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 Why The JI? Submit online. by guest on September 25, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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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 receptor (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 transcription factor 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 gene 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 proteins 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