) or AC-264613 (10 μM) for 6 h and IRF5 protein was measured in whole-cell lysates by ELISA. Data were obtained from cells of three donors and represent the mean ± SE. *P < 0.05; **P < 0.01 (with Bonferroni’s correction); N.S., not significant.
SIGIRR protein (Fig. 7). 4. Discussion The present study demonstrated that stimulation of M1 macrophages (on day 9 of culture) with HNE and LPS led to synergistic upregulation of IL-12p40 production. IL-12 and IL-23 share the IL-12p40 subunit, but only IL-23 targets the p19 subunit. Unexpectedly, there was no synergistic increment of IL-23 production after stimulation of macrophages with both HNE and LPS, so we investigated the detailed influence of HNE and LPS on IL-23 production by M1 macrophages. While LPS (a TLR4 agonist) induced a slight increase of IL-23 production by macrophages, we found that Resiquimod (a TLR7/8 agonist) significantly upregulated IL-23 production. IRF5 is a downstream mediator of TLR7/8 signaling [23]. We found that HNE or AC264613 (a PAR-2 agonist) attenuated the IRF5 protein level in macrophages, while
GB83 (a PAR-2 antagonist) restored IRF5 after stimulation with HNE or AC264613. Pretreatment of macrophages with HNE or AC264613 diminished IL-23 production in response to stimulation with Resiquimod, whereas GB83 restored the response of IL-23 to Resiquimod. We also found that the level of SIGIRR protein was much higher in M1 macrophages than in monocytes. GM-CSF has been reported to activate transcription factor SP1 [9]. The proximal promoter region of SIGIRR has a binding site for the transcription factor SP1, a zinc finger protein that binds directly to DNA and enhances SIGIRR gene transcription [8]. This study showed that transfection of macrophages with 3
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Fig. 6. Effect of SIGIRR siRNA, IRF4 siRNA, or SP1 siRNA on IL-23 production by macrophages after stimulation with LPS and/or Resiquimod M1 macrophages (day 9) transfected with SIGIRR siRNA, IRF4 siRNA, or SP1 siRNA were stimulated with LPS (10 ng) and/or Resiquimod (5 μM) and IL-23 protein was measured by ELISA. Data were obtained from cells of three donors and represent the mean ± SE. **P < 0.01 (with Bonferroni’s correction); N.S., not significant.
Fig. 4. Effect of HNE or AC-264613 on IL-23 production by macrophages after stimulation with Resiquimod M1 macrophages (day 9) pretreated with HNE (50 μM) or AC264613 (10 μM) for 6 h in presence or absence of GB83 (4 μM) were stimulated with Resiquimod (5 μM) for 6 h and IL-23 protein was measured by ELISA. Data were obtained from cells of three donors and represent the mean ± SE. **P < 0.01 (with Bonferroni’s correction; N.S., not significant.
SIGIRR deficiency has been shown to significantly enhance the expression of IRF4 [22], which represses IRF5 [25]. In the present study, transfection of macrophages with SIGIRR siRNA or SP1 siRNA led to upregulation of IRF4 protein expression as determined by western blotting or ELISA. LPS reduced SIGIRR protein expression compared to that in untreated M1 macrophages. This finding is in agreement with the observation that stimulation by LPS reduces binding of SP1 to the SIGIRR promoter [26]. IRF4 is known to serve as an inhibitor of TLR signaling via binding to MyD88, which impairs its interaction with IRF5 and other downstream signaling elements [15]. Interestingly, we found that small interfering RNA for IRF4 dramatically restored IL-23 production by SIGIRR siRNA-transfected macrophages stimulated with Resiquimod. LPS signaling has also been reported to counteract SP1dependent promoter activation of SIGIRR via the TLR4-p38MAPK pathway [26], while GM-CSF induces the activation of SP1 [9]. Interestingly, we found that IL-23 production was significantly enhanced in response to stimulation of macrophages with Resiquimod alone, while it was blunted after stimulation with both LPS and Resiquimod. GM-CSF has been reported to induce IRF4 [27], and IRF4 also is induced by LPS through activation of TLR4 [13]. Indeed, LPS has been shown to upregulate IRF4 mRNA expression [15]. We also found that LPS enhanced the level of IRF4 protein, as determined by western blotting or ELISA. In this study, we demonstrated that small interfering RNA for IRF4 dramatically restored IL-23 production after exposure of macrophages to LPS and Resiquimod, while silencing of SP1 dramatically attenuated the response of IL-23 to Resiquimod. SIGIRR deficiency has been shown to significantly enhance IRF4 expression [25]. This study also demonstrated that transfection of macrophages with SIGIRR siRNA led to upregulation of IRF4 protein, as determined by western blotting or ELISA. Kruppel-like Factor 2 (KLF2) is a member of a large family of SP1-related transcription factors. It was reported that the SP1binding site is the functional responsive element of KLF2 [28]. KLF2 is involved in direct transactivation of the IRF4 promoter, so KLF2 is a direct target of IRF4 [12]. In the present study, transfection of macrophages with KLF2 siRNA led to a decrease of IRF4 expression, as
Fig. 5. SIGIRR protein levels in monocytes and M1 macrophages SIGIRR protein levels were measured by ELISA on day 9 of culture. Data were obtained from cells of three donors and represent the mean ± SE. **P < 0.01 (with Bonferroni’s correction).
SP1 siRNA reduced SIGIRR protein expression compared to that in untreated M1 macrophages. SIGIRR is a membrane protein with a critical role in the negative regulation of TLR4-mediated signaling [24]. Thus, stimulation of macrophages with LPS alone induced a slight increment of IL-23 production, whereas SIGIRR siRNA further increased the response of IL-23 to LPS. Unexpectedly, we found that SIGIRR siRNA significantly attenuated IL-23 production after stimulation of macrophages with Resiquimod, unlike the response to LPS. Thus, SIGIRR may be a positive regulator of TLR7/8-mediated signaling.
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Fig. 7. Western blotting and ELISA for detection of IRF4 or SIGIRR After transfection of M1 macrophages (day 9) with siRNA for SIGIRR, KLF2 or SP1, IRF4 or SIGIRR protein was detected by western blotting or ELISA of whole-cell lysates. Whole-cell lysates of M1 macrophages stimulated with LPS (10 ng) for 6 h were utilized as the positive control. The density of each IRF4 or SIGIRR band was normalized to that of GAPDH. Samples were tested in triplicate and three separate experiments were performed. Data were obtained from cells of three donors and represent the mean ± SE. *P < 0.05; **P < 0.01 (with Bonferroni’s correction); N.S., not significant (a) Representative western blot. (b) Densitometry data. (c) ELISA for IRF4. (d) ELISA for SIGIRR.
transactivation of the IRF4 promoter by KLF2 in the absence of an SP1binding site. Accordingly, IL-23 production was suppressed by IRF4.
determined by western blotting or ELISA. We also found that SIGIRR siRNA significantly attenuated IL-23 production by macrophages exposed to Resiquimod, whereas silencing of IRF4 dramatically restored IL-23 production by SIGIRR siRNA-transfected macrophages stimulated with Resiquimod. In addition, we found that silencing of SP1 significantly attenuated the response of IL-23 to stimulation with Resiquimod. These data suggests that SP1 deficiency results in increased
5. Conclusion We examined the influence of LPS and Resiquimod (TLR4 and TLR7/8 agonists) on IL-23 production by M1 macrophages. GM-CSF 5
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axis maintains myeloma cell survival, Nat. Commun. 7 (2016) 10258–10273. [13] J. Eguchi, X. Kong, M. Tenta, X. Wang, S. Kang, E.D. Rosen, Interferon regulatory factor 4 regulates obesity-induced inflammation through regulation of adipose tissue macrophage polarization, Diabetes 62 (2013) 3394–3403. [14] K. Honma, H. Udono, T. Kohno, K. Yamamoto, A. Ogawa, T. Takemori, A. Kumatori, S. Suzuki, T. Matsuyama, K. Yui, Interferon regulatory factor 4 negatively regulates the production of proinflammatory cytokines by macrophages in response to LPS, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 16001–16006. [15] H. Negishi, Y. Ohba, H. Yanai, A. Takaoka, K. Honma, K. Yui, T. Matsuyama, T. Taniguchi, K. Honda, Negative regulation of Toll-like-receptor signaling by IRF-4, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 15989–15994. [16] A. Schoenemeyer, B.J. Barnes, M.E. Mancl, E. Latz, N. Goutagny, P.M. Pitha, K.A. Fitzgerald, D.T. Golenbock, The interferon regulatory factor, IRF5, is a central mediator of toll-like receptor 7 signaling, J. Biol. Chem. 280 (2005) 17005–17012. [17] F. Gerosa, B. Baldani-Guerra, L.A. Lyakh, G. Batoni, S. Esin, R.T. Winkler-Pickett, M.R. Consolaro, M. De Marchi, D. Giachino, A. Robbiano, M. Astegiano, A. Sambataro, R.A. Kastelein, G. Carra, G. Trinchieri, Differential regulation of interleukin 12 and interleukin 23 production in human dendritic cells, J. Exp. Med. 205 (2008) 1447–1461. [18] R.S. Tan, B. Ho, B.P. Leung, J.L. Ding, TLR cross-talk confers specificity to innate immunity, Int. Rev. Immunol. 33 (2014) 443–453. [19] T.K. Ghosh, D.J. Mickelson, J.C. Solberg, K.E. Lipson, J.R. Inglefield, S.S. Alkan, TLR-TLR cross talk in human PBMC resulting in synergistic and antagonistic regulation of type-1 and 2 interferons, IL-12 and TNF-alpha, Int. Immunopharmacol. 7 (2007) 1111–1121. [20] R. Yamaguchi, T. Yamamoto, A. Sakamoto, S. Narahara, H. Sugiuchi, Y. Yamaguchi, Neutrophil elastase enhances IL-12p40 production by lipopolysaccharide-stimulated macrophages via transactivation of the PAR-2/EGFR/TLR4 signaling pathway, Blood Cells Mol. Dis. 59 (2016) 1–7. [21] R.M. Strieter, D.G. Remick, J.P.3rd Lynch, M. Genord, C. Raiford, R. Spengler, S.L. Kunkel, Differential regulation of tumor necrosis factor-alpha in human alveolar macrophages and peripheral blood monocytes: a cellular and molecular analysis, Am. J. Respir. Cell. Mol. Biol. 1 (1989) 57–63. [22] R. Yamaguchi, T. Yamamoto, A. Sakamoto, Y. Ishimaru, S. Narahara, H, E. Hirose, Y. Yamaguchi, Mechanism of interleukin-13 production by granulocyte-macrophage colony-stimulating factor-dependent macrophages via protease-activated receptor-2, Blood Cells Mol. Dis. 55 (2015) 21–26. [23] H.J. Martin, J.M. Lee, D. Walls, S.D. Hayward, Manipulation of the toll-like receptor 7 signaling pathway by Epstein-Barr virus, J. Virol. 81 (2007) 9748–9758. [24] M.F. Gulen, Z. Kang, K. Bulek, W. Youzhong, T.W. Kim, Y. Chen, C.Z. Altuntas, K. Sass Bak-Jensen, M.J. McGeachy, J.S. Do, H. Xiao, G.M. Delgoffe, B. Min, J.D. Powell, V.K. Tuohy, D.J. Cua, X. Li, The receptor SIGIRR suppresses Th17 cell proliferation via inhibition of the interleukin-1 receptor pathway and mTOR kinase activation, Immunity 32 (2010) 54–66. [25] D. Xu, F. Meyer, E. Ehlers, L. Blasnitz, L. Zhang, Interferon regulatory factor 4 (IRF4) targets IRF-5 to regulate Epstein-Barr virus transformation, J. Biol. Chem. 286 (2011) 18261–18267. [26] K. Ueno-Shuto, K. Kato, Y. Tasaki, M. Sato, K. Sato, Y. Uchida, H. Sakai, T. Ono, M.A. Suico, K. Mitsutake, N. Tokutomi, H. Kai, T. Shuto, Lipopolysaccharide decreases single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR) expression by suppressing specificity protein 1 (Sp1) via the Toll-like receptor 4 (TLR4)-p38 pathway in monocytes and neutrophils, J. Biol. Chem. 289 (2014) 18097–18109. [27] D.C. Lacey, A. Achuthan, A.J. Fleetwood, H. Dinh, J. Roiniotis, G.M. Scholz, M.W. Chang, S.K. Beckman, A.D. Cook, J.A. Hamilton, Defining GM-CSF- and macrophage-CSF-dependent macrophage responses by in vitro models, J. Immunol. 188 (2012) 5752–5765. [28] J. Wu, J.B. Lingrel, KLF2 inhibits Jurkat T leukemia cell growth via upregulation of cyclin-dependent kinase inhibitor p21WAF1/CIP1, Oncogene 23 (2004) 8088–8096.
upregulated SIGIRR expression by macrophages. The influence of crosstalk between TLR4 and TLR7/8 on IL-23 production was differentially regulated by SIGIRR. SIGIRR is both a negative regulator of TLR4 and a positive regulator of TLR7/8. Acknowledgement This study was partly supported by a Kumamoto Health Science University special fellowship grant (No. 27-A-1). Conflict of interest declaration The authors declare that there are no conflicts of interest. References [1] C.L. Langrish, B.S. McKenzie, N.J. Wilson, R. de Waal Malefyt, R.A. Kastelein, D.J. Cua, IL-12 and IL-23: master regulators of innate and adaptive immunity, Immunol. Rev. 202 (2004) 96–105. [2] C.A. Hunter, New IL-12-family members: IL-23 and IL-27, cytokines with divergent functions, Nat. Rev. Immunol. 5 (2005) 521–531. [3] R.A. Kastelein, C.A. Hunter, D.J. Cua, Discovery and biology of IL-23 and IL-27: related but functionally distinct regulators of inflammation, Annu. Rev. Immunol. 25 (2007) 221–242. [4] S. Goriely, M.F. Neurath, M. Goldman, How microorganisms tip the balance between interleukin-12 family members, Nat. Rev. Immunol. 8 (2008) 81–86. [5] N.J. Mason, J. Fiore, T. Kobayashi, K.S. Masek, Y. Choi, C.A. Hunter, TRAF6-dependent mitogen-activated protein kinase activation differentially regulates the production of interleukin-12 by macrophages in response to Toxoplasma gondii, Infect. Immun. 72 (2004) 5662–5667. [6] T. Krausgruber, K. Blazek, T. Smallie, S. Alzabin, H. Lockstone, N. Sahgal, T. Hussell, M. Feldmann, I.A. Udalova, IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses, Nat. Immunol. 12 (2011) 231–238. [7] F.Y. Liew, D. Xu, E.K. Brint, L.A. O'Neill, Negative regulation of toll-like receptormediated immune responses, Nat. Rev. Immunol. 5 (2005) 446–458. [8] C. Kadota, S. Ishihara, M.M. Aziz, M.A. Rumi, N. Oshima, Y. Mishima, I. Moriyama, T. Yuki, Y. Amano, Y. Kinoshita, Down-regulation of single immunoglobulin interleukin-1R-related molecule (SIGIRR)/TIR8 expression in intestinal epithelial cells during inflammation, Clin. Exp. Immunol. 162 (2010) 348–361. [9] Y. Ohoka, A. Yokota-Nakatsuma, N. Maeda, H. Takeuchi, M. Iwata, Retinoic acid and GM-CSF coordinately induce retinal dehydrogenase 2 (RALDH2) expression through cooperation between the RAR/RXR complex and Sp1 in dendritic cells, PLoS One. 9 (2014) e96512. [10] A. Achuthan, A.D. Cook, M.C. Lee, R. Saleh, H.W. Khiew, M.W. Chang, C. Louis, A.J. Fleetwood, D.C. Lacey, A.D. Christensen, A.T. Frye, P.Y. Lam, H. Kusano, K. Nomura, N. Steiner, I. Förster, S.L. Nutt, M. Olshansky, S.J. Turner, J.A. Hamilton, Granulocyte macrophage colony-stimulating factor induces CCL17 production via IRF4 to mediate inflammation, J. Clin. Invest. 126 (2016) 3453–3466. [11] H. Das, A. Kumar, Z. Lin, W.D. Patino, P.M. Hwang, M.W. Feinberg, P.K. Majumder, M.K. Jain, Kruppel-like factor 2 (KLF2) regulates proinflammatory activation of monocytes, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 6653–6658. [12] H. Ohguchi, T. Hideshima, M.K. Bhasin, G.T. Gorgun, L. Santo, M. Cea, M.K. Samur, N. Mimura, R. Suzuki, Y.T. Tai, R.D. Carrasco, N. Raje, P.G. Richardson, N.C. Munshi, H. Harigae, T. Sanda, J. Sakai, K.C. Anderson, The KDM3A-KLF2-IRF4
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