IL-23-induced macrophage polarization and its pathological roles in mice with imiquimod-induced psoriasis
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Macrophages acquire distinct phenotypes during tissue stress and inflammatory responses. Macrophages are roughly categorized into two different subsets named inflammatory M1 and anti-inflammatory M2 macrophages. We herein identified a unique pathogenic macrophage subpopulation driven by IL-23 with a distinct gene expression profile including defined types of cytokines. The freshly isolated resting mouse peritoneal macrophages were stimulated with different cytokines in vitro, the expression of cytokines and chemokines were detected by microarray, real-time PCR, ELISA and multiple colors flow cytometry. Adoptive transfer of macrophages and imiquimod-induced psoriasis mice were used. In contrast to M1- and M2-polarized macrophages, IL-23-treated macrophages produce large amounts of IL-17A, IL-22 and IFN-γ. Biochemical and molecular studies showed that IL-23 induces IL-17A expression in macrophages through the signal transducer and activator of transcription 3 (STAT3)-retinoid related orphan receptor-γ T (RORγT) pathway. T-bet mediates the IFN-γ production in IL-23-treated macrophages. Importantly, IL-23-treated macrophages significantly promote the dermatitis pathogenesis in a psoriasis-like mouse model. IL-23-treated resting macrophages express a distinctive gene expression prolife compared with M1 and M2 macrophages. The identification of IL-23-induced macrophage polarization may help us to understand the contribution of macrophage subpopulation in Th17-cytokines-related pathogenesis.
Keywordsinterferon-gamma interleukin-17 interleukin-23 imiquimod-induced psoriasis macrophage polarization
Macrophages demonstrate significant plasticity and are able to modify their phenotype and function in response to the surrounding microenvironments (Murray and Wynn, 2011). It is well known that macrophage polarization display tremendous heterogeneity and is involved in tissue remodeling and pathogenesis. Recently, an elegant study evaluated the transcriptome of human macrophages induced by a variety of stimuli and revealed an extraordinary spectrum of macrophage activation states that far extend the current M1 versus M2-polarization model (Xue et al., 2014). Importantly, the diverse macrophage subsets can have drastic effects on health and disease within the tissues where they reside (Labonte et al., 2014).
IL-23, one member of the IL-12 cytokine family, is crucial in the pathogenesis of psoriasis, experimental autoimmune encephalomyelitis (EAE), collagen-induced arthritis (CIA), inflammatory bowel disease (IBD) (Tonel et al., 2010; Teng et al., 2015) and leukocyte adhesion deficiency type 1 (LAD1) (Moutsopoulos et al., 2017). Polymorphisms in the gene encoding the IL-23 receptor (IL-23R) are important susceptibility factors for Behcet’s disease, ankylosing spondylitis, and IBDs like Crohn’s disease and ulcerative colitis (Remmers et al., 2010; Kadi et al., 2013). It is known that IL-23 is essential for the terminal differentiation of IL-17-producing T effector cells (Park et al., 2005; McGeachy et al., 2009), which were initially shown to be a chief pathogenic cell population in EAE and CIA (Duerr et al., 2006; Remmers et al., 2010), human psoriasis (Wilson et al., 2007; Lubberts, 2015) and LAD1 (Moutsopoulos et al., 2017). However, in addition to acting on Th17 cells, IL-23 also regulates the function of innate lymphocytes (Guo et al., 2012). IL-23R is predominantly found on activated memory T cells, natural killer (NK) cells, and innate lymphoid cells (ILCs), and at lower levels on monocytes, macrophages, and dendritic cells (DCs) in humans; whereas mouse IL-23R is expressed on activated T cells, ILCs, γδ T cells, macrophages and DCs (Kastelein et al., 2007; Awasthi et al., 2009; Aychek et al., 2015). Importantly, studies have demonstrated that IL-23 induces these innate cells to secrete IL-17 and/or IL-22, although it remains unknown whether IL-23 affects the functional development of IL-23R-expressing innate cells in vivo (Cella et al., 2009; Guo et al., 2012; Paget et al., 2012).
We herein demonstrate that IL-23-treated monocyte/macrophages selectively produce IL-17A, IL-22 and IFN-γ, and display a distinct lineage gene expression profile in sharply contrast to M1 and M2 subsets. Importantly, M(IL-23) macrophages significantly promote the severity of dermatitis pathogenesis in a mouse psoriasis-like model. Thus, our findings reveal a previously unappreciated macrophage polarization driven by IL-23 with unique cell surface markers and cytokine-producing gene profile.
IL-23 induces a distinct macrophage gene expression profile
M(IL-23), M1 and M2 polarizations are reciprocally regulated
The involvement of STAT3-RORγT and T-bet in M(IL-23) polarization
To understand the intracellular signal pathway for IFN-γ production in M(IL-23) macrophages, we detected the Th1-related key transcription factor T-bet (Robinson and O’Garra, 2002). The expression of T-bet was significantly up-regulated in macrophages after IL-23 treatment as determined by real-time PCR and Western blots (Fig. 5G and 5H). Macrophages isolated from T-bet KO mice expressed significantly lower IFN-γ after IL-23 treatment (P < 0.001, Fig. 5I). However, the T-bet deficiency failed to impact the IL-17A, IL-17F and IL-22 expression in macrophages induced by IL-23 (Fig. S14). These results suggest that the enhanced T-bet in macrophages by IL-23 is involved in the IFN-γ but not IL-17A, IL-17F and IL-22 expression.
The roles of M(IL-23) macrophages in a psoriasis model
Macrophage polarization is determined by genetic and environmental factors. Macrophage polarizations play a critical role in mastering the amplitudes and types of host immunity. In the present study, we identified a previously unappreciated macrophage polarization, that is, M(IL-23) macrophage subpopulation with the following characteristics and supporting evidences: 1) IL-23-treated resting macrophages display distinct gene expression profiling than M1 and M2 macrophages; 2) IL-23-treated resting macrophages selectively produce IL-17A, IL-17F, IL-22 and IFN-γ, but not M1 and M2-related cytokines and molecules including TNF-ɑ, IL-12, iNOS, Arg1, YM1, and FIZZ1; 3) Resting macrophages are susceptible to M(IL-23) induction, while polarized M1 and M2 macrophages are highly resistant to IL-23 treatment. 4) IL-23-treated resting macrophages present in psoriasis-like dermatitis and promote pathogenesis; and 5) IL-23-induced IL-17A, IL-17F and IL-22 expression in macrophages is dependent on STAT3-RORγT pathway, while the expression of IFN-γ in IL-23-treated macrophages was likely mediated by T-bet pathway. Therefore, IL-23-treated macrophages display distinct phenotype and cytokine production compared with M1 and M2 macrophages.
It is reported that IL-23 significantly contributes to inflammatory disease risk in humans (Duerr et al., 2006; Genetic Analysis of Psoriasis et al., 2010). Mice deficient in IL-23 but not IL-12 are resistant to experimental immune-mediated disease like EAE, RA, and IBD (Cua et al., 2003; Murphy et al., 2003). The promotion of Th17 subset is highly recognized to be the key player to mediate the critical role of IL-23 in inflammatory diseases and infection-induced pathological consequences like Lyme disease and toxoplasma encephalitis (Weaver et al., 2013). Our present study shows that IL-23 acts directly on macrophages to induce IL-17A, IL-17F, IL-22 and IFN-γ productions which likely promote the severity of psoriasis-like dermatitis in mice. The ability of IL-23 to induce IL-17 production in macrophages is consistent with the recent observations showing that IL-17 production by macrophages contributes to allergic asthma and that IL-23 protection against plasmodium berghei infection in mice is partially dependent on IL-17 from macrophages (Song et al., 2008; Ishida et al., 2013). The significance of M(IL-23) macrophage polarization in Th17 cytokines-related inflammatory diseases requires to be clarified.
IL-23, an IL-12 cytokine family member, is a heterodimeric molecule composed of p40 and p19 subunits (Langrish et al., 2005). The known biological roles and the pro-inflammatory activities of IL-23 in inflammation and autoimmune diseases include but not limit to the induction of Th17-induced secretion of IL-17 and suppression of CD4+CD25+ regulatory T cells (Iwakura and Ishigame, 2006; Izcue et al., 2008). IL-23 signals through IL-23R and IL-12Rβ1 to activate JAK and predominantly the phosphorylation and activation of STAT3 (Oppmann et al., 2000), which acts to promote transcription of Il23r and Rorc (encoding RORγ), establishing a positive feedback loop and stabilizing expression of genes encoding pro-inflammatory effector molecules including Il17a, Il17f, Il22 and Csf2 (Parham et al., 2002; Codarri et al., 2011). In macrophages, IL-23 uses the classical STAT3-RORγT pathway to induce Th17 cytokines gene expression profile. On the other hand, IFN-γ is characteristically produced by NK, T and NKT cells. It is reported that monocytes/macrophages can express IFN-γ by IL-12/IL-18 and LPS/ATP stimulations, respectively (Raices et al., 2008). In the present study, LPS + IFN-γ and IL-4 failed to induce detectable IFN-γ expression in resting macrophages, but IL-23 drove resting macrophages to express high levels of IFN-γ via T-bet pathway.
In summary, we identified a unique macrophage subpopulation M(IL-23) induced by IL-23 with a distinct gene expression profile in contrast to M1 and M2 macrophages. Importantly, IL-23-induced M(IL-23) macrophage polarization is closely involved in the pathogenesis in an IMQ-induced psoriasis mouse model. The physiological function of M(IL-23) macrophages in tissue repair and remodeling, as well as the role of M(IL-23) macrophages in pathogenesis caused by infections, tumors and graft rejection need to be explored in the future.
Materials and methods
Animals and reagents
C57BL/6(B6) mice were purchased from Beijing University Experimental Animal Center. ROR-γt knock-out (KO) mice (B6.129P2(Cg)-Rorctm2Litt/J; JAX; Stock No.: 007572) were purchased from The Jackson Laboratory. All mice were maintained in a specific pathogen-free facility. All experimental manipulations were undertaken in accordance with the Institutional Guidelines for the Care and Use of Laboratory Animals, Institute of Zoology.
Recombinant mouse cytokines were purchased from PeproTech (Rocky Hill, NJ). RmIL-21 and IL-23 were obtained from R&D Systems (Minneapolis, MN). Bacterial lipopolysaccharide (LPS; E. coli 055:B5) was purchased from Sigma-Aldric (St Louis, MO). Selective STAT3 inhibitor (NSC74859; 4655/10) were obtained from R&D Systems and RORγT inverse agonist (SR2211; 557353) were from The Merck Group (Darmstadt, Germany). The reagents were used at the indicated or following concentrations based on our previous studies (Hou et al., 2013; Hou et al., 2014): recombinant mouse IL-1β (100 ng/mL), IL-2 (100 U/mL), IL-4 (1000 U/mL), IL-6 (20 ng/mL), IL-10 (20 ng/mL), IL-12 (10 ng/mL), IL-13 (20 ng/mL), IL-17A (100 ng/mL), IL-21 (100 ng/mL), IL-23 (100 ng/mL), IL-33 (100 ng/mL), TNF-α (100 ng/mL), IFN-γ (50 ng/mL) TGF-β1 (5 ng/mL); and recombinant human IL-23 (100 ng/mL), M-CSF (10 ng/mL); LPS (500 ng/mL), NSC74859 (100 μmol/L), SR2211 (10 μmol/L).
Anti-mF4/80-PE-Cy5, anti-mCD11b-PE-Cy5, anti-mTNF-α-FITC and anti-mCXCR5-PE were purchased from BD Biosciences Pharmingen (San Diego, CA, US). Anti-mIL-17A-PE was purchased from Biolegend (San Diego, CA, US). Anti-mIL-23R-AF488 mAb was purchased from R&D Systems. The primary antibodies against p-STAT3 (Tyr705), p-STAT3 (Ser727), STAT3, IRF4 and BATF were purchased from Cell Signaling Technology (Beverly, MA, US). The primary antibodies against RORγT were from Millipore Biotechnology (Billerica, MA, US).
Primary mouse peritoneal macrophages were obtained from B6 mice as described previously (Zhu et al., 2014). The purity of macrophages was more than 90% of CD11b+F4/80+macrophages as analyzed by flow cytometry (Yang et al., 2016). For real-time PCR and ELISA assays, CD11b+F4/80+ macrophages were further sorted by a MoFlo XDP High Speed Cell Sorter (Beckman Coulter).
Microarray hybridization and data analysis
Total RNA was amplified and labeled by Low Input Quick Amp Labeling Kit, One-Color (Cat#5190-2305, Agilent technologies, Santa Clara, CA, US). Differentially expressed genes were defined as genes with at least 2 fold variance of expression levels in M1, M2 and M(IL-23) polarized macrophages compared to M0 macrophages.
Cells were cultured on coverslips for the indicated time and then fixed in 4% paraformaldehyde for 10 min and stored in PBS at 4°C (Hou et al., 2014). Cells were permeabilized and blocked and then were incubated with the indicated mAbs (1:100 dilution) overnight at 4°C. Following PBS washes, the secondary Ab was applied for 1 h and HOECHST33342 (2 μg/mL) for 10 min. Photomicrographs were taken using an LSM510META Laser Scanning Microscope (Zeiss, Germany).
It was performed as described (Sun et al., 2012). Protein bands were visualized by adding HRP membrane substrate (Millipore) and then scanned using the Tanon 1600R Gel Image System (Tanon Co., Ltd., Shanghai, China). GAPDH mAb (Proteintech Group, Inc) was used to normalize for the amount of loaded protein.
IL-17A, IL-17F, IL-22 and IFN-γ ELISA assays were performed following the manufacturer’s instructions (Biolegend).
Real-time PCR was performed using multiple kits (SYBR Premix Ex TaqTM, DRR041A, Takara Bio) on CFX96 (Bio-Rad) (Li et al., 2017). The primers are listed in Table S1. Housekeeping gene hypoxanthine phosphoribosyl transferase (HPRT) was used as an internal control.
The day that psoriasis was first induced by imiquimod (IMQ) was defined as day 0. On days −1 and 1, B6 mice were injected i.v. with 1 × 106 IL-23-induced macrophages which were treated with IL-23 for 72 h in vitro as described above. B6 mice were used to induce psoriasis by IMQ as described previously (van der Fits et al., 2009). Mice were evaluated daily. Back redness (erythema), presence of scales (scaling), and hardness of the skin were scored using a semiquantitative scoring system from 0 to 4 based on their external physical appearance: 0 = no skin abnormalities, 1 = slight, 2 = moderate, 3 = marked, and 4 = severe. In addition, mice were weighed, and dorsal skin thickening was assessed by measuring double-skin fold thickness using a digital micrometer (Mitutoyo). At the end of the experiment, back skin samples were fixed in 4% formaldehyde and stained with H&E. Parakeratosis, acanthosis and leukocyte infiltration were assessed to evaluate scores in a blinded way. Scores from 0 to 2 were given, as follows: 0 = no abnormalities; 1 = psoriasis-like dermatitis: epidermal acanthosis, reduction of granulose layer, and hyperkeratosis with modest leukocyte infiltration; 2 = psoriasis-like dermatitis: higher epidermal acanthosis, absence of granulose layer, and higher hyperkeratosis with leukocyte infiltration enriched in neutrophils.
Intracellular cytokine staining
Macrophages were treated with GolgiPlug (BD Pharmingen) for the last 6 h of incubation (Zhu et al., 2014). Cells were fixed and permeabilized with fixation and permeabilization solution (BD; 553722) and Perm/Wash buffer (BD; 554723). Cells were analyzed for the intracellular production of cytokines by staining with anti-mTNF-ɑ-FITC, anti-mIFN-γ-PE, or anti-mIL-17A-PE, respectively. The cells were then detected by a flow cytometry.
Data are presented as mean ± SD. Student’s unpaired t test for comparison of means was used. For multiple group comparison, significant difference was calculated using the non-parametric Mann-Whitney U test. A P value less than 0.05 was considered significant.
The authors thank Dr. Lianjun Zhang for his reading the manuscript, Mrs. Qing Meng, Mrs. Xiaoqiu Liu, and Mr. Yabing Liu for their expert technical assistance, Mrs. Ling Li for her excellent laboratory management. This work was supported by grants from the National Natural Science Foundation for General and Key Programs (C81530049, C81130055, C31470860, Y.Z.), Knowledge Innovation Program of Chinese Academy of Sciences (XDA04020202-19, Y.Z.), and the CAS/SAFEA International Partnership Program for Creative Research Teams (Y.Z.).
CIA, collagen-induced arthritis; DCs, dendritic cells; EAE, experimental autoimmune encephalomyelitis; IBD, inflammatory bowel disease; ILCs, innate lymphoid cells; IMQ, imiquimod; LAD1, leukocyte adhesion deficiency type 1; NK, natural killer; RORγT, retinoid related orphan receptor-γ T; STAT3, signal transducer and activator of transcription 3.
COMPLIANCE WITH ETHICS GUIDELINES
Yuzhu Hou, Linnan Zhu, Hongling Tian, Hai-Xi Sun, Ruoyu Wang, Lianfeng Zhang, and Yong Zhao declare that they have no conflict of interest.
All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000 (5). Informed consent was obtained from all patients for being included in the study. All institutional and national guidelines for the care and use of laboratory animals were followed.
Yuzhu Hou, Linnan Zhu and Hongling Tian designed and carried out the major experiments, and analyzed data; Hai-Xi Sun performed bioinformatics assays. Ruoyu Wang provided experimental supervision and analyzed data; Linnan Zhu provided animal models and revised manuscript; and Yong Zhao designed experiments, analyzed data, wrote manuscript and provided overall supervision.
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