Twist1 and Twist2 Contribute to Cytokine Downregulation following Chronic NOD2 Stimulation of Human Macrophages through the Coordinated Regulation of This information is current as Transcriptional Repressors and Activators of September 25, 2021. Shasha Zheng, Matija Hedl and Clara Abraham J Immunol 2015; 195:217-226; Prepublished online 27 May 2015; doi: 10.4049/jimmunol.1402808 Downloaded from http://www.jimmunol.org/content/195/1/217

Supplementary http://www.jimmunol.org/content/suppl/2015/05/27/jimmunol.140280 http://www.jimmunol.org/ Material 8.DCSupplemental References This article cites 49 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/195/1/217.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Twist1 and Twist2 Contribute to Cytokine Downregulation following Chronic NOD2 Stimulation of Human Macrophages through the Coordinated Regulation of Transcriptional Repressors and Activators

Shasha Zheng, Matija Hedl, and Clara Abraham

Proper regulation of microbial-induced cytokines is critical to intestinal immune homeostasis. Acute stimulation of nucleotide- binding oligomerization domain 2 (NOD2), the Crohn’s disease–associated sensor of bacterial peptidoglycan, induces cytokines. However, chronic NOD2 stimulation in macrophages decreases cytokines upon pattern recognition (PRR) restimulation; cytokine attenuation to PRR stimulation is similarly observed in intestinal macrophages. The role for the transcriptional repres- sors Twist1 and Twist2 in regulating PRR-induced cytokine outcomes is poorly understood and has not been reported for NOD2. Downloaded from We found that Twist1 and Twist2 were required for optimal cytokine downregulation during acute and, particularly, chronic NOD2 stimulation of human macrophages. Consistently, Twist1 and Twist2 expression was increased after chronic NOD2 stim- ulation; this increased expression was IL-10 and TGF-b dependent. Although Twist1 and Twist2 did not coregulate each other’s expression, they cooperated to enhance binding to cytokine promoters after chronic NOD2 stimulation. Moreover, Twist1 and Twist2 contributed to enhance expression and promoter binding of the proinflammatory inhibitor c-Maf and the transcriptional http://www.jimmunol.org/ repressor Bmi1. Restoring c-Maf and Bmi1 expression in Twist-deficient macrophages restored NOD2-induced cytokine down- regulation. Furthermore, with chronic NOD2 stimulation, Twist1 and Twist2 contributed to the decreased expression and cytokine promoter binding of the transcriptional activators activating 4, C/EBPa, Runx1, and Runx2. Knockdown of these transcriptional activators in Twist-deficient macrophages restored cytokine downregulation after chronic NOD2 stimulation. Finally, NOD2 synergized with additional PRRs to increase Twist1 and Twist2 expression and Twist-dependent pathways. There- fore, after chronic NOD2 stimulation Twist1 and Twist2 coordinate the regulation of both transcriptional activators and repress- ors, thereby mediating optimal cytokine downregulation. The Journal of Immunology, 2015, 195: 217–226.

uman nucleotide-binding oligomerization domain 2 in vivo attenuates subsequent experimental colitis (6), thereby dem- by guest on September 25, 2021 (NOD2), an intracellular sensor of bacteria-derived muramyl onstrating the beneficial effects of chronic NOD2 stimulation in in- H dipeptide (MDP; a component of peptidoglycan), confers the testinal immune regulation. greatest genetic risk of developing Crohn’s disease (CD), a disease of Mechanisms contributing to cytokine downregulation after chronic intestinal inflammation (1). When peripheral monocytes enter chronic NOD2 stimulation in human myeloid–derived cells include mucosal sites, such as the intestinal lamina propria, they are contin- the upregulation of the intracellular inhibitors IRAK-M (4), Tollip uously exposed to bacterial products, including the NOD2 ligand, (3), and IRF4 (6, 9), and the Tyro3, Axl, and Mer tyrosine peptidoglycan/MDP (2, 3). Initial NOD2 stimulation results in cyto- kinase receptors (10); the NF-kB1–dependent upregulation of the kine secretion (3–5). However, ongoing NOD2 stimulation signifi- transcriptional repressor activating transcription factor (ATF)3 (3); cantly downregulates cytokine secretion upon restimulation through and the secretion of the inhibitory mediators IL-10 and TGF-b (5). pattern recognition receptors (PRRs) (3–7). This downregulation is Each of these mechanisms contributes only partially to cytokine impaired in individuals with CD-associated NOD2 polymorphisms (4, downregulation and is operational to varying degrees in different 5). Cytokine secretion in intestinal macrophages is similarly attenu- individuals (e.g., IRAK-M) (4, 11). Given the dramatic alterations ated upon PRR stimulation (8), which is important for intestinal im- in macrophage functions and importance of downregulating PRR- mune homeostasis. Moreover, chronic MDP administration to mice initiated pathways upon chronic microbial stimulation, we hy- pothesized that additional critical mechanisms mediating these changes have yet to be identified, including those involving tran- Department of Internal Medicine, Yale University, New Haven, CT 06520 scriptional regulators. Received for publication November 5, 2014. Accepted for publication April 27, 2015. Twist1 and Twist2 are basic helix-loop-helix transcriptional This work was supported by National Institutes of Health Grants R01DK099097, repressors that bind to E boxes in promoters, acting as master R01DK077905, DK-P30-34989, U01-DK062422, and T32-DK007017. regulators in a variety of biological processes, including organo- Address correspondence and reprint requests to Dr. Clara Abraham, Department of genesis, osteogenesis, cancer progression, and hematopoietic cell Internal Medicine, Section of Digestive Diseases, Yale University, 333 Cedar Street 2/2 (LMP 1080), New Haven, CT 06520. E-mail address: [email protected] development (12–14). As such, Twist1 mice are embryonic The online version of this article contains supplemental material. lethal because of a variety of developmental defects (15). Muta- Abbreviations used in this article: ATF, activating transcription factor; CD, Crohn’s tions in Twist1 are associated with Saethre–Chotzen syndrome disease; ChIP, chromatin immunoprecipitation; MDM, monocyte-derived macro- (16, 17), an autosomal dominant disorder characterized by cra- phage; MDP, muramyl dipeptide; NOD2, nucleotide-binding oligomerization domain niofacial and limb anomalies. Although the major focus on Twist 2; PRR, pattern recognition receptor; siRNA, small interfering RNA. function has been in the context of the critical cellular processes Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 described above, there is evidence for a role for Twists in regu- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402808 218 Twist REGULATES CHRONIC NOD2 STIMULATION OUTCOMES lating inflammation (14, 18–22). Twist22/2 mice demonstrate Transfection of small interfering RNAs and plasmids increased myeloid lineage development (14). Moreover, Twist22/2 +/2 +/2 Primary human MDMs were transfected with 100 nM scrambled or ON- mice or Twist1 /Twist2 mice demonstrate increased proin- TARGETplus SMARTpool small interfering RNA (siRNA) against flammatory cytokines associated with increased NF-kB pathway Twist1, Twist2, c-Maf, Bmi1, ATF4, C/EBPa, Runx1, or Runx2 (Dharmacon, signaling and perinatal death and defects in the type I IFN–me- Lafayette, CO) (four pooled siRNAs for each gene) or with 2 mg pT3-EF1a- diated suppression of proinflammatory cytokines in macrophages Bmi1 [Addgene plasmid 31783 kindly deposited by X. Chen (23)], c-Maf (GeneCopoeia, Rockville, MD), or empty vector using Amaxa nucleofector (18, 22). In T cells, Twist1 limits Th1, Th17, and T follicular helper technology (Amaxa, San Diego, CA). cell development (20, 21). However, the role and mechanisms through which Twist1 and Twist2 regulate PRR-induced cytokine Chromatin immunoprecipitation outcomes is poorly understood; Twist regulation of NOD2-induced Chromatin immunoprecipitation (ChIP) analysis of MDMs was performed outcomes has not been reported. Given the repressive functions of the according to a modified protocol (3). Primers were designed to amplify Twist and evidence for their ability to limit inflammation in select situations, we hypothesized that Twist1 and/or Twist2 would contribute to downregulating cytokines during both acute and chronic NOD2 stimulation in primary human macrophages. We found that Twist1 and Twist2 were required for the down- regulation of cytokines during both acute and, in particular, chronic NOD2 stimulation of human monocyte–derived macrophages

(MDMs). Consistently, Twist1 and Twist2 were each significantly Downloaded from upregulated after prolonged NOD2 stimulation; this upregulation required autocrine IL-10 and TGF-b and was defective in MDMs from CD-associated NOD2 risk carriers. Binding of the upregu- lated Twist1 and Twist2 transcriptional repressors to cytokine promoters was increased in a cooperative manner after chronic

NOD2 stimulation. Moreover, Twist1 and Twist2 upregulated the http://www.jimmunol.org/ expression and promoter binding of additional inhibitory tran- scriptional regulators, including c-Maf and Bmi1, while decreasing the expression and promoter binding of essential activating tran- scription factors, including ATF4, C/EBPa, Runx1, and Runx2. Complementation of the reduced c-Maf and Bmi1 expression, or reduction in the upregulated ATF4, C/EBPa, Runx1, and Runx2 under Twist-deficient conditions were each sufficient to restore cy- tokine downregulation under chronic NOD2 stimulation conditions. Therefore, Twist1 and Twist2 contribute to cytokine downregulation by guest on September 25, 2021 during chronic NOD2 stimulation through the coordination of the reciprocal expression and cytokine promoter binding of additional transcriptional repressors and activators.

Materials and Methods Primary MDM culture and genotyping Informed consent was obtained as approved by the Yale University Insti- tutional Review Board. Monocytes were purified from healthy individuals or CD patients (Supplemental Fig. 1E) and differentiated to MDMs for 7 d with 10 ng/ml M-CSF (Shenandoah Biotechnology, Warwick, PA) as in Ref. 4. We performed NOD2 genotyping by TaqMan (Applied Biosystems, Foster City, CA) or Sequenom platform (Sequenom, San Diego, CA). mRNA expression Total RNA was isolated and reverse transcribed, and quantitative PCR was performed as previously described (4) with normalization to GAPDH. Primers are available upon request. Myeloid cell stimulation FIGURE 1. Twist1 and Twist2 are required for optimal cytokine For tolerance induction in vitro, human MDMs (0.5 3 106) were pretreated downregulation upon chronic NOD2 stimulation. (A) Timeline schematic with 100 mg/ml MDP (Bachem, King of Prussia, PA) for 48 h prior to for MDP pretreatment of MDMs, Twist1/2 knockdown, and subsequent m extensive wash and retreated for 24 h with 100 g/ml MDP. In some cases, acute MDP treatment. (B) Human MDMs were left untreated (for acute) or anti–IL-10 or anti–TGF-b–neutralizing Abs (R&D Systems, Minneapolis, pretreated with 100 mg/ml MDP for 24 h, then transfected with scrambled, MN) or lipid A (Peptide International, Louisville, KY) was used. Super- natants were assayed for cytokine secretion per manufacturer instructions Twist1, or Twist2 siRNA alone or in combination, and 24 h later (total 48-h m using the following Abs: TNF, IL-6, IL-8, IL-10 (BD Biosciences, San MDP pretreatment), MDMs were treated with 100 g/ml MDP for an Jose, CA), or IL-12 (eBioscience, San Diego, CA). additional 24 h (acute). Supernatants were examined for cytokines. Mean + SEM for n = 4. Similar results were observed for combined Twist1 and expression analysis Twist2 siRNA for an additional n = 8. Numbers on the bars are the ratios of Western blot analysis was performed as in Ref. 4 using Abs to Twist1/ cytokine secretion upon MDP treatment of pretreated versus nonpretreated Twist2 (ab50887; Abcam, Cambridge, MA), c-Maf (sc7866; Santa Cruz MDMs. Statistical significance above the knockdown sample bars is Biotechnology, Santa Cruz, CA), Bmi1 (05-1322; EMD Millipore, Billerica, compared with its corresponding scrambled siRNA sample. *p , 0.05, MA), or anti-GAPDH (EMD Millipore). **p , 0.01, ***p , 0.001, †p , 1 3 1024. scr, scrambled; tx, treatment. The Journal of Immunology 219 genomic sequences at the cytokine gene promoter region (available upon used by us and others, and results in optimal cytokine downregula- request). Abs used included the following: Twist1 (ab50887), Twist2 tion after chronic NOD2 stimulation (3–5). Upon knockdown of (ab66031), Runx1 (ab23980), ATF4 (ab85049), Bmi1 (ab14389) (Abcam), Twist1 or Twist2 after initiation of chronic NOD2 stimulation and Runx2 (sc10785x), C/EBPa (sc-61x), or c-Maf (sc-7866x) (Santa Cruz Biotechnology). then restimulating through NOD2 (Fig. 1A), there was significant reversal in the downregulation of both proinflammatory cytokines Statistical analysis and anti-inflammatory cytokines (Fig. 1B). Reversal was further Significance was assessed using a two-tailed Student t test. A p value , 0.05 enhanced when Twist1 and Twist2 were knocked down in combi- was considered significant. A Bonferroni correction was applied for mul- nation (Fig. 1B). Taken together, Twist1 and Twist2 cooperate to tiple comparisons. To maintain a consistent picogram per milliliter scale for downregulate cytokines upon both acute and chronic NOD2 stimu- cytokine concentration measures, the higher levels observed for IL-8 and lation of human MDMs. IL-6 (nanogram per milliliter levels) are shown with a multiplication factor. Expression of Twist1 and Twist2 increases with chronic Results NOD2 stimulation in human MDMs in an IL-10– and Twist1 and Twist2 are required for optimal downregulation TGF-b–dependent manner and fails to increase in CD of cytokines upon acute and chronic NOD2 stimulation NOD2 risk carriers To elucidate the role of Twist1 and Twist2 in cytokine downreg- Given the role for Twist1 and Twist2 in downregulating cytokines ulation with acute NOD2 stimulation in human MDMs, we used upon chronic NOD2 stimulation, we questioned whether Twist siRNA to reduce the expression of Twist1 and Twist2, alone and in expression is upregulated with chronic NOD2 stimulation of human combination. We assessed expression of each Twist with each MDMs. Twist1 and Twist2 mRNA expression was enhanced in Downloaded from knockdown condition and found selective downregulation of the 48-h MDP-pretreated MDMs and further increased 4 h after targeted Twist (Supplemental Fig. 1A), such that Twist1 and restimulation (Fig. 2A). Twist1/2 protein expression was similarly Twist2 did not coregulate the expression of the other. We further increased after restimulation of 48-h MDP-pretreated MDMs confirmed knockdown of Twist1 and Twist2 proteins (Supplemental (Fig. 2B). We next sought to define the mechanisms upregulating Fig. 1B). Knockdown of Twist1 and Twist2, alone and in combi- Twist1 and Twist2 expression upon chronic NOD2 stimulation in

nation, led to increased secretion of the proinflammatory cytokines human MDMs. We have previously shown that the early secretion http://www.jimmunol.org/ TNF, IL-6, IL-8, IL-12p40 (Fig. 1), and IL-1b (Supplemental Fig. of IL-10 and TGF-b is required for subsequent pro- and anti- 1C), as well as the anti-inflammatory mediators IL-10 (Fig. 1) and inflammatory cytokine downregulation during chronic NOD2 IL-1Ra (Supplemental Fig. 1C), upon acute NOD2 stimulation of stimulation (5). Furthermore, recent studies have demonstrated human MDMs. We next evaluated the role of Twist1 and Twist2 in a necessity for IL-10R on intestinal macrophages for the genera- the cytokine downregulation observed after chronic NOD2 stimu- tion of anti-inflammatory macrophages (25, 26), thereby high- lation. We previously found that stimulation of NOD2 for 48 h is lighting the importance of IL-10 in conditioning the inhibitory optimal for downregulating cytokine secretion upon restimulation mechanisms generated in intestinal macrophages. We found that through NOD2 or other PRRs (4). We used 100 mg/ml MDP as this autocrine IL-10 and TGF-b were each required, and moreover concentration approximates muramic acid stool levels (24), has been cooperated with each other, for optimal upregulation in Twist1 and by guest on September 25, 2021

FIGURE 2. Twist1 and Twist2 expression is increased in MDMs after chronic NOD2 stimulation in an IL-10– and TGF-b–dependent manner, whereas this increase is impaired in MDMs from CD NOD2 risk carriers. (A) MDMs (n = 4) were left untreated or pretreated with 100 mg/ml MDP for 48 h and then stimulated with 100 mg/ml MDP for 4 h (acute). Fold mRNA expression of Twist1 and Twist2 normalized to untreated cells + SEM. (B) MDMs were left untreated or pretreated with 100 mg/ml MDP for 48 h and then treated with 100 mg/ml MDP for 8 h and assessed for Twist1/2 expression by Western blot. Representative Western blot from one of three individuals. GAPDH was used as a loading control. (C) MDMs were cultured with isotype control, neu- tralizing TGF-b (25 mg/ml), or neutralizing IL-10 (5 mg/ml) Abs, alone or in combination, for 1 h, then left untreated (for acute) or pretreated with 100 mg/ ml MDP for 48 h, and then treated with 100 mg/ml MDP for an additional 4 h (acute). Fold mRNA induction of Twist1 and Twist2 normalized to untreated cells (n = 4) + SEM. Statistical significance above the neutralizing Ab sample bars is compared with its corresponding isotype treated sample. (D) MDMs from wild-type (WT) NOD2 healthy controls (WT HC, n = 8), WT NOD2 CD patients (WT CD, n = 8), or CD NOD2 risk carriers [risk single nucleotide polymorphism (SNP), n = 3; as per Supplemental Fig. 1E] were left untreated or pretreated with 100 mg/ml MDP for 48 h, and then stimulated with 100 mg/ml MDP for 4 h (acute). Included is pretreatment and acute stimulation with 0.1 mg/ml lipid A to ensure that MDMs from NOD2 risk patients are responsive to other stimuli. Fold Twist1 and Twist2 mRNA expression is represented by normalizing treated samples to untreated samples + SEM. *p , 0.05, **p , 0.01, †p , 1 3 1024. tx, treatment. 220 Twist REGULATES CHRONIC NOD2 STIMULATION OUTCOMES

Twist2 expression upon chronic NOD2 stimulation in human cytokine downregulation after chronic NOD2 stimulation (Fig. 1). MDMs (Fig. 2C). Finally, we questioned whether MDMs from CD We therefore questioned whether the binding of these transcrip- patients similarly upregulate Twist1 and Twist2 upon chronic tional repressors to cytokine promoters was enhanced after chronic NOD2 stimulation (see Supplemental Fig. 1E for patient charac- NOD2 stimulation, and if so, whether Twist1 and Twist2 cooper- teristics). Twist1 and Twist2 expression in MDMs from wild-type ated with each other for this enhanced binding. Twist1 and Twist2 NOD2 CD patients was induced to a similar degree as in MDMs binding to both proinflammatory and anti-inflammatory cytokine from healthy controls (Fig. 2D). Furthermore, the range of Twist1 promoters increased particularly 48 h after NOD2 stimulation and and Twist2 expression upon chronic NOD2 stimulation in these even more so upon restimulation of MDP-pretreated MDMs (Fig. individuals was not significantly different (Supplemental Fig. 1D). 3A). Upon examining restimulated MDP-pretreated MDMs where In contrast, Twist1 and Twist2 expression failed to increase after Twist1 and Twist2 binding to cytokine promoters was most enriched, chronic NOD2 stimulation of MDMs from CD loss-of-function we observed that knockdown of each Twist affected binding not only NOD2 risk carriers (Fig. 2D). MDMs from these NOD2 risk of itself but also of the other Twist protein to cytokine promoters carriers were responsive to stimulation through alternative PRRs, (Fig. 3B). Taken together, chronic NOD2 stimulation results in such that Twist1 and Twist2 expression increased in these MDMs significantly enriched and cooperative binding of the transcrip- upon chronic lipid A treatment (Fig. 2D). Therefore, Twist1 and tional repressors Twist1 and Twist2 to proinflammatory and anti- Twist2 increase in an IL-10– and TGF-b–dependent manner upon inflammatory cytokine promoters. chronic NOD2 stimulation. Twist1- and Twist2-dependent upregulation of c-Maf and Bmi1 Twist1 and Twist2 cooperate with each other for the enhanced is critical for cytokine downregulation after chronic NOD2 Downloaded from binding to cytokine promoters observed after chronic NOD2 stimulation stimulation In addition to their direct suppressor activity, transcriptional repressors, Twist1 and Twist2 transcriptional repressors were upregulated with including the Twist proteins, can both increase the expression of ad- chronic NOD2 stimulation (Fig. 2) and were required for optimal ditional inhibitory molecules and suppress the expression of essential http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. Twist1 and Twist2 cooperate with each other for enhanced binding to cytokine promoters after chronic NOD2 stimulation. (A) MDMs (n =4) were left untreated or pretreated with 100 mg/ml MDP for 48 h and then treated with 100 mg/ml MDP for an additional 4 h (acute). (B) MDMs (n = 4) were left untreated (for acute) or pretreated with 100 mg/ml MDP for 24 h and then transfected with scrambled, Twist1, or Twist2 siRNA alone or in combination, and 24 h later (total 48-h MDP pretreatment), MDMs were treated with 100 mg/ml MDP for an additional 4 h (acute). (A and B) Recruitment of Twist1 and Twist2 to cytokine gene promoters was assessed by ChIP. Fold enrichment normalized to untreated, scrambled siRNA–transfected cells + SEM. (B) Statistical significance above the knockdown sample bars is compared with its corresponding scrambled siRNA sample. *p , 0.05, **p , 0.01, ***p , 0.001. tx, treatment. The Journal of Immunology 221 activating molecules; in combination, these enable optimal suppressive therefore questioned whether inhibitory molecules upregulated by outcomes (12). We therefore first investigated whether Twist1 and Twist1 and/or Twist2 identified through these other processes might be Twist2 might be contributing to cytokine downregulation after chronic similarly Twist dependent upon chronic NOD2 stimulation in human NOD2 stimulation through upregulating inhibitory molecules previ- MDMs and thereby contribute to the cytokine downregulation ob- ously identified to play a role in NOD2-induced cytokine downreg- served. During myeloid lineage development, Twist2 can upregulate ulation. However, Twist1 and Twist2 knockdown, either alone or in the proinflammatory transcriptional inhibitor c-Maf (14); Twist2 can combination, did not impair the upregulation of NFkB1, ATF3, also bind to the c-Maf promoter with LPS treatment in mouse mac- IRAK-M, or Tollip after chronic NOD2 stimulation (Supplemental rophages (29). However, c-Maf regulation of PRR-initiated pathways Fig. 2A). We further examined proximal signaling pathways that are has not been well defined. Twist1 regulates Bmi1 expression and induced upon acute NOD2 stimulation and found that combined cooperates with Bmi1 in repressor functions during epithelial–mes- Twist1 and Twist2 knockdown did not regulate acute NOD2–induced enchymal transition relevant to cancer metastasis (30); a role for Bmi1 activation of the ERK, p38, or JNK pathways (Supplemental Fig. 2B) in PRR signaling and outcomes is poorly understood. Furthermore, or the early secretion of IL-1b (Supplemental Fig. 2C), which we have Twist regulates PI3K pathways (31), and Akt1 can contribute to an found to be a measure of NOD2-induced caspase-1 activation (27, 28). anti-inflammatory macrophage phenotype (32). We found that c-Maf, Twist1 and Twist2 have not been well studied in PRR-induced Bmi1, and Akt1 were upregulated with chronic NOD2 stimulation cytokine regulation. However, they have been well investigated in (Fig. 4A). However, only c-Maf and Bmi1 transcript upregulation was carcinogenesis, osteogenesis, and hematopoietic cell development. We Twist1 and Twist2 dependent (Fig. 4B); c-Maf and Bmi1 protein Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. Upon chronic NOD2 stimulation, expression and cytokine promoter binding of the anti-inflammatory transcription factors c-Maf and Bmi1 are upregulated in a Twist-dependent manner. (A) MDMs (n = 8) were left untreated or pretreated with 100 mg/ml MDP for 48 h and then treated with 100 mg/ml MDP for 4 h (acute). Fold increase in c-Maf, Bmi1, and Akt1 mRNA was normalized to untreated MDMs + SEM. (B) MDMs (n = 8) were left untreated (for acute) or pretreated with 100 mg/ml MDP for 24 h and then transfected with scrambled, Twist1, or Twist2 siRNA, alone or in combination, and 24 h later (total 48 h after MDP pretreatment), MDMs were treated with 100 mg/ml MDP for 4h (acute) and assessed for c-Maf, Bmi1, and Akt1 mRNA expression. Fold mRNA expression normalized to untreated, scrambled siRNA–transfected MDMs + SEM. (C) MDMs were left untreated or pretreated with 100 mg/ml MDP for 24 h and then transfected with scrambled or a combination of Twist1 and Twist2 siRNA 6 vectors expressing c-Maf or Bmi1, and 24 h later (total 48-h MDP pretreatment), MDMs were treated with 100 mg/ml MDP for an additional 8 h (acute), and c-Maf or Bmi1 expression was assessed by Western blot. GAPDH was assessed as a loading control. (D) MDMs were left untreated (for acute) or pretreated with 100 mg/ml MDP for 24 h and then transfected with scrambled or a combination of Twist1 and Twist2 siRNA, and 24 h later (total 48 h after MDP pretreatment), MDMs were treated with 100 mg/ml MDP for an additional 4 h (acute). Recruitment of c-Maf (n = 4) and Bmi1 (n = 5) to cytokine gene promoters was assessed by ChIP. Fold enrichment normalized to untreated, scrambled siRNA–transfected MDMs + SEM. (B and C) Statistical significance above the knockdown sample bars is compared with its corresponding scrambled siRNA sample. *p , 0.05, **p , 0.01, ***p , 0.001, †p , 1 3 1024, ††p , 1 3 1025. tx, treatment. 222 Twist REGULATES CHRONIC NOD2 STIMULATION OUTCOMES upregulation was similarly Twist dependent (Fig. 4C). Furthermore, osteogenesis, carcinogenesis, and myeloid differentiation (12). We the binding of c-Maf and Bmi1 to both proinflammatory and anti- therefore hypothesized that Twist1 and Twist2 might be similarly inflammatory cytokine promoters was enriched with chronic NOD2 downregulating activating transcription factors upon chronic stimulation (Fig. 4D); this enhanced promoter binding was Twist1 and NOD2 stimulation where cytokines are dramatically reduced. Twist2 dependent (Fig. 4D). Importantly, through c-Maf and Bmi1 How activating transcription factors are regulated with chronic knockdown (Fig. 5A), we established that c-Maf and Bmi1 each NOD2 stimulation has not been well defined. We therefore con- contributed to the cytokine downregulation observed after chronic sidered the Twist-regulated activating transcription factors defined NOD2 stimulation (Fig. 5B). Moreover, they cooperated with each in other cellular processes. Twist1 can attenuate ATF4 binding to other for optimal cytokine downregulation (Fig. 5B). Finally, to the osteocalcin promoter in osteoclasts (33). Furthermore, Twist1 clearly establish the role of c-Maf and Bmi1 in Twist-dependent and Twist2 can inhibit Runx2 transactivation functions during os- contributions to chronic NOD2-induced cytokine downregulation in teoblast differentiation (34). Moreover, Twist2 can negatively MDMs,werestoredbothc-MafandBmi1expressioninTwist- regulate myeloid lineage differentiation by interacting with and deficient MDMs to levels similar to those observed after chronic inhibiting the transcription factors Runx1 and C/EBPa (14). Twist NOD2 stimulation (Fig. 4C). Despite the absence of Twist1 and proteins can also regulate Akt2 expression in breast cancer cells Twist2, reconstitution of each c-Maf and Bmi1 could partially restore (35). To our knowledge, roles for ATF4, C/EBPa, Runx1, and the cytokine downregulation observed after chronic NOD2 stimula- Runx2 in NOD2-induced outcomes have not been reported, and we tion (Fig. 5C). Moreover, the restoration was further improved with therefore first examined the regulation of each of these transcrip- combined expression of both c-Maf and Bmi1 (Fig 5C). Therefore, tion factors with acute and chronic NOD2 stimulation of MDMs. one mechanism through which Twist1 and Twist2 downregulate With NOD2 stimulation of MDMs, ATF4, C/EBPa, Runx1, Downloaded from cytokines after chronic NOD2 stimulation is through the upregulation Runx2, and Akt2 were each upregulated acutely but downregu- in expression and cytokine promoter binding of the transcriptional lated by 48 h, and they failed to increase on restimulation of repressors c-Maf and Bmi1. NOD2 (Fig. 6A), consistent with the attenuated cytokines at this time. We further assessed the Twist dependency for these down- Twist1 and Twist2 downregulate the expression and binding to regulated transcription factors and found that ATF4, C/EBPa, cytokine promoters of the transcriptional activators ATF4, Runx1, and Runx2 downregulation was dependent on the com- http://www.jimmunol.org/ a C/EBP , Runx1, and Runx2 bined expression of Twist1 and Twist2 (Fig. 6B). Akt2 downreg- Twist1 and Twist2 can both decrease the expression and compete ulation after chronic NOD2 stimulation, in contrast, was Twist with promoter binding of activating transcription factors in independent (Fig. 6B). We therefore pursued the role and regu-

FIGURE 5. Upregulated Bmi1 and c-Maf cooperate to mediate Twist-dependent cytokine downregulation upon chronic NOD2 stimulation of human MDMs. (A) MDMs (n = 4) were by guest on September 25, 2021 transfected with scrambled, Bmi1, or c-Maf siRNA for 24 h. Fold mRNA expression normalized to scrambled siRNA–transfected MDMs + SEM. (B) MDMs (n = 4) were left untreated (for acute) or pretreated with 100 mg/ ml MDP for 24 h and then transfected with scrambled, c-Maf, or Bmi1 siRNA, alone or in combination, and 24 h later (total 48-h MDP pretreatment), MDMs were stimulated with 100 mg/ml MDP for 24 h (acute), and cytokine se- cretion was assessed + SEM. Numbers on the bars are the ratios of cytokine secretion upon MDP treatment of the corresponding pretreated versus nonpretreated MDMs. Statistical signifi- cance above the knockdown sample bars is compared with its corresponding scrambled siRNA sample. (C) MDMs were left untreated (for acute) or pretreated with 100 mg/ml MDP for 24 h and then transfected with scrambled or a combination of Twist1 and Twist2 siRNA 6 vectors expressing c-Maf or Bmi1 (or empty vector control), and 24 h later (total 48-h MDP pretreatment), MDMs were treated with 100 mg/ ml MDP for an additional 24 h (acute), and supernatants were examined for cytokines. Mean concentration + SEM for n = 4. Numbers on the bars are the ratios of cytokine secretion upon MDP treatment of pretreated versus nonpre- treated MDMs. *p , 0.01, **p , 0.01, ***p , 0.001, †p , 1 3 1024, ††p , 1 3 1025. scr, scrambled; tx, treatment. The Journal of Immunology 223 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 6. Expression and cytokine promoter binding of the transcriptional activators ATF4, C/EBPa, Runx1, and Runx2 is reduced in a Twist-de- pendent manner during chronic NOD2 stimulation. (A) MDMs (n = 8) were left untreated or pretreated with 100 mg/ml MDP for 48 h, and then MDMs were treated with 100 mg/ml MDP for 4 h (acute). Fold change in ATF4, C/EBPa, Runx1, Runx2, and Akt2 mRNA normalized to untreated MDMs + SEM. (B) MDMs (n = 8) were left untreated (for acute) or pretreated with 100 mg/ml MDP for 24 h and then transfected with scrambled, Twist1, or Twist2 siRNA, alone or in combination, and 24 h later (total 48-h MDP pretreatment), MDMs were stimulated with 100 mg/ml MDP for 4 h (acute) and assessed for ATF4, C/EBPa, Runx1, Runx2, and Akt2 mRNA expression. Fold mRNA expression normalized to untreated, scrambled siRNA–transfected MDMs + SEM. (C) MDMs (n = 4–6) were left untreated (for acute) or pretreated with 100 mg/ml MDP for 24 h and then transfected with scrambled or a combination of Twist1 and Twist2 siRNA, and 24 h later (total 48-h MDP pretreatment), MDMs were treated with 100 mg/ml MDP for an additional 4 h (acute). Recruitment of ATF4, C/EBPa, Runx1, and Runx2 to cytokine gene promoters was assessed by ChIP. Fold enrichment normalized to untreated, scrambled siRNA– transfected cells + SEM. (B and C) Statistical significance above the knockdown sample bars is compared with its corresponding scrambled siRNA sample. *p , 0.01, **p , 0.01, ***p , 0.001, †p , 1 3 1024. tx, treatment. lation of the Twist-dependent transcription factors. ATF4, expression and binding of ATF4, C/EBPa, Runx1, and Runx2 to C/EBPa, Runx1, and Runx2 bound to cytokine promoters with acute cytokine promoters in Twist-deficient MDMs was contributing to NOD2 stimulation, and this binding was dramatically reduced the failure to downregulate cytokines upon chronic NOD2 stim- after chronic NOD2 stimulation, consistent with the reduced ulation, we used siRNA to each of the transcription factors cytokines observed under these conditions (Fig. 6C). Moreover, (Fig. 7A), alone and in combination. Reduced expression of each upon Twist1 and Twist2 knockdown during chronic NOD2 stim- ATF4, C/EBPa, Runx1, and Runx2 partially restored cytokine ulation, the binding of these transcription factors to cytokine downregulation, and combined knockdown of these Twist- promoters failed to undergo reduction relative to scrambled regulated transcription factors fully restored cytokine downregu- knockdown cells, consistent with the impaired cytokine down- lation in Twist-deficient macrophages after chronic NOD2 stim- regulation (Fig. 6C). Finally, to ensure that the relative increase in ulation (Fig. 7B). Cells were viable under these conditions (data 224 Twist REGULATES CHRONIC NOD2 STIMULATION OUTCOMES not shown). Therefore, Twist1 and Twist2 contribute to the whether these other PRRs cooperate with NOD2 to regulate Twist1- downregulation in expression and promoter binding of various and Twist2-mediated outcomes. Similar to chronic NOD2 stimu- transcription factors, in particular ATF4, C/EBPa, Runx1, and lation, cytokines are downregulated upon restimulation of MDM Runx2, which in turn is necessary for optimal cytokine down- after chronic stimulation of TLR2, TLR4, or TLR9 (Supplemental regulation after chronic NOD2 stimulation in MDMs. Fig. 3A). We found that upon pretreatment through TLR2, TLR4, or TLR9, and then restimulation of each respective TLR, Twist1 NOD2 synergizes with TLR2, TLR4, and TLR9 to enhance and Twist2 expression was increased (Supplemental Fig. 3B). The Twist1 and Twist2 upregulation and induction of Twist-dependent pathways we had identified were similarly reg- Twist-dependent pathways ulated under these conditions, with upregulation of the transcrip- Because intestinal macrophages are exposed to multiple PRRs, we tional repressors c-Maf and Bmi1 (Supplemental Fig. 3C) and next assessed whether chronic stimulation of other PRRs similarly downregulation of the transcriptional activators ATF4, C/EBPa, upregulates Twist1 and Twist2 expression in human MDMs, and Runx1, and Runx2 (Supplemental Fig. 3D). Although low-dose MDP treatment synergized with each of the TLR ligands to in- crease expression of cytokines during acute treatment, cytokines were downregulated upon chronic stimulation of NOD2 with each of the TLRs (Supplemental Fig. 3A). Moreover, chronic MDP treatment synergized with each TLR to upregulate Twist1 and Twist2 and the transcriptional repressors c-Maf and Bmi1

(Supplemental Fig. 3B, 3C). Taken together, under the chronic Downloaded from microbial ligand exposure that occurs in the intestinal environ- ment, NOD2 can synergize with other PRRs to upregulate Twist1 and Twist2 expression and Twist-dependent mechanisms that lead to cytokine downregulation.

Discussion http://www.jimmunol.org/ In this study, we demonstrate that Twist1 and Twist2 contribute to the downregulation of proinflammatory cytokines upon acute and particularly chronic NOD2 stimulation of human MDMs. Twist1 and Twist2 expression increased with chronic NOD2 stimula- tion, consistent with the important role for these transcriptional repressors in the downregulation of cytokines observed after prolonged NOD2 stimulation. Twist upregulation is similarly ob- served in MDMs from CD patients but is impaired in MDMs from CD NOD2 risk carriers. Early secreted IL-10 and TGF-b were by guest on September 25, 2021 critical for Twist1 and Twist2 expression upregulation with chronic NOD2 stimulation. Binding of Twist1 and Twist2 to cy- tokine promoters was particularly enhanced after chronic NOD2 stimulation, and the two proteins cooperated with each other for optimal promoter binding. In addition to the direct binding of the Twist1 and Twist2 transcriptional repressors to cytokine pro- moters, they also orchestrated a coordinated program in which the expression and cytokine promoter binding of additional tran- scriptional repressors including c-Maf and Bmi1 were increased, whereas the expression and cytokine promoter binding of various activating transcription factors including ATF4, C/EBPa, Runx1, and Runx2 were reduced. These combined Twist-mediated mechanisms contributed to the ability of Twist1 and Twist2 to downregulate NOD2-induced cytokines (Supplemental Fig. 4). Furthermore, NOD2 could synergize with various TLRs to me- FIGURE 7. ATF4, C/EBPa, Runx1, and Runx2 downregulation con- tributes to Twist-mediated cytokine downregulation after chronic NOD2 diate these outcomes, consistent with the cooperative role for stimulation. (A) MDMs (n = 4) were transfected with scrambled, ATF4, NOD2 with other PRRs and cytokines in enhancing downstream C/EBPa, Runx1, or Runx2 siRNA for 24 h. Fold mRNA expression nor- immune outcomes (36–42). Therefore, we now elucidate a clear malized to scrambled, siRNA–transfected MDMs + SEM. (B) MDMs (n = role for Twist1- and Twist2-mediated mechanisms in down- 4) were left untreated (for acute) or pretreated with 100 mg/ml MDP for regulating cytokines upon acute and chronic NOD2 stimulation of 24 h and then transfected with scrambled or a combination of Twist1 and primary human MDMs. Twist2 siRNA 6 ATF4, C/EBPa, Runx1, or Runx2 siRNA alone or in A role for Twist repressors in PRR-induced outcomes has been combination, and 24 h later (total 48-h MDP pretreatment), MDMs were relatively unexplored. However, Twist1- and Twist2-mediated m stimulated with 100 g/ml MDP for 24 h (acute), and cytokine secretion transcriptional mechanisms identified to regulate suppression in was assessed. Numbers on the bars are the ratios of cytokine secretion biological processes such as osteogenesis, hematopoiesis, and upon MDP treatment of pretreated versus the corresponding nonpretreated MDMs. Significance comparisons are between MDP treatment of pre- carcinogenesis provided the opportunity to now dissect if these treated, Twist1/2 siRNA–treated cells relative to the same condition in pathways are Twist dependent and similarly or differentially combination with siRNA to each of the transcriptional activators or as regulate outcomes upon PRR stimulation of human macrophages. indicated. *p , 0.01, **p , 0.01, ***p , 0.001, †p , 1 3 1024. scr, A number of the downstream molecules contributing to Twist- scrambled; tx, treatment. dependent regulation in these other cell processes have not been The Journal of Immunology 225 previously implicated in NOD2-induced outcomes. One such ex- 2. Feng, B. S., S. H. He, P. Y. Zheng, L. Wu, and P. C. Yang. 2007. Mast cells play ample is the role we now identify for Twist-dependent c-Maf reg- a crucial role in Staphylococcus aureus peptidoglycan-induced diarrhea. Am. J. Pathol. 171: 537–547. ulation in downregulating cytokines after chronic NOD2 stimulation. 3. Zheng, S., and C. Abraham. 2013. NF-kB1 inhibits NOD2-induced cytokine c-Maf has been predominantly studied in the context of T cell secretion through ATF3-dependent mechanisms. Mol. Cell. Biol. 33: 4857–4871. 4. Hedl, M., J. Li, J. H. Cho, and C. Abraham. 2007. Chronic stimulation of Nod2 differentiation, in particular Tr1 cells (43). However, a few stud- mediates tolerance to bacterial products. Proc. Natl. Acad. Sci. USA 104: 19440– ies have also identified a role for c-Maf as an anti-inflammatory 19445. transcription factor in myeloid cells; c-Maf downregulates tran- 5. Hedl, M., and C. Abraham. 2011. Secretory mediators regulate Nod2-induced tolerance in human macrophages. 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