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. 2015 Jul 1;195(1):217-26.
doi: 10.4049/jimmunol.1402808. Epub 2015 May 27.

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  1 , Matija Hedl  1 , Clara Abraham  2
Affiliations

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 et al. J Immunol. .

Abstract

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 repressors Twist1 and Twist2 in regulating PRR-induced cytokine outcomes is poorly understood and has not been reported for NOD2. 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 stimulation; this increased expression was IL-10 and TGF-β 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 repressor Bmi1. Restoring c-Maf and Bmi1 expression in Twist-deficient macrophages restored NOD2-induced cytokine downregulation. 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/EBPα, 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. Therefore, after chronic NOD2 stimulation Twist1 and Twist2 coordinate the regulation of both transcriptional activators and repressors, thereby mediating optimal cytokine downregulation.

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Figures

Figure 1
Figure 1. Twist1 and Twist2 are required for optimal cytokine downregulation upon chronic NOD2 stimulation
(A) Timeline schematic for MDP pre-treatment of MDMs, Twist1/2 knockdown and subsequent acute MDP treatment. (B) Human MDMs were left untreated (for acute) or pre-treated with 100μg/ml MDP for 24h, then transfected with scrambled, Twist1 or Twist2 siRNA alone or in combination, and 24h later (total 48h MDP pre-treatment), MDMs were treated with 100μg/ml MDP for an additional 24h (acute). Supernatants were examined for cytokines. Mean + SEM for n=4. Similar results were observed for combined Twist1 and Twist2 siRNA for an additional n=8. Numbers on the bars are the ratios of cytokine secretion upon MDP treatment of pre-treated versus non-pretreated MDMs. Statistical significance above the knockdown sample bars is compared to its corresponding scrambled siRNA sample. *, p<0.05; **, p<0.01; ***, p<0.001; †, p<1×10−4. Tx, treatment; scr, scrambled.
Figure 2
Figure 2. Twist1 and Twist2 expression is increased in MDMs after chronic NOD2 stimulation in an IL-10- and TGFβ-dependent manner whereas this increase is impaired in MDMs from Crohn's disease NOD2 risk carriers
(A) MDMs (n=4) were left untreated or pre-treated with 100μg/ml MDP for 48h, then stimulated with 100μg/ml MDP for 4h (acute). Fold mRNA expression of Twist1 and Twist2 normalized to untreated cells + SEM. (B) MDMs were left untreated or pre-treated with 100 μg/ml MDP for 48h, then treated with 100μg/ml MDP for 8h and assessed for Twist1/2 expression by Western blot. Representative Western blot from 1 of 3 individuals. GAPDH was used as a loading control. (C) MDMs were cultured with isotype control, neutralizing TGFβ (25μg/ml) or neutralizing IL-10 (5μg/ml) antibodies, alone or in combination, for 1h, then left untreated (for acute) or pre-treated with 100μg/ml MDP for 48h, and then treated with 100μg/ml MDP for an additional 4h (acute). Fold mRNA induction of Twist1 and Twist2 normalized to untreated cells (n=4) + SEM. Statistical significance above the neutralizing antibody sample bars is compared to its corresponding isotype treated sample. (D) MDMs from WT NOD2 healthy controls (WT HC, n=8), WT NOD2 Crohn's disease patients (WT CD, n=8) or Crohn's disease NOD2 risk carriers (Risk SNP, n=3; as per Supplemental Fig 1E) were left untreated or pre-treated with 100μg/ml MDP for 48h, then stimulated with 100μg/ml MDP for 4h (acute). Included is pre-treatment and acute stimulation with 0.1μg/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<1×10−4. Tx, treatment.
Figure 3
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 pre-treated with 100μg/ml MDP for 48h, and then treated with 100μg/ml MDP for an additional 4h (acute). (B) MDMs (n=4) were left untreated (for acute) or pre-treated with 100μg/ml MDP for 24h, then transfected with scrambled, Twist1 or Twist2 siRNA alone or in combination, and 24h later (total 48h MDP pre-treatment) MDMs were treated with 100μg/ml MDP for an additional 4h (acute). (A-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 to its corresponding scrambled siRNA sample. *, p<0.05; **, p<0.01; ***, p<0.001. Tx, treatment.
Figure 4
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 pre-treated with 100μg/ml MDP for 48h, and then treated with 100μg/ml MDP for 4h (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 pre-treated with 100μg/ml MDP for 24h, then transfected with scrambled, Twist1 or Twist2 siRNA, alone or in combination, 24h later (total 48h after MDP pre-treatment) MDMs were treated with 100μg/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 pre-treated with 100μg/ml MDP for 24h, then transfected with scrambled, or a combination of Twist1 and Twist2 siRNA ± vectors expressing c-Maf or Bmi1, and 24h later (total 48h MDP pre-treatment), MDMs were treated with 100μg/ml MDP for an additional 8h (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 pre-treated with 100μg/ml MDP for 24h, then transfected with scrambled or a combination of Twist1 and Twist2 siRNA, and 24h later (total 48h after MDP pre-treatment) MDMs were treated with 100μg/ml MDP for an additional 4h (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-C) Statistical significance above the knockdown sample bars is compared to its corresponding scrambled siRNA sample. *, p<0.05; **, p<0.01; ***, p<0.001; †, p<1×10−4; ††, p<1×10−5. Tx, treatment.
Figure 5
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 transfected with scrambled, Bmi1 or c-Maf siRNA for 24h. Fold mRNA expression normalized to scrambled siRNA-transfected MDMs + SEM. (B) MDMs (n=4) were left untreated (for acute) or pre-treated with 100μg/ml MDP for 24h, then transfected with scrambled, c-Maf or Bmi1 siRNA, alone or in combination, and 24h later (total 48h MDP pre-treatment), MDMs were stimulated with 100μg/ml MDP for 24h (acute) and cytokine secretion was assessed + SEM. Numbers on the bars are the ratios of cytokine secretion upon MDP treatment of the corresponding pre-treated versus non-pretreated MDMs. Statistical significance above the knockdown sample bars is compared to its corresponding scrambled siRNA sample. (C) MDMs were left untreated (for acute) or pre-treated with 100μg/ml MDP for 24h, then transfected with scrambled, or a combination of Twist1 and Twist2 siRNA ± vectors expressing c-Maf or Bmi1 (or empty vector control), and 24h later (total 48h MDP pre-treatment), MDMs were treated with 100μg/ml MDP for an additional 24h (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 pre-treated versus non-pretreated MDMs. *, p<0.01; **, p<0.01; ***, p<0.001; †, p<1×10−4; ††, p<1×10−5. Tx, treatment; scr, scrambled.
Figure 6
Figure 6. Expression and cytokine promoter binding of the transcriptional activators ATF4, C/EBPα, Runx1 and Runx2 is reduced in a Twist-dependent manner during chronic NOD2 stimulation
(A) MDMs (n=8) were left untreated or pre-treated with 100μg/ml MDP for 48h, and then MDMs were treated with 100μg/ml MDP for 4h (acute). Fold change in ATF4, C/EBPα, Runx1, Runx2 and Akt2 mRNA normalized to untreated MDMs + SEM. (B) MDMs (n=8) were left untreated (for acute) or pre-treated with 100μg/ml MDP for 24h, then transfected with scrambled, Twist1 or Twist2 siRNA, alone or in combination, and 24h later (total 48h MDP treatment), MDMs were stimulated with 100μg/ml MDP for 4h (acute) and assessed for ATF4, C/EBPα, 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 pre-treated with 100μg/ml MDP for 24h, then transfected with scrambled or a combination of Twist1 and Twist2 siRNA, and 24h later (total 48h MDP pre-treatment) MDMs were treated with 100μg/ml MDP for an additional 4h (acute). Recruitment of ATF4, C/EBPα, Runx1 and Runx2 to cytokine gene promoters was assessed by ChIP. Fold enrichment normalized to untreated, scrambled siRNA-transfected cells + SEM. (B-C) Statistical significance above the knockdown sample bars is compared to its corresponding scrambled siRNA sample. *, p<0.01; **, p<0.01; ***, p<0.001; †, p<1×10−4. Tx, treatment.
Figure 7
Figure 7. ATF4, C/EBPα, Runx1, and Runx2 downregulation contributes to Twist-mediated cytokine downregulation after chronic NOD2 stimulation
(A) MDMs (n=4) were transfected with scrambled, ATF4, C/EBPα, Runx1 or Runx2 siRNA for 24h. Fold mRNA expression normalized to scrambled, siRNA-transfected MDMs + SEM. (B) MDMs (n=4) were left untreated (for acute) or pre-treated with 100μg/ml MDP for 24h, then transfected with scrambled or a combination of Twist1 and Twist2 siRNA + ATF4, C/EBPα, Runx1 or Runx2 siRNA, alone or in combination, and 24h later (total 48h MDP pre-treatment), MDMs were stimulated with 100μg/ml MDP for 24h (acute) and cytokine secretion was assessed. Numbers on the bars are the ratios of cytokine secretion upon MDP treatment of pre-treated versus the corresponding non-pretreated MDMs. Significance comparisons are between MDP treatment of pre-treated, Twist1/2 siRNA-treated cells relative to the same condition in combination with siRNA to each of the transcriptional activators or as indicated. *, p<0.01; **, p<0.01; ***, p<0.001; †, p<1×10−4. Tx, treatment; scr, scrambled.
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