Pathogen-specific TLR2 Protein Activation Programs Macrophages to Induce Wnt-β-Catenin Signaling*

Innate immunity recognizes and resists various pathogens; however, the mechanisms regulating pathogen versus nonpathogen discrimination are still imprecisely understood. Here, we demonstrate that pathogen-specific activation of TLR2 upon infection with Mycobacterium bovis BCG, in comparison with other pathogenic microbes, including Salmonella typhimurium and Staphylococcus aureus, programs macrophages for robust up-regulation of signaling cohorts of Wnt-β-catenin signaling. Signaling perturbations or genetic approaches suggest that infection-mediated stimulation of Wnt-β-catenin is vital for activation of Notch1 signaling. Interestingly, inducible NOS (iNOS) activity is pivotal for TLR2-mediated activation of Wnt-β-catenin signaling as iNOS−/− mice demonstrated compromised ability to trigger activation of Wnt-β-catenin signaling as well as Notch1-mediated cellular responses. Intriguingly, TLR2-driven integration of iNOS/NO, Wnt-β-catenin, and Notch1 signaling contributes to its capacity to regulate the battery of genes associated with TReg cell lineage commitment. These findings reveal a role for differential stimulation of TLR2 in deciding the strength of Wnt-β-catenin signaling, which together with signals from Notch1 contributes toward the modulation of a defined set of effector functions in macrophages and thus establishes a conceptual framework for the development of novel therapeutics.

Innate immunity recognizes and resists various pathogens; however, the mechanisms regulating pathogen versus nonpathogen discrimination are still imprecisely understood. Here, we demonstrate that pathogen-specific activation of TLR2 upon infection with Mycobacterium bovis BCG, in comparison with other pathogenic microbes, including Salmonella typhimurium and Staphylococcus aureus, programs macrophages for robust up-regulation of signaling cohorts of Wnt-␤-catenin signaling. Signaling perturbations or genetic approaches suggest that infection-mediated stimulation of Wnt-␤-catenin is vital for activation of Notch1 signaling. Interestingly, inducible NOS (iNOS) activity is pivotal for TLR2-mediated activation of Wnt-␤-catenin signaling as iNOS ؊/؊ mice demonstrated compromised ability to trigger activation of Wnt-␤-catenin signaling as well as Notch1-mediated cellular responses. Intriguingly, TLR2-driven integration of iNOS/NO, Wnt-␤-catenin, and Notch1 signaling contributes to its capacity to regulate the battery of genes associated with T Reg cell lineage commitment. These findings reveal a role for differential stimulation of TLR2 in deciding the strength of Wnt-␤-catenin signaling, which together with signals from Notch1 contributes toward the modulation of a defined set of effector functions in macrophages and thus establishes a conceptual framework for the development of novel therapeutics.
Macrophages as important sentinels of the innate immunity utilize a limited number of pattern recognition receptors, including TLR2 to discriminate among various microbial pathogens and also to mount immune responses commensurate with each pathogen (1,2). However, the immune cells cannot exclusively utilize "pattern recognition" as the basis of pathogen-specific defense, because a plethora of microbial pathogens share the molecules involved in such recognition with innocuous commensal flora (3,4). In this regard, macrophages must be able to distinguish the nature and scope of microbial threats to tailor specific transcriptional responses. In this perspective, intensive interplay between signaling pathways can act as an important regulatory mechanism by which the immune responses are tailored against the type of pathogen encountered. However, information about the signaling cascades regulating pathogen-specific TLR2 responses is still imprecisely understood. Interestingly, TLR2 triggering in contrast to current doctrine could lead to tolerogenic or regulatory responses thus conjecturing a role for pathogen-specific activation of TLR2 in immune evasion mechanisms (5)(6)(7). Thus, pathogen-specific TLR2-mediated signaling pathways could contribute to unique tailor-made defense responses to invading pathogens. Among signaling events, Wnt-␤-catenin signaling plays a critical role in regulation of various cellular processes, including cell polarity, motility, differentiation, apoptosis, and carcinogenesis in both nonvertebrates and vertebrates (8,9). Wnt ligands secreted from various cell types typically bind to transmembrane receptors of the Frizzled family. This results in activation of multifaceted signaling cascade culminating in marked inhibition of a negative regulator of ␤-catenin protein levels, glycogen synthase kinase-3␤ (GSK-3␤). The inhibition of GSK-3␤ leads to nuclear translocation of ␤-catenin and subsequent activation of lymphoid enhancer factor/T cell factor (LEF1/TCF) 5 target genes (8,10,11). Despite reported effects of Wnt signaling on various cell fate decisions, the participation of other vital signaling events involved in defining the inflammatory signatures during ensuing immunity remains to be identified. In this regard, involvement of Notch signaling in conferring specific phenotypical attributes to macrophages and dendritic cells assumes critical importance. Notch signaling is generally initiated by binding of Jagged or Delta, specific ligands of Notch receptor. Upon binding of cognate ligand, Notch protein undergoes a proteolytic cleavage that releases Notch intracellular domain (NICD/Cleaved Notch) that translocates to the nucleus and forms a complex with DNA-binding protein CSL/ RBP-Jk and activates specific gene transcription (12). Interestingly, Wnt and Notch signaling pathways are intimately intertwined during self-renewal of stem cells and tumor development. Furthermore, physical binding of Notch to ␤-catenin or their association with common co-factors has been demonstrated in various cellular systems. In addition, accurate coordination of Notch and Wnt signals is critical during normal development (13)(14)(15). Intriguingly, many Notch target genes such as cyclooxygenase-2 (COX-2), Jagged1, and Hes1 are also targeted by Wnt-␤-catenin, suggesting a functional overlap between Notch and Wnt-␤-catenin pathways (16 -20).
In view of the above observations, we set out to unravel the molecular mechanisms contributing toward pathogen-specific Toll-like receptor responses, principally with respect to the role of Wnt-␤-catenin and Notch signaling axis. We demonstrate that "pathogenic" TLR2 stimulation confers differential activation of Wnt-␤-catenin signaling in macrophages. Infection with Mycobacterium bovis BCG in comparison with Salmonella typhimurium and Staphylococcus aureus resulted in consistent activation of Wnt-␤-catenin signaling at multiple levels, including up-regulation of Wnt5a and FzD4 transcript levels, stabilization of ␤-catenin, and activation of Wnt-␤-catenin transcriptional activity, thus culminating in the expression of a multitude of genetic signatures crucial for mounting appropriate regulatory or tolerogenic responses, including COX-2 and suppressor of cytokine signaling-3 (SOCS-3). Intriguingly, nonpathogenic bacterial strains Mycobacterium smegmatis or Escherichia coli failed to induce consistent up-regulation of signaling cohorts of the Wnt-␤-catenin signaling cascade. M. bovis BCG-triggered stabilization of ␤-catenin led to an increase in its occupancy on genomic targets, including Jagged1 culminating in induced activation of Notch1 signaling. We present the evidence that inducible nitric-oxide synthase (iNOS) activity is a critical factor in TLR2-mediated activation of Wnt-␤-catenin signaling as macrophages derived from iNOS knock-out (iNOS Ϫ/Ϫ ), but not from wild type (WT) mice, failed to activate Wnt5a/FzD4 expression as well as Notch1 signaling upon M. bovis BCG infection. The loss of TLR2-mediated Wnt5a/FzD4 expression or Notch1 activation in iNOS Ϫ/Ϫ macrophages could be rescued by treatment with the NO donor, 3-morpholinosydnonimine (SIN-1). Correlative evidence infers that this mechanism operates in vivo as immunohistochemical expression of ␤-catenin, Jagged1, activated Notch1, or its target gene products COX-2 and SOCS-3 could be detected in brains derived from wild type (WT) but not iNOS Ϫ/Ϫ mice that were intracerebrally infected with M. bovis BCG. Consistent with these results, activation of Wnt-␤-catenin/Notch1 signaling in vivo could be demonstrated only in granulomatous lesions in brains derived from human tuberculous meningitis patients as opposed to healthy individuals validating the role of TLR2-dependent activation of the Wnt-Notch signaling axis in mycobacterial pathogenesis. Interestingly, Wnt-␤-catenin/Notch signaling dictates T Reg lineage commitment via reprogramming of the gene expression pattern in macrophages, including induced expression of COX-2 and SOCS-3. Thus, these studies establish the Wnt-␤-catenin/Notch signaling axis as a determinant of pathogen-specific regulatory TLR2 responses that may play a major role in dictating the functional outcomes of tuberculosis infection.

EXPERIMENTAL PROCEDURES
Cells, Mice, and Bacteria-Peritoneal macrophages were isolated from peritoneal exudates of C57BL/6 or iNOS Ϫ/Ϫ C57BL/6 or TLR2 Ϫ/Ϫ C57BL/6 mice that were maintained at the central animal facility, Indian Institute of Science. CD4 ϩ T cells were enriched from splenocytes obtained from C57BL/6 mice. The RAW 264.7 mouse macrophage cell line was cultivated in DMEM (Sigma) supplemented with 10% heat-inactivated FBS (Sigma). All studies involving mice were carried out after the approval from the Institutional Ethics Committee for Animal Experimentation and from Institutional Biosafety Committee. M. bovis BCG Pasteur 1173P2, S. typhimurium, and S. aureus were grown to mid-log phase, and batch cultures were aliquoted followed by storage at Ϫ70°C. Representative vials were thawed and enumerated for viable colony-forming units and used at 10 multiplicities of infection for infection in all the experiments.
Treatment with Pharmacological Reagents-All the pharmacological reagents were procured from Calbiochem and were reconstituted in sterile DMSO (Sigma) and used at the following concentrations: ␤-catenin inhibitor (7. In all experiments involving pharmacological reagents, a tested concentration was used after careful titration experiments assessing the viability of the macrophages using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assay. In experiments with inhibitors, the cells were treated with a given inhibitor for 60 min before experimental treatment.
Immunoblotting-Macrophages were washed twice with PBS, scraped off the culture dish, and collected by centrifugation. Cell lysates were prepared in RIPA buffer consisting of 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 and incubated on ice for 30 min. Whole cell lysate was collected by centrifuging lysed cells at 13,000 ϫ g for 10 min at 4°C. An equal amount of protein from each cell lysate was subjected to SDS-PAGE and transferred onto PVDF membranes (Millipore) by semidry Western blotting (Bio-Rad) method. Nonspecific binding was blocked with 5% nonfat dry milk powder in TBST (20 mM Tris-HCl (pH 7.4), 137 mM NaCl, and 0.1% Tween 20) for 60 min. The blots were incubated overnight at 4°C with primary antibodies diluted in TBST with 5% BSA. After washing with TBST, blots were incubated with anti-rabbit or anti-mouse IgG secondary antibodies conjugated to HRP for 2 h. After further washing in TBST, the immunoblots were developed with enhanced chemiluminescence detection system (PerkinElmer Life Sciences) as per manufacturer's instructions.
Nuclear and Cytosolic Subcellular Fractionation-Macrophages were treated as indicated, harvested by centrifugation, and gently resuspended in ice-cold Buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF). After incubation on ice for 15 min, cell membranes were disrupted with 10% Nonidet P-40, and the nuclear pellets were recovered by centrifugation 13,000 ϫ g for 15 min at 4°C. The supernatants from this step were used as cytosolic extracts. Nuclear pellets were lysed with ice-cold Buffer C (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF), and nuclear extracts were collected after centrifugation at 13,000 ϫ g for 20 min at 4°C.
Enzyme Immunoassay for PGE 2 -Enzyme immunoassays for quantification of PGE 2 in culture supernatant of treated samples were carried out in 96-well microtiter plates using PGE 2 Express EIA kit (Cayman Chemical). Assay plates were incubated with culture supernatants and processed according to the manufacturer's instructions. The absorbance was measured at 420 nm using enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices).
Measurement of Nitric Oxide-To measure the amount of NO produced by macrophages, macrophages were treated as indicated. At the end of experiment, culture supernatants were harvested by centrifugation and subjected to assay for NO production using Griess reagent according to the manufacturer's instructions (Promega).
␥-Secretase Activity Assay-To measure ␥-secretase activity, solubilized cell membranes were incubated in 150 l of assay buffer containing 50 mM Tris-HCl (pH 6.8), 2 mM EDTA, 0.25% CHAPSO (w/v), and 8 M fluorogenic ␥-secretase peptide substrate (Calbiochem) at 37°C for 12 h. After incubation, samples were centrifuged at 13,000 ϫ g for 15 min followed by measurement of fluorescence using a fluorometer with excitation wavelength at 355 nm and emission wavelength at 440 nm.
Transfection Studies-RAW 264.7 macrophage cells were transfected with 100 nM siRNA using Oligofectamine (Invitrogen) according to the manufacturer's instructions. Transfection efficiency has been more than 50% through all the experiments as determined by counting the number of siGLO Lamin A/C-positive cells in a microscopic field using a fluorescent microscope. 72 h post-transfection, the cells were treated as indicated and processed for expression analysis. Wnt5a, ␤-catenin, Notch1, MyD88, and control siRNAs were obtained from Dharmacon as siGENOME TM SMARTpool reagents, which contains a pool of four different double-stranded RNA oligonucleotides (siRNA). RAW 264.7 macrophages were transiently transfected with PKC␣, PKC␤, and PKC␦ dominant negative cDNA constructs using low molecular weight polyethyleneimine (Sigma).
Chromatin Immunoprecipitation Assay-Chromatin immunoprecipitation (ChIP) assays were carried out using protocol provided by Upstate Biotechnology, Inc., with certain modifications. Briefly, mouse macrophages were infected with M. bovis BCG for 6 h. The cells were fixed with 1.42% formaldehyde for 15 min at room temperature followed by inactivation of formaldehyde with addition of 125 mM glycine. Chromatin extracts containing DNA fragments with an average size of 500 bp were immunoprecipitated using anti-␤-catenin antibodies. Purified DNA was analyzed by quantitative PCR using the SYBR Green method (Finnzymes, Finland). Regions with ␤-catenin/TCF-binding site in mouse Jagged1 promoter were amplified using primer pairs, ␤-catenin/TCF forward, 5Ј-cctccccgcgtttcatg-3Ј, ␤-catenin/TCF reverse, 5Ј-gcaaagagcccggcctc-3Ј; 28 S rRNA was used as control in the PCR and the primers were forward 5Ј-ctgggtataggggcgaaagac-3Ј and reverse 5Ј-ggccccaagacctctaatcat-3Ј. All results were normalized either by respective input values or by amplification of 28 S rRNA. All ChIP experiments were repeated at least three times.
Tuberculosis Patients and Healthy Subjects-The study population was comprised of tuberculous meningitis patients (TBM, n ϭ 24), pulmonary tuberculosis patients (n ϭ 11), and healthy controls (n ϭ 4) reporting to the National Institute of Mental Health and Neurosciences, Bangalore, India. TBM patients were described as having clinical meningitis along with culture positivity for acid-fast bacilli and/or M. tuberculosis cultured from the cerebrospinal fluid. Active pulmonary tuberculosis disease in patients was established by detection of acid-fast bacilli in sputum smear examinations or growth of bacilli in BACTEC cultures. Patients with active pulmonary tuberculosis infection were also examined for radiological abnormalities by chest x-ray. The healthy subjects included in the study were recruited after radiological and clinical examination to exclude individuals with active tuberculosis disease. The study subjects had given written consent, and the study was approved by the Institutional Bioethics Committee.
In Vivo Challenge of Mice with M. bovis BCG or SIN-1-C57BL/6 and iNOS Ϫ/Ϫ C57BL/6 mice used in the current investigation were 5-6 weeks old, and each in vivo experiment involved three animals per group. For intracerebral infection, 1 ϫ 10 6 M. bovis BCG Pasteur 1173P2 bacteria were washed in PBS, resuspended in 50 l of sterile PBS, followed by intracranial inoculation using 1-ml syringes and a 26-gauge needle. Control mice received 50 l of sterile PBS using the same protocol. One set of iNOS Ϫ/Ϫ mice were inoculated with 20 g of SIN-1. Before intracranial inoculation, mice were anesthetized with intraperitoneal injection of ketamine (6 mg). In experiments involving TLR2 antibody, WT mice received anti-TLR2 or control IgG antibody (200 g/kg) 24 h prior to infection with M. bovis BCG (18,21). For in vivo knockdown of MyD88, WT mice were injected with 0.6 nmol of MyD88 or control siRNA complexed with low molecular weight polyethyleneimine 24 h before intracranial inoculation with M. bovis BCG (18,(22)(23)(24). After 5 days of inoculation, brains were harvested from experimental mice and processed for either tissue sectioning or RNA isolation.
Immunohistochemistry-Microtome sections (4m) were sliced from formalin-fixed, decalcified, and paraffin-embedded tissue samples. These paraffin-embedded sections were first deparaffinized, followed by antigen retrieval with boiling 10 mM citrate buffer (pH 6.0) in a boiling water bath for 10 min, treated with 1% H 2 O 2 for 10 min, and blocked with 5% BSA for 1 h at room temperature. The tissue sections were incubated with primary antibodies for 12 h and HRP-conjugated secondary antibodies for 90 min. The horseradish peroxidase reaction was detected with 0.05% diaminobenzidine and 0.03% H 2 O 2 . Sections were counterstained with hematoxylin, dehydrated, and mounted. Stained tissue sections were analyzed with a Leica DMLB microscope (Leica Microsystems, Wetzlar, Germany). All experiments were performed with appropriate isotypematched control Abs.
Detection of CD4 ϩ CD25 ϩ FoxP3 ϩ T Reg Cells by Flow Cytometry-Macrophages were treated as indicated for 24 h followed by co-culture with enriched CD4 ϩ T cells for 5 days at 37°C in a humidified 5% CO 2 atmosphere. Surface and intracellular staining to detect CD4 ϩ CD25 ϩ Foxp3 ϩ T Reg cells was performed with specifically labeled mAbs, and samples were proceeded for flow cytometry (LSR II, BD Biosciences). Cells were gated for CD4 ϩ lymphocytes followed by determination of the percentages of CD25 ϩ Foxp3 ϩ cells. For each sample, five thousand events were recorded. Data were analyzed using BD FACS DIVA software (BD Biosciences).
Statistical Analysis-Levels of significance for comparison between samples were determined by the Student's t test distribution. The data in the graphs are expressed as the mean Ϯ S.E. p values Ͻ 0.05 were defined as significant. Graphpad Prism 3.0 software (Graphpad Software) was used for all the statistical analysis.

Pathogen-specific TLR2 Signaling Controls Activation of Wnt-␤-Catenin and Notch1
Signaling-Wnt5a, a prototypical member of Wnt family, is induced by LPS/IFN-␥ in human macrophages and has been critically implicated in inflammatory macrophage signaling in sepsis and various other pathophysiological diseases, including tuberculosis (25,26). Interestingly, Wnt5a has long been considered to be a representative noncanonical Wnt in several cell types. However, recent reports have indicated that Wnt5a can activate discrete ␤-catenin signaling in the presence of FzD4 and LRP5 (27,28). Furthermore, during the initial stages of this study, we had performed extensive screening in regard to expression levels of various members of Wnt family upon infection of macrophages with M. bovis BCG in comparison with nonpathogenic bacterial strains M. smegmatis or E. coli. As demonstrated in supplemental  Fig. S1, B-D). Activation of canonical Wnt signaling is characterized by increased levels of phosphorylated GSK-3␤ with a concomitant decrease in phosphorylated ␤-catenin (8). Accordingly, infection of macrophages with M. bovis BCG triggered significant increase and concomitant decrease in phosphorylation of GSK-3␤ and ␤-catenin, respectively, compared with S. typhimurium or S. aureus challenge (Fig. 1B).
As described previously, Wnt signaling-regulated cell fate decisions often involve the activation of Notch1 signaling, and we have previously demonstrated that M. bovis BCG challenge provokes spectrum of cellular signaling, including Notch signaling activation (16,18,19). Evidently, Notch target genes are often regulated by Wnt-␤-catenin, thus suggesting a role for integrated signaling circuits in modulation of immune responses (17,20). Consistent with these observations, M. bovis BCG infection compared with S. typhimurium or S. aureus triggered robust activation of Notch1 signaling as evidenced by induced expression of Notch1, its cognate receptor Jagged1, generation of NICD, and enhanced expression of Notch1 target gene products, COX-2 and SOCS-3 (Fig. 1, A and C, and supplemental Fig. S1E).
Activation of Wnt-␤-Catenin and Notch1 Signaling during Human Tuberculosis Infection-To bring relevance to the biology of Mycobacterium infection in vivo, we investigated the expression of various cohorts of Wnt-␤-catenin and Notch1 signaling in peripheral blood mononuclear cells derived from tuberculosis patients. Transcript level analysis demonstrated higher levels of Wnt5a, FzD4, Notch1, and Jag-ged1 as well as its target genes COX-2 and SOCS-3 in M.

Pathogen-specific Activation of Wnt-␤-Catenin
tuberculosis-infected individuals in comparison with healthy subjects (Fig. 2A). In addition, immunohistochemical expression analysis of brain tissue samples from TBM patients exhibited significantly increased expression levels of ␤-catenin, Jagged1, or NICD compared with healthy subjects (Fig. 2, B-D). Similarly, COX-2 expression levels were   OCTOBER  significantly enhanced in brain samples of TBM patients compared with healthy subjects (Fig. 2E).

Pathogenic TLR2-MyD88 Axis Is an Essential Link in M. bovis BCG-triggered Activation of Wnt-␤-Catenin and Notch1
Signaling in Vivo in Mice-To establish contribution of pathogenic TLR2-MyD88 axis in activation of Wnt-␤-catenin and Notch1 signaling in vivo, a suggested murine model for the study of CNS tuberculosis or tuberculous meningitis involving intracranial inoculation of M. bovis BCG was utilized (29). Selective interference with TLR2 signaling in vivo by neutralizing antibodies against TLR2 clearly abrogated M. bovis BCG-triggered Wnt5a, FzD4, Notch1, and Jagged1 expression as well as expression of Notch1 signaling target genes COX-2 and SOCS-3 (Fig. 3A). Accordingly, siRNA-mediated knockdown of MyD88 in vivo abolished M. bovis BCG-induced expression of signaling intermediates of Wnt-␤catenin and Notch1 signaling, including Wnt5a, FzD4, Jag-ged1, and Notch1 or its target genes COX-2 and SOCS-3 (Fig. 3B). These results strongly advocate a critical role for TLR2-MyD88 axis in activation of Wnt-␤-catenin and Notch1 signaling in vivo.

Jagged1 Is Pathogenic Link between M. bovis BCG-triggered Wnt-␤-Catenin and Notch1
Pathways-Wnt-␤-catenin pathway can directly influence diverse signaling cascades through activation of specific genetic signatures, including Jagged1driven activation of Notch1 (20). In view of robust activation of Wnt-␤-catenin and Notch1 pathways during pathogen-specific activation of TLR2 signaling by mycobacteria, identification of possible cross-talk between these signaling pathways assumes novel significance. In this perspective, pharmacological inhibition of ␤-catenin activity significantly abolished M. bovis BCGtriggered Jagged1 expression as well as activation of Notch1 (Fig. 4, A and B). Accordingly, expression of Notch1 target genes, COX-2, SOCS-3, as well as PGE 2 , an immunosuppressive product of COX-2 activity, was inhibited by ␤-catenin inhibitor ( Fig. 4C and supplemental Fig. S2A). Furthermore, activation of Notch1 signaling is tightly regulated by protease activity executed by ␥-secretase complex that releases the active NICD (12). Significantly, pharmacological inhibition of ␤-catenin strongly repressed M. bovis BCG-triggered ␥-secretase activity (Fig. 4D). Furthermore, siRNA-mediated knockdown of Wnt5a and ␤-catenin severely compromised M. bovis BCG-elicited expression of Jagged1, NICD, as well as COX-2 and SOCS-3 (Fig. 4, E and F, and supplemental Fig. S2B and data not shown). Moreover, stabilization of ␤-catenin in macrophages by treatment with the GSK-3␤ inhibitor LiCl was sufficient to induce Jagged1 expression and enhance ␥-secretase complex activity as well as NICD generation (Fig. 4, G and H). Similarly, LiCl treatment augmented COX-2, SOCS-3, and PGE 2 expression, and infection with M. bovis BCG further potentiated LiCl-induced expression of COX-2 and SOCS-3 ( Fig. 4I and supplemental Fig. S2C).
To further validate this, we identified eight sites for ␤-catenin/TCF binding consensus in mouse Jagged1 promoter, and chromatin immunoprecipitation analysis revealed that M. bovis BCG infection results in enhanced recruitment of ␤-catenin at the Jagged1 promoter in vivo (Fig. 4J). Furthermore, gel shift experiments showed an increased binding of nuclear proteins to ␤-catenin/TCF consensus in Jagged promoter upon infection with M. bovis BCG, which was compromised upon pretreatment of macrophages with ␤-catenin inhibitor (supplemental Fig. S2D). Concomitantly, LiCl treatment also induced enhanced binding of ␤-catenin/TCF to its consensus (supplemental Fig. 2D). These findings noticeably support the critical participation of the Wnt-␤-catenin pathway in M. bovis BCG-induced Jagged1 expression and activation of Notch1 signaling in macrophages.

Requirement of iNOS/NO in M. bovis BCG-driven Activation of Wnt-Notch1
Signaling Axis-Nitric oxide (NO), a catalytic product of iNOS, frequently executes critical cell fate decisions by functioning as an important molecular signal in regulation of specific proinflammatory responses in macrophages (30,31). In this regard, we had earlier reported that iNOS/NO could trigger the activation of Notch1 signaling as well as expression of its target genes, including COX-2 and MMP-9, during M. bovis BCG infection (16,18). Interestingly, iNOS/NO was reported to act as a critical regulator of Wnt signaling responses in a murine model of colitis (32). In this perspective, we addressed whether M. bovis BCG-specific activation of TLR2/iNOS/NO axis participates in activation of Wnt-Notch1 signaling. When analyzed, infection with M. bovis BCG triggered robust increase in NO levels in comparison with M. smegmatis-and E. coli-infected macrophages (supplemental Fig. S3A). Furthermore, as shown in Fig. 5, A and B, and supplemental Fig. S3, B and C, macrophages from iNOS null mice exhibited marked deficiency in M. bovis BCG-triggered expression of Wnt5a, FzD4, LRP5, and Jagged1 as well as Notch1 target genes SOCS-3, COX-2, and its bioactive product PGE 2 , when compared with WT macrophages. Accordingly, deficiency in iNOS expression resulted in marked inhibition in phosphorylation of GSK-3␤, concomitant decrease in phosphorylation of ␤-catenin, and recruitment of ␤-catenin at Jagged1 promoter (Fig. 5, C and D). These results are consistent with decreased activity of ␥-secretase complex in iNOS Ϫ/Ϫ macrophages ( Fig. 5E and supplemental Fig. S3D). Importantly, deficiency in activation of Wnt-Notch1 signaling was not due to global impairment in diverse cellular functions in iNOS null macrophages as analogues of the iNOS downstream mediator, SIN-1 (NO donor), could restore the activation of various signaling cohorts of Wnt-Notch1 signaling as well as ␥-secretase complex in iNOS Ϫ/Ϫ macrophages (Fig. 5, A and C, and supplemental Fig. S3, B, D, and E). Moreover, inhibition of GSK-3␤ by LiCl in iNOS null macrophages not only activated canonical Wnt-␤-catenin signaling as evidenced by stabilized ␤-catenin levels (supplemental Fig. S3F), but it also enhanced expression of Notch1 ligand, Jagged1, and Notch1 target genes COX-2 and SOCS-3 (Fig. 5B). Similarly, we observed augmented activation of ␥-secretase complex in iNOS Ϫ/Ϫ macrophages upon inhibition of GSK-3␤ by LiCl (Fig.  5E). These results indicate the decisive role of iNOS/NO axis during mycobacterium-specific integration of Wnt-␤-catenin and Notch1 signaling.

iNOS/NO Is a Critical Link in Mycobacterium-specific Activation of Wnt-Notch1
Signaling in Vivo-To ascertain the critical role of iNOS/NO axis in TLR2-driven activation of Wnt-Notch1 signaling in vivo, WT and iNOS Ϫ/Ϫ mice were challenged intracranially with M. bovis BCG, and the activation status of Wnt-␤-catenin and Notch1 signaling was analyzed. In accordance with results obtained with macrophages, iNOS deficiency in iNOS Ϫ/Ϫ mice severely compromised the M. bovis BCG potential to trigger augmented expression of Wnt5a, FzD4, ␤-catenin, Jagged1, and Notch1/NICD as evaluated by quantitative real time PCR or immunohistochemistry-based quantifications in the brain sections (Fig. 6, A-D). Similarly, expression levels of Notch1 target genes COX-2 or SOCS-3 were markedly reduced in brain sections derived from infected iNOS Ϫ/Ϫ mice compared with WT mice (Fig. 6, A, E, and F). Importantly, NO donor (SIN-1) treatment of iNOS Ϫ/Ϫ mice in vivo restored expression of Wnt5a, FzD4, ␤-catenin, Jagged1, and Notch1 as well as Notch1 target genes COX-2 and SOCS-3 (Fig. 6, A-F). These results serve as correlative evidence for the role of iNOS/NO in regulation of TLR2-mediated cellular responses, including activation of Wnt-␤-catenin and Notch1 signaling.
PKC-MAPK-NF-B Axis Orchestrates Mycobacterium-specific TLR2 Responses in a Wnt-Notch1-dependent Manner-Protein kinase C (PKC) is suggested to act as a critical regulatory kinase and to effect profound changes in cell physiology by eliciting a transcriptional response and altering the mRNA pro- file of the cells (33,34). In addition, recent reports advocate strong correlation between Notch and PKC activity in important regulatory functions within various immune cells (35). Furthermore, the steady expansion of innate immune responses initiated by innate immune receptors often involves regulatory action of PKC that acts upstream of activation of MAPK (36,37). In this perspective, we assessed whether PKC-MAPK and Wnt-Notch1 signaling axis collaborated functionally to regulate the defined set of effector functions in macrophages. As an example, M. bovis BCG-triggered expression of Notch1 target gene COX-2 was significantly reduced by pharmacological inhibition of PKC (supplemental Fig. S4A). Furthermore, among specific PKC isoforms, inhibition of PKC␣, PKC␤, and PKC␦ activity by specific pharmacological inhibitors or dominant negative constructs markedly abolished M. bovis BCGstimulated expression of COX-2 (Fig. 7, A and B). Accordingly, M. bovis BCG challenge led to significant activation of specific PKC isoforms in comparison with M. smegmatis and E. coli, which could be blocked by pharmacological inhibition of ␤-catenin by ␤-catenin inhibitor or Notch1 by GSI-I (supplemental Fig. S4, B and C, and Fig. 7, C and D). Furthermore, inhibition of GSK-3␤ by LiCl induced robust activation of specific PKC isoforms (Fig. 7E). We had earlier reported that M. bovis BCG-triggered expression of Notch1 target gene, COX-2, involves robust activation of Raf1, ERK1/2, and p38 MAPK (16). In this regard, pharmacological inhibition of PKC␣, PKC␤, and PKC␦ abrogated infection-triggered activation of Raf1, ERK1/2, and p38 MAPK, suggesting PKC-MAPK axis could act as a critical determinant of Wnt-Notch1-mediated cellular responses (supplemental Fig. S4D).
NF-B is a unique yet a general transcription factor, which in concert with receptor proximal signaling cohorts regulates a range of cellular functions. Interestingly, NF-B often acts as "gain control" for Wnt-␤-cateninand Notch1-mediated signals (16,18,19,38). In this regard, M. bovis BCG infection triggered marked activation of NF-B as evident by nuclear translocation of the p65 subunit of NF-B as well as increased binding of nuclear proteins to NF-B consensus (Fig. 7F and supplemental Fig. S4E). Interestingly, interference in ␤-catenin, Notch1, or PKC activity reversed M. bovis BCGmediated nuclear translocation of NF-B from cytosol (Fig.  7F). These results suggest that TLR2-activated Wnt-Notch1 and PKC-MAPK signaling axis integrate together to activate NF-B, which drives activation/expression of the factors involved in regulation of pathogen-specific macrophage functions.
TLR2/iNOS-dependent Activation of Wnt-␤-Catenin/ Notch1 Signaling Controls T Reg Cell Lineage Commitment-Macrophages tailor immune responses to microbial pathogens by stimulating differentiation of CD4 ϩ T cells into inflammatory or immunoregulatory phenotypes (39 -41). Interestingly, recent reports corroborate positive influence of Wnt-␤catenin and Notch1 signaling as well as COX-2 activity in T Reg cell lineage commitment (42)(43)(44). It is noteworthy that T Reg cells have been shown to dampen the immune response to a wide range of intracellular pathogens, including mycobacteria (42,45). In this perspective, we assessed the contribution of pathogen-specific activation of TLR2 signaling in Wnt-␤-catenin/Notch1-dependent differentiation of T Reg cells. As shown in Fig. 8, A and B, priming of splenic T cells with M. bovis BCG-infected macrophages resulted in steady increase in percentage of CD4 ϩ CD25 ϩ FoxP3 ϩ T Reg cells. Interestingly, interference with TLR2/iNOS signaling axis in iNOS null macrophages compromised the ability of M. bovis BCG to favor differentiation of T Reg cells (Fig. 8A). Importantly, inhibition of ␤-catenin by the ␤-catenin inhibitor, secretion of Wnt5a by IWP-II, or activity of Notch1 and COX-2 by GSI-I and NS-398, respectively, led to ablation of M. bovis BCG-induced differentiation of T Reg cells (Fig. 8B). Together, these results eloquently suggest that pathogenspecific activation of TLR2 signaling exerts cooperative regulation of a distinct set of effector functions in macrophages by virtue of signaling integration involving cross-talk of Wnt-␤-catenin and Notch1 signaling.

DISCUSSION
The discriminating responses of innate immunity to invading pathogens often require careful orchestration of signaling events that tailor pathogen-specific defensive measures. This study identifies differential activation of Wnt-␤-catenin and Notch1 signaling in response to pathogen-specific activation of TLR2 during infection with M. bovis BCG, S. typhimurium, and S. aureus. Among the tested pathogens, M. bovis BCG triggered robust expression of Wnt5a, FzD4, and LRP5, a heightened stabilization and nuclear translocation of ␤-catenin, thus effectuating the transcriptional activation of Jagged1, and a functional overlap between Wnt-␤-catenin and Notch1 signaling.
Bringing correlation with the clinical manifestations of M. tuberculosis infection in vivo, we could detect augmented expression of signaling cohorts of the Wnt-␤-catenin-Notch1 cascade as well as COX-2 and SOCS-3 in peripheral blood mononuclear cells of pulmonary tuberculosis patients or brain samples derived from TBM patients. Induced expression of COX-2 and SOCS-3 acts as a significant factor in influencing the initiation and strength of the mounted innate immune response. The functional attributes of PGE 2 , product of COX-2 activity, include restrained production of IL-12, IFN-␥, reactive oxygen intermediates, and increased expression of IL-10, thus polarizing skewed acquired immune responses toward immunoregulatory phenotype (46,47). SOCS-3, a negative regulator of multiple cytokines and Toll-like receptor-induced signaling, is often associated with down-modulation of pro-inflammatory responses during infection with pathogenic microbes (48,49).
During intensive interplay between signaling pathways, NO serves as a pathological link that modulates direct cooperation of TLR2 with Notch1 signaling to regulate specific components of TLR2 responses (16,18). Significantly, NO was shown to regulate Wnt-mediated responses in colitis (32). In view of these observations, we explored whether TLR2-triggered activation of Wnt-␤-catenin signaling could fill in the capacity of iNOS/NO to regulate Notch1 responses in iNOS null macrophages. We show that stabilization of ␤-catenin in iNOS Ϫ/Ϫ macrophages could trigger the activation of Notch1 signaling as evidenced by activation of ␥-secretase complex as well as expression of Notch1 ligand, Jagged1, and Notch1 target gene products COX-2 and SOCS-3.
A series of recent studies have eloquently addressed the relevance of Wnt signaling for lymphocyte development (50,51). However, information on the role of Wnt signaling in antigenpresenting cells and its impact on differentiation of T cells remains scanty. A seminal study by Manicassamy et al. (43) revealed an attractive potential role for Wnt-␤-catenin signaling pathway in secretion of immunosuppressive cytokines from dendritic cells, essential for T Reg cell production and generation of tolerance (52). Interestingly, production of T Reg cells, purveyors of immune suppression, is critical for pathogenesis of various intracellular pathogens, including mycobacteria. CD4 ϩ CD25 ϩ FoxP3 ϩ regulatory T cells have been suggested to suppress immunity against M. tuberculosis in patients with active disease (42,45). Furthermore, in vitro studies suggest that PGE 2 , a biosynthetic product of COX-2 activity, paves a way for the development of T Reg cells (42). In view of these observations, we hypothesized that pathogenic TLR2activated Wnt-␤-catenin signaling could hold the key for T Reg cell lineage commitment. Accordingly, inhibition of Wnt-␤-catenin as well as downstream Notch1 signaling and COX-2 activity in macrophages prominently suppressed mycobacterium-induced T Reg differentiation. Furthermore, iNOS null macrophages failed to promote T Reg differentiation upon mycobacterial infection. These results clearly establish that pathogen-specific TLR2 signaling orchestrates differentiation of T Reg cells by virtue of signaling integration through cross-talk of Wnt-␤-catenin and Notch1 signaling and induced expression of COX-2.
Overall, the current investigation identifies Wnt-␤catenin as a critical regulator of pathogen-specific TLR2 responses, which in conjunction with Notch1 controls the expression of a battery of genes that could foster the generation of T Reg cells.