Ac2PIM-responsive miR-150 and miR-143 Target Receptor-interacting Protein Kinase 2 and Transforming Growth Factor Beta-activated Kinase 1 to Suppress NOD2-induced Immunomodulators*

Background: Ac2PIM signals via TLR2 to direct both pro- and anti-inflammatory responses. Results: Ac2PIM induces miR-150/143 via SRC-FAK-PYK2-CREB-P300 signaling to target RIP2 and TAK1 and subdues MDP-stimulated PI3K-PKC-MAPK-β-catenin axis. Conclusion: Ac2PIM-mediated TLR2 signaling suppresses the NOD2-induced immunomodulators viz. COX-2, SOCS-3, and MMP-9. Significance: TLR2-NOD2 crosstalk accentuated the utilities of Ac2PIM and MDP as vaccine adjuvant. Specific and coordinated regulation of innate immune receptor-driven signaling networks often determines the net outcome of the immune responses. Here, we investigated the cross-regulation of toll-like receptor (TLR)2 and nucleotide-binding oligomerization domain (NOD)2 pathways mediated by Ac2PIM, a tetra-acylated form of mycobacterial cell wall component and muramyl dipeptide (MDP), a peptidoglycan derivative respectively. While Ac2PIM treatment of macrophages compromised their ability to induce NOD2-dependent immunomodulators like cyclooxygenase (COX)-2, suppressor of cytokine signaling (SOCS)-3, and matrix metalloproteinase (MMP)-9, no change in the NOD2-responsive NO, TNF-α, VEGF-A, and IL-12 levels was observed. Further, genome-wide microRNA expression profiling identified Ac2PIM-responsive miR-150 and miR-143 to target NOD2 signaling adaptors, RIP2 and TAK1, respectively. Interestingly, Ac2PIM was found to activate the SRC-FAK-PYK2-CREB cascade via TLR2 to recruit CBP/P300 at the promoters of miR-150 and miR-143 and epigenetically induce their expression. Loss-of-function studies utilizing specific miRNA inhibitors establish that Ac2PIM, via the miRNAs, abrogate NOD2-induced PI3K-PKCδ-MAPK pathway to suppress β-catenin-mediated expression of COX-2, SOCS-3, and MMP-9. Our investigation has thus underscored the negative regulatory role of Ac2PIM-TLR2 signaling on NOD2 pathway which could broaden our understanding on vaccine potential or adjuvant utilities of Ac2PIM and/or MDP.

In the current study, we attempted to unravel the crosstalk, if any, between Ac 2 PIM-mediated TLR2 signaling and MDP-induced NOD2 pathway. Interestingly, we found that macrophages that were stimulated with Ac 2 PIM displayed marked reduction in its ability to express NOD2-responsive immunomodulators like COX-2, SOCS-3, and MMP-9 but not NO, TNF-␣, VEGF-A, or IL-12. This underscores the differential regulatory abilities of Ac 2 PIM-induced TLR2 pathway on NOD2 signaling. Ac 2 PIM indeed suppressed NOD2 responses by downregulating the expression of RIP2 and TAK1, adaptors of NOD2 signaling. Importantly, we identified Ac 2 PIMinduced post-transcriptional mechanism presented by microRNAs, miR-150 and miR-143, that targeted RIP2 and TAK1, respectively. Deciphering the molecular mechanism, we found that Ac 2 PIM signals via the TLR2-SRC-focal adhesion kinase (FAK)-protein tyrosine kinase 2 (PYK2)-cAMP response element-binding protein (CREB) pathway to mediate the recruitment of a coactivator complex with intrinsic histone acetyltransferase (HAT) functions, CREB-binding protein (CBP)/P300 to the promoters of miR-150 and miR-143. Further, NOD2-induced PI3K-PKC-MAPK-␤-catenin signaling axis, that was found to be crucial for the expression of the immunomodulators, was significantly inhibited in the presence of Ac 2 PIM. Together, this study has generated avenues to evaluate the vaccine potential and adjuvant utilities of Ac 2 PIM and/or MDP.

Experimental Procedures
Cells and Mice-Brewer thioglycollate (8%)-elicited primary macrophages were obtained from peritoneal exudates of wildtype (C3H/HeJ or C57BL/6J) or tlr2-KO mice. Murine RAW 264.7 macrophages cell line was obtained from National Center for Cell Sciences, Pune, India. Macrophages were cultured in DMEM (Gibco, Life Technologies) supplemented with 10% heat-inactivated FBS (Gibco, Life Technologies) and maintained at 37°C in 5% CO 2 incubator. All strains of mice were purchased from The Jackson Laboratory and maintained in the Central Animal Facility (CAF), Indian Institute of Science (IISc). All studies involving mice were performed after the approval from the Institutional Ethics Committee for animal experimentation as well as from Institutional Biosafety Committee.
Treatment with Pharmacological Reagents-Cells were treated with the given pharmacological inhibitors (all from Calbiochem) 1 h prior to the experimental treatments at  (10 M). DMSO at 0.1% concentration was used as the vehicle control. In all experiments involving pharmacological reagents, a tested concentration was used after careful titration experiments assessing the viability of the macrophages using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.
Luciferase Assay-Cells were lysed in Reporter lysis buffer (Promega) and assayed for luciferase activity using Luciferase Assay Reagent (Promega) as per the manufacturer's instructions. The results were normalized for transfection efficiencies measured by ␤-galactosidase activity. O-nitrophenol ␤-D-galactopyranoside (HiMedia) was utilized for the ␤-galactosidase assay.
RNA Isolation and Real-Time qRT-PCR-Total RNA from macrophages was isolated using TRI reagent (Sigma-Aldrich). First strand cDNA synthesis was done with 1 g of total RNA using First Strand cDNA synthesis kit (Applied Biological Materials Inc.). Expression of target gene was assessed by Real-Time quantitative Reverse Transcription-PCR (qRT-PCR) using SYBR Green PCR mix (KAPA Biosystems). All the experiments were repeated at least three times independently to ensure the reproducibility of the results. Gapdh was used as internal control. The primers used for Real-Time qRT-PCR amplification were as follows: Gapdh forward 5Ј-gagccaaacgggtcatcatct-3Ј, reverse 5Ј-gaggggccatccacagtctt-3Ј; Ripk2 forward 5Ј-gccattgagattccgcatcct-3Ј, reverse 5Ј-aacttcgtgattgagagagtgac-3Ј and Map3k7 forward 5Ј-cggatgagccgttacagtatc, reverse 5Ј-actccaagcgtttaatagtgtcg-3Ј. All the primers were purchased from Eurofins Genomics.
miRNA Expression Profiling-Total RNA was isolated from untreated, MDP-treated, and Ac 2 PIM and MDP co-treated macrophages (n ϭ 2). Sample and reference RNAs were labeled with Hy3 and Hy5, respectively using miRCURY LNA TM array power labeling kit (Exiqon). Sample and reference RNA hybridization was carried out in Tecan HS4800 hybridization station (Tecan). The miRCURY LNA TM array microarray slides were scanned using a G2565BA microarray scanner system (Agilent) and ImaGene (version 7.0) software (BioDiscovery) was used for image analysis. The log median ratio of Hy3/Hy5 intensity for replicative spots of each miRNA and the fold change in the log median ratio for each sample was calculated. miRNAs that exhibited increased fold expression in the Ac 2 PIM-MDP cotreated samples when compared with MDP alone were clustered and represented in the heat map. Data obtained were analyzed by significance analysis of microarrays (SAM) to identify differentially regulated miRNAs.
Quantification of miRNA Expression-Total RNA was isolated from macrophages using TRI reagent. Real-Time qRT-PCR for miR-26a, miR-150, and miR-143 was performed using specific TaqMan miRNA assays (Ambion, Life Technologies) as per manufacturer's instructions. U6 snRNA was used as internal control.
Immunoblotting Analysis-Cells were washed with 1ϫ PBS and lysed in RIPA buffer (50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 g/ml each of aprotinin, leupeptin, pepstatin, 1 mM Na 3 VO 4 , 1 mM NaF) on ice for 30 min. Whole cell lysates were collected. After estimation of total protein by Bradford reagent, equal amount of protein from each cell lysate was resolved on 12% SDS-PAGE and transferred onto PVDF membranes (Millipore) by semi-dry immunoblotting method (Bio-Rad). 5% nonfat dry milk powder in TBST (20 mM Tris-HCl (pH 7.4), 137 mM NaCl, and 0.1% Tween 20) was used for blocking nonspecific binding for 60 min. After washing with TBST, the blots were incubated overnight at 4°C with primary antibody diluted in TBST with 5% BSA. After washing with TBST, blots were incubated with anti-rabbit secondary antibody conjugated to HRP for 2 h. The immunoblots were developed with enhanced chemiluminescence detection system (PerkinElmer) as per manufacturer's instructions. ␤-Actin was used as loading control.
Enzyme Immunoassay (ELISA)-Cell-free culture supernatants were used for performing ELISA for TNF-␣, VEGF-A, and IL-12 (kits from PeproTech) as per the manufacturer's instructions. Briefly, 96-well flat bottom plates (Nunc MaxiSorp, Thermo Scientific) were coated with specific capture antibodies overnight at 4°C followed by three washes with 1ϫ PBST (1ϫ PBS with 0.05% Tween 20). After blocking with 1% BSA for 1 h at room temperature, wells were washed, and incubated with cell-free culture supernatants for 2 h. After three washes with 1ϫ PBST, wells were incubated with respective detection antibodies for 2 h at room temperature. Further, the wells were washed and incubation with streptavidin-HRP antibody for 30 min at room temperature. The reactions were developed with 3,3Ј,5,5Ј-tetramethylbenzidine (Sigma-Aldrich) and the absorbance was measured at 450 nm using an ELISA reader (Molecular Devices).
Estimation of NO-Cell-free culture supernatants were used for estimating NO produced by macrophage. Greiss reagent (Promega) was used to assay NO production according to the manufacturer's instructions. Briefly, nitrite standards and cellfree supernatants were added to 96-well flat bottom plates (Nunc MaxiSorp, Thermo Scientific). The samples were incubated with equilibrated sulfanilamide solution for 10 min in dark at room temperature. Further, N-1-napthylethylenediamine dihydrochloride solution was added and incubated in dark for 10 min before measuring the absorbance at 520 nm.
Statistical Analysis-Levels of significance for comparison between samples were determined by the Student's t test distribution and one-way ANOVA. The data in the graphs are expressed as the mean Ϯ S.E. for values from three independent experiments and p values Ͻ 0.05 were defined as significant. GraphPad Prism 5.0 software (GraphPad Software) was used for all the statistical analysis.

Results
Ac 2 PIM Inflects NOD2-induced Immunomodulators-Confounding to several studies where PIM 2 was found to be proinflammatory in its function (10,12), several members of PIM family that are TLR2 agonists are majorly anti-inflammatory in nature and can suppress pro-inflammatory responses mediated by other PRRs (13)(14)(15). We sought to analyze the cross-regulation, if any, between PIM-induced TLR2 signaling and another PRR signaling like NOD2 pathway. While Ac 2 PIM, one of the abundant PIMs of mycobacteria (9), was utilized as TLR2 agonist, MDP was utilized as the NOD2 agonist. Canonical NOD2 activation leads to the recruitment and active phosphorylation of the cytosolic adaptor proteins, RIP2 and TAK1. Interestingly, prior activation of TLR2 signaling by Ac 2 PIM significantly suppressed MDP-induced active phosphorylation of RIP2 and TAK1 (Fig. 1A). To establish if such inhibition had effects on the downstream responses, we chose to analyze the known NOD2-responsive immunomodulatory genes such as COX-2, SOCS-3, MMP-9. In accordance with the existing literature, MDP-NOD2 failed to induce the expression of COX-2, SOCS-3, and MMP-9 in presence of the RIP2 inhibitor, PP2 (Fig. 1B). Surprisingly, though MDP or Ac 2 PIM treatment alone induced the expression of COX-2, SOCS-3, and MMP-9 in macrophages, prior engagement of TLR2 with Ac 2 PIM significantly down-regulated the ability of NOD2 to induce these genes (Fig. 1C). Ability of Ac 2 PIM to signal through TLR2 to mediate such functions was confirmed in macrophages derived from tlr2-KO mice wherein pretreatment of Ac 2 PIM failed to suppress the expression of the candidate genes (Fig. 1D). However, other NOD2-responsive immunomodulators like NO, TNF-␣, VEGF-A, and IL-12 remained unchanged during co-treatment with Ac 2 PIM (Fig. 1, E-H). These results collectively indicate a differential regulation of NOD2 responses by Ac 2 PIM-induced TLR2 signaling.
Ac 2 PIM-responsive miRNAs Target RIP2 and TAK1 to Downregulate NOD2 Responses-Further, we investigated the molecular mechanism that govern the Ac 2 PIM-induced suppression of NOD2 signaling. As Ac 2 PIM abrogated the active phosphorylation of NOD2-induced RIP2 and TAK1 (Fig. 1A), transcript and total protein of RIP2 and TAK1 were assessed. Interestingly, while no significant difference in Ripk2 and Map3k7 transcripts were observed with individual or co-treatment of Ac 2 PIM and MDP ( Fig. 2A), marked reduction in the total RIP2 and TAK1 protein was observed when the cells were treated with Ac 2 PIM prior to MDP (Fig. 2B). This indicated a possible FIGURE 2. miR-150 and miR-143 target RIP2 and TAK1 kinases. A and B, peritoneal macrophages were pretreated with Ac 2 PIM followed by MDP treatment for 2 h. Transcript (A) and protein (B) levels of RIP2 and TAK1 were determined by Real-Time qRT-PCR and immunoblotting respectively. C, genome-wide miRNA microarray profiling was done in macrophages treated as indicated. A heat map comparison of miRNAs that exhibited increased fold expression in the Ac 2 PIM-MDP co-treated samples when compared with MDP alone (n ϭ 2). D, putative miR-26a, miR-150 and miR-143 binding sites in the CDS of Ripk2, 3Ј-UTR of Ripk2 and 3Ј-UTR of Map3k7, respectively. E and F, peritoneal macrophages were treated with Ac 2 PIM alone for 4 h (E) or with Ac 2 PIM for 2 h prior to 2 h MDP treatment (F). Real-Time qRT-PCR was performed on total RNA isolated using miRNA-specific primers. G, RAW 264.7 macrophages were transfected with specific miRNA mimics as indicated to assess the total expression levels of RIP2 and TAK1 by immunoblotting. H and I, RAW 264.7 macrophages were transfected with WT Ripk2 3Ј-UTR or miR-150⌬ Ripk2 3Ј-UTR (H) or WT Map3k7 3Ј-UTR or miR-143⌬ Map3k7 3ЈUTR (I) with miR-150 mimics (H) or miR-143 mimics (I) as indicated. Transfected macrophages were further treated with MDP or Ac 2 PIM or both and luciferase assay was performed. All data represent the mean Ϯ S.E. from three independent experiments, *, p Ͻ 0.05; **, p Ͻ 0.005; ***, p Ͻ 0.001; ns, non-significant (t test in E, one-way ANOVA in F, H, and I). All blots are representative of three independent experiments. The cells were treated with 2 g/ml Ac 2 PIM for 2 h unless mentioned otherwise followed by 200 ng/ml MDP. Med, medium; NC, negative control. involvement of post-transcriptional regulation, like those mediated by miRNAs, of RIP2 and TAK1 by Ac 2 PIM-induced TLR2 signaling. In this regard, we carried out a genome-wide expression profiling of miRNAs in macrophages treated with MDP alone or co-treated with Ac 2 PIM. Among the various differentially regulated miRNAs, we identified and clustered the miRNAs that exhibited increased expression in the Ac 2 PIM-MDP co-treated samples when compared with MDP alone (Fig.  2C). Extensive bioinformatic analysis (TargetScan, miRWalk, miRanda and RNAhybrid) identified Ripk2 as a potential target of miR-26a and miR-150 and Map3k7 as a potential target of miR-143 (Fig. 2D). The target sites located at the residues spanning from 867 to 873 of the coding sequence of Ripk2 (for miR-26a), 12 to 18 of the 3Ј-UTR of Ripk2 (for miR-150) and 380 to 387 of the 3Ј-UTR of Map3k7 (for miR-143) were identified as critical for miRNA-CDS/3Ј-UTR interactions.
Validating the microarray results, macrophages treated with Ac 2 PIM alone (Fig. 2E) or co-treated with MDP (Fig. 2F) displayed increased expression of miR-26a, miR-150, and miR-143. Importantly, MDP treatment alone did not alter the expression of these miRNAs (Fig. 2F). To establish the effect of these miRNAs on the identified targets, miRNA-specific mimics were utilized. While miR-26a mimic failed to down-regulate RIP2 expression, miR-150 and miR-143 were identified as the Ac 2 PIM-responsive miRNAs that targeted RIP2 and TAK1 respectively (Fig. 2G). To further establish that Ripk2 and Map3k7 are the bonafide targets of miR-150 and miR-143, we utilized the classical 3Ј-UTR luciferase assays. In line with the previous results, Ac 2 PIM, or co-treatment of Ac 2 PIM with MDP or transfection with miR-150 (in case of Ripk2) or miR-143 (in case of Map3k7) mimics markedly reduced WT Ripk2 and Map3k7 3Ј-UTR luciferase activity. However, the reduction was not significant when mutant constructs for miR-150 binding on Ripk2 3ЈUTR and miR-143 binding site on Map3k7 3Ј-UTR were utilized (Fig. 2, H and I). These results thus validate that Ripk2 and Map3k7 are direct targets of miR-150 and miR-143 respectively. Further, macrophages transfected with miR-150-or miR-143-specific inhibitors failed to down-regulate RIP2 and TAK1 expression in presence of Ac 2 PIM (Fig. 3, A  and B). Corroborating these results, macrophages transfected with miR-150-or miR-143-specific inhibitors also failed to exhibit Ac 2 PIM-mediated suppression of MDP-NOD2 signaling-induced expression of COX-2, SOCS-3, and MMP-9 (Fig.  3, C and D).

SRC-FAK-PYK2-CREB Signaling Mediates Ac 2 PIM-induced Expression of miR-150 and miR-143 via CBP/p300
Recruitment-Further, we explored the possible molecular mechanism of Ac 2 PIM-induced miR-150 and miR-143 expression. Role for TLR2 in mediating the expression of these miRNAs was validated in primary macrophages obtained from tlr2-KO mice. Ac 2 PIM stimulation failed to induce both miR-150 and miR-143 in tlr2-KO macrophages (Fig. 4A). Of note, TLR2 activates multiple signaling cascades to regulate immune responses in macrophages including several tyrosine kinase receptor family receptors like SRC, FAK, and PYK2 (30). Interesting reports also indicate that FAK signaling could mediate the activation of a cellular transcription factor, CREB and its binding to the DNA (31,32). Hence, we explored the role of TLR2-dependent acti-vation of the SRC-FAK-PYK2 complex and a possible downstream CREB-dependent CBP/P300 functions in the current scenario. While macrophages obtained from WT mice exhibited activation of SRC-FAK-PYK2 complex and CREB on Ac 2 PIM stimulation as assessed by the respective activatory phosphorylations, tlr2-KO macrophages failed to do so (Fig.  4B). Importantly, stimulation of macrophages with MDP alone did not induce the pathway (Fig. 4B). We also confirmed the Ac 2 PIM-induced FAK-dependent CREB activation by utilizing FAK-specific pharmacological inhibitor (Fig. 4C). Activation of CREB leads to its binding to the CBP/P300 coactivator complex that is recruited to the DNA to bring about transcriptional activation via its HAT functions (33). To establish the role for the above mentioned pathway during the Ac 2 PIM-induced expression of miR-150 and miR-143, macrophages were treated with FAK-or HAT-specific pharmacological inhibitors prior to Ac 2 PIM treatment. Macrophages failed to induce the expression of both miR-150 and miR-143 on Ac 2 PIM stimulation in presence of these inhibitors (Fig. 4D). The role for CREB-CBP/ P300 in expression of Ac 2 PIM-induced miR-150 and miR-143 was further validated by ChIP experiments. Corroborating the previous results, Ac 2 PIM-stimulation of macrophages resulted in significant recruitment of pCREB, P300, and corresponding increased H3K18 acetylation at both miR-150 and miR-143 promoters (Fig. 4E). This suggests that Ac 2 PIM activates TLR2-SRC-FAK-PYK2 complex, which in turn effectuates CREB activation, recruitment of CREB-CBP/P300 at the promoters of miR-150 and miR-143 and their expression. Analyzing the functional significance of the identified pathway, we found that primary macrophages pretreated with FAK or HAT inhibitors failed to exhibit Ac 2 PIM-mediated suppression of MDP-NOD2 signaling-induced expression of COX-2, SOCS-3, and MMP-9 (Fig. 4F).  were treated with Ac 2 PIM for 4 h and real-time qRT-PCR was performed on total RNA isolated using miRNA-specific primers. B, peritoneal macrophages from C57BL/6J WT and tlr2-KO mice were treated with Ac 2 PIM or MDP for 1 h as indicated. Total cell lysates were assessed for pFAK, pPYK2, pSRC, and pCREB by immunoblotting. C, macrophages were treated with FAK-specific inhibitor for 1 h prior to 1 h Ac 2 PIM treatment and lysates were analyzed for pCREB by immunoblotting. D, primary macrophages pretreated with either FAK inhibitor or HAT inhibitor for 1 h were treated with Ac 2 PIM for 4 h. Real-Time qRT-PCR was performed using miRNA-specific primers. E, pCREB and P300 recruitment and H3K18ac modification at the promoters of miR-150 and miR-143 was evaluated by ChIP in macrophages treated with Ac 2 PIM for 4 h. F, peritoneal macrophages were treated with indicated inhibitors for 1 h prior to 2 h Ac 2 PIM treatment followed by a 12 h MDP treatment. Lysates were assessed for COX-2, SOCS-3, and MMP-9 by immunoblotting. All data represent the mean Ϯ S.E. from three independent experiments, *, p Ͻ 0.05; **, p Ͻ 0.005; ***, p Ͻ 0.001 (one-way ANOVA in A and D, t test in E). All blots are representative of three independent experiments. All blots are representative of three independent experiments. The cells were treated with 2 g/ml Ac 2 PIM for 2 h unless mentioned otherwise followed by 200 ng/ml MDP. Med, medium; KO, knock-out; Inhi., inhibitor.

Discussion
Commonly, a concerted inter-regulatory network of PRR signaling determines the immune responses mounted against the pathogen/PAMPs (1,34). In the current investigation, we ana-FIGURE 6. Ac 2 PIM regulates NOD2-␤-catenin-mediated COX-2, SOCS-3, and MMP-9 expression. A, peritoneal macrophages were pretreated with a pharmacological inhibitor of JAK kinase (AG490) followed by treatment with MDP or IFN-␥ (200 units/ml) to analyze phosphorylation status of STAT1 and STAT3. B and C, inhibitory phosphorylation status of ␤-catenin and GSK-3␤ during the following conditions: treatment of macrophages with MDP for the indicated time points (B), pretreatment of macrophages with PI3K-MAPK pathway-specific pharmacological inhibitors followed by MDP treatment for 6 h (C). D, peritoneal macrophages were treated with AG490, ␤-catenin inhibitor or LiCl (GSK-3␤ inhibitor) prior to 12 h MDP treatment. Lysates were assessed for COX-2, SOCS-3, and MMP-9 by immunoblotting. E and F, Phosphorylation status of ␤-catenin and GSK-3␤ was assessed by immunoblotting under following conditions: Peritoneal macrophages were pretreated with the indicated inhibitors for 1 h followed by Ac 2 PIM and MDP treatment for 6 h (E), miR-150-(left panel) or miR-143-(right panel) specific miRNA inhibitor-transfected RAW 264.7 macrophages were treated with Ac 2 PIM prior to MDP treatment for 6 h (F). All blots are representative of three independent experiments. The cells were treated with 2 g/ml Ac 2 PIM for 2 h followed by 200 ng/ml MDP. Med, medium; DMSO, dimethyl sulfoxide; NC, negative control; Inhi., inhibitor; LiCl, lithium chloride. lyzed the crosstalk of two important families of PRR, TLRs, and NLRs. Specifically, a mycobacterial cell wall glycolipid Ac 2 PIM and component of bacterial peptidoglycan MDP were utilized as cognate ligands for TLR2 and NOD2 pathway. Interestingly, Ac 2 PIM-stimulated TLR2 signaling was found to abrogate NOD2-responsive immune modulators. This was in accordance with the previous observations of antagonistic regulation between TLR2-NOD2 signaling wherein NOD2 was found to negatively regulate TLR2-induced Th1 responses (6,7). The agonist used in the study was peptidoglycan, a bacterial PAMP common for both pathways. However, NOD2-TLR2 can also synergistically induce inflammatory responses (16,35). Further, there exists a cooperative regulation of NOD2-and TLR2specific ligands for mediating immune cytokines (8,36,37). NOD2-TLR2 synergy was also found in different cellular contexts (38,39). Thus, the present study underscores the fact that though multiple PAMPs recognize a single PRR, there exist regulatory mechanisms to orchestrate the ligand-specific immune responses.
Ac 2 PIM was potent to inhibit COX-2, SOCS-3, and MMP-9 expression but did not alter the levels of NO, TNF-␣, VEGF-A, and IL-12 during co-treatment with MDP. This was surprising as Ac 2 PIM alone, like MDP, could upregulate these immunomodulators. Supporting this observation, monoacyl form of PIM 2 was previously reported to have induced the expression of these immunomodulators (40,41).
Of note, though classical NOD2 responses via RIP2 and TAK1 are well established, various previous investigations have suggested that MDP-induced NOD2 responses could be RIP2/ TAK1-independent (42,43). However, in the current study, we found role for the classical NOD2 responses. Deciphering the mechanism of Ac 2 PIM-arbitrated inhibition of MDP-NOD2 responses, we found miRNA-mediated regulation of RIP2 and TAK1 expression. Extensive studies on TLR-responsive miRNAs suggest a role for miRNAs in not only orchestrating innate immune responses or a negative feed-back loop (44 -48) but also negatively regulate responses mediated by other PRRs including NOD2 (49). Here, miR-150 and miR-143 were found to be induced by Ac 2 PIM-stimulation of macrophages that targeted the NOD2 adapters, RIP2 and TAK1, respectively. Though several investigations have implicated miR-150 in regulating innate immune responses (45, 50 -52), no reports on miR-150 and regulation of RIP2 or NOD2 signaling exists. Interestingly, supporting our data, miR-143 has been previously reported to regulate immune responses in various cellular contexts (53)(54)(55) and target TAK1 in mesenchymal stem cells (56) and adipocytes (57).
Though few reports have indicated the crosstalk between TLR2 signaling and SRC-FAK-PYK2 complex (30,58,59), no reports exist on the molecular mechanism induced by Ac 2 PIM via TLR2. We found Ac 2 PIM activated TLR2-SRC-FAK-PYK2 cascade to induce expression of miR-150 and miR-143. FAK and PYK2 kinases were previously reported for their possible abilities to activate CREB responses (31,32,60). Other investigations have implicated CREB activation to mediate TLR2-mediated immune functions (47,61). In line with these, we found SRC-FAK-PYK2 signals to activate CREB and induce CREB-CBP/P300 recruitment to miR-150 and miR-143 promoters and epigenetically regulate their expression via the intrinsic HAT activity.