Expression signature of human endogenous retroviruses in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is one of the most diagnosed forms of leukemia worldwide and it is usually classified into two forms: indolent and aggressive. These two forms are characterized by distinct molecular features that drive different responses to treatment and clinical outcomes. In this context, a better understanding of the molecular landscape of the CLL forms may potentially lead to the development of new drugs or the identification of novel biomarkers. Human endogenous retroviruses (HERVs) are a class of transposable elements that have been associated with the development of different human cancers, including different forms of leukemias. However, no studies about HERVs in CLL have ever been reported so far. Here, we present the first locus-specific profiling of HERV expression in both the aggressive and indolent forms of CLL. Our analyses revealed several dysregulations in HERV expression occurring in CLL and some of them were specific for either the aggressive or indolent form of CLL. Such results were also validated by analyzing an external cohort of CLL patients and by RT-qPCR. Moreover, in silico analyses have shown relevant signaling pathways associated with them suggesting a potential involvement of the dysregulated HERVs in these pathways and consequently in CLL development.

Small cell lung cancer: Subtypes and therapeutic implications.

Small cell lung cancer (SCLC) is an extremely aggressive neuroendocrine tumor, accounting for approximated 13% of all lung cancer cases. SCLC is characterized by rapid growth and early metastasis. Despite marked improvements in the number and efficacy of targeted, therapeutic options and overall survival rates in SCLC have remained nearly unchanged for almost three decades. The lack of significant progress can be attributed to our poor understanding of the biology of SCLC. Although immune checkpoint inhibitors were recently approved as front-line therapies for SCLC, we still need to better understand the mechanisms responsible for the selective vulnerability of some SCLCs to these inhibitors. Recent work utilizing sequencing data and single cell analyses identified four distinct subsets of SCLC, based on the expression levels of the transcription factors ASCL1, NEUROD1, POU2F3 and YAP1. Each subset was found to have its own distinct biology and therapeutic vulnerabilities. However, these subsets appear to be phenotypically unstable, representing snapshots in the gradual evolution of a tumor that exhibits significant plasticity. Tumor evolution, a product of this plasticity, results in the emergence of significant intratumoral heterogeneity which plays an important role in multiple aspects of SCLC development and progression, including cell survival and proliferation, metastasis and angiogenesis. The recent paradigm shifting discoveries in the biology of SCLC are now beginning to inform the design of new therapeutic strategies for the management of this intractable disease.

AKT Isoforms in Macrophage Activation, Polarization, and Survival.

Macrophages display an array of activation phenotypes depending on the activation signal and the cellular microenvironment. The type and magnitude of the response depend on signaling molecules as well as on the epigenetic and metabolic status of the cells at the time of activation. The AKT family of kinases consists of three isoforms encoded by independent genes possessing similar functions and structures. Generation of research tools such as isoform-specific gene deletion mutant mice and cells and isoform-specific antibodies has allowed us to understand the role of each kinase isoform in macrophage activation and homeostasis. This chapter discusses the current evidence on the role of AKT kinases in macrophage activation, polarization, and homeostasis, highlighting the gaps in knowledge and future challenges in the field.

Akt3 induces oxidative stress and DNA damage by activating the NADPH oxidase via phosphorylation of p47phox.

Akt activation up-regulates the intracellular levels of reactive oxygen species (ROS) by inhibiting ROS scavenging. Of the Akt isoforms, Akt3 has also been shown to up-regulate ROS by promoting mitochondrial biogenesis. Here, we employ a set of isogenic cell lines that express different Akt isoforms, to show that the most robust inducer of ROS is Akt3. As a result, Akt3-expressing cells activate the DNA damage response pathway, express high levels of p53 and its direct transcriptional target miR-34, and exhibit a proliferation defect, which is rescued by the antioxidant N-acetylcysteine. The importance of the DNA damage response in the inhibition of cell proliferation by Akt3 was confirmed by Akt3 overexpression in p53-/- and INK4a-/-/Arf-/- mouse embryonic fibroblasts (MEFs), which failed to inhibit cell proliferation, despite the induction of high levels of ROS. The induction of ROS by Akt3 is due to the phosphorylation of the NADPH oxidase subunit p47phox, which results in NADPH oxidase activation. Expression of Akt3 in p47phox-/- MEFs failed to induce ROS and to inhibit cell proliferation. Notably, the proliferation defect was rescued by wild-type p47phox, but not by the phosphorylation site mutant of p47phox In agreement with these observations, Akt3 up-regulates p53 in human cancer cell lines, and the expression of Akt3 positively correlates with the levels of p53 in a variety of human tumors. More important, Akt3 alterations correlate with a higher frequency of mutation of p53, suggesting that tumor cells may adapt to high levels of Akt3, by inactivating the DNA damage response.

PHENSIM: Phenotype Simulator.

Despite the unprecedented growth in our understanding of cell biology, it still remains challenging to connect it to experimental data obtained with cells and tissues' physiopathological status under precise circumstances. This knowledge gap often results in difficulties in designing validation experiments, which are usually labor-intensive, expensive to perform, and hard to interpret. Here we propose PHENSIM, a computational tool using a systems biology approach to simulate how cell phenotypes are affected by the activation/inhibition of one or multiple biomolecules, and it does so by exploiting signaling pathways. Our tool's applications include predicting the outcome of drug administration, knockdown experiments, gene transduction, and exposure to exosomal cargo. Importantly, PHENSIM enables the user to make inferences on well-defined cell lines and includes pathway maps from three different model organisms. To assess our approach's reliability, we built a benchmark from transcriptomics data gathered from NCBI GEO and performed four case studies on known biological experiments. Our results show high prediction accuracy, thus highlighting the capabilities of this methodology. PHENSIM standalone Java application is available at https://github.com/alaimos/phensim, along with all data and source codes for benchmarking. A web-based user interface is accessible at https://phensim.tech/.

Phosphoproteomics screen reveals akt isoform-specific signals linking RNA processing to lung cancer.

The three Akt isoforms are functionally distinct. Here we show that their phosphoproteomes also differ, suggesting that their functional differences are due to differences in target specificity. One of the top cellular functions differentially regulated by Akt isoforms is RNA processing. IWS1, an RNA processing regulator, is phosphorylated by Akt3 and Akt1 at Ser720/Thr721. The latter is required for the recruitment of SETD2 to the RNA Pol II complex. SETD2 trimethylates histone H3 at K36 during transcription, creating a docking site for MRG15 and PTB. H3K36me3-bound MRG15 and PTB regulate FGFR-2 splicing, which controls tumor growth and invasiveness downstream of IWS1 phosphorylation. Twenty-one of the twenty-four non-small-cell-lung carcinomas we analyzed express IWS1. More importantly, the stoichiometry of IWS1 phosphorylation in these tumors correlates with the FGFR-2 splicing pattern and with Akt phosphorylation and Akt3 expression. These data identify an Akt isoform-dependent regulatory mechanism for RNA processing and demonstrate its role in lung cancer.

The combination of TPL2 knockdown and TNFα causes synthetic lethality via caspase-8 activation in human carcinoma cell lines.

Most normal and tumor cells are protected from tumor necrosis factor α (TNFα)-induced apoptosis. Here, we identify the MAP3 kinase tumor progression locus-2 (TPL2) as a player contributing to the protection of a subset of tumor cell lines. The combination of TPL2 knockdown and TNFα gives rise to a synthetic lethality phenotype via receptor-interacting serine/threonine-protein kinase 1 (RIPK1)-dependent and -independent mechanisms. Whereas wild-type TPL2 rescues the phenotype, its kinase-dead mutant does not. Comparison of the molecular events initiated by small interfering RNA for TPL2 (siTPL2) ± TNFα in treatment-sensitive and -resistant lines revealed that the activation of caspase-8, downstream of miR-21-5p and cFLIP, is the dominant TPL2-dependent event. More important, comparison of the gene expression profiles of all of the tested cell lines results in the clustering of sensitive and resistant lines into distinct groups, providing proof of principle for the feasibility of generating a predictive tool for treatment sensitivity.

RNAdetector: a free user-friendly stand-alone and cloud-based system for RNA-Seq data analysis.

BACKGROUND: RNA-Seq is a well-established technology extensively used for transcriptome profiling, allowing the analysis of coding and non-coding RNA molecules. However, this technology produces a vast amount of data requiring sophisticated computational approaches for their analysis than other traditional technologies such as Real-Time PCR or microarrays, strongly discouraging non-expert users. For this reason, dozens of pipelines have been deployed for the analysis of RNA-Seq data. Although interesting, these present several limitations and their usage require a technical background, which may be uncommon in small research laboratories. Therefore, the application of these technologies in such contexts is still limited and causes a clear bottleneck in knowledge advancement. RESULTS: Motivated by these considerations, we have developed RNAdetector, a new free cross-platform and user-friendly RNA-Seq data analysis software that can be used locally or in cloud environments through an easy-to-use Graphical User Interface allowing the analysis of coding and non-coding RNAs from RNA-Seq datasets of any sequenced biological species. CONCLUSIONS: RNAdetector is a new software that fills an essential gap between the needs of biomedical and research labs to process RNA-Seq data and their common lack of technical background in performing such analysis, which usually relies on outsourcing such steps to third party bioinformatics facilities or using expensive commercial software.

The epigenetic factor KDM2B regulates cell adhesion, small rho GTPases, actin cytoskeleton and migration in prostate cancer cells.

The histone demethylase KDM2B is an epigenetic factor with oncogenic properties that is regulated by the basic fibroblasts growth factor (FGF-2). It has recently been shown that KDM2B co-operates with Polycomb Group proteins to promote cell migration and angiogenesis in tumors. In the present study we addressed the role of KDM2B in regulating actin cytoskeleton signaling, cell-cell adhesion and migration of prostate tumor cells. We report here that KDM2B is functionally expressed in DU-145 prostate cancer cells, activated by FGF-2 and regulates EZH2. KDM2B knockdown induced potent up-regulation of gene transcription and protein expression of the epithelial markers E-cadherin and ZO-1, while KDM2B overexpression down-regulated the levels of both markers, suggesting control of cell adhesion by KDM2B. RhoA and RhoB protein expression and activity were diminished upon KDM2B-knockdown and upregulated in KDM2B-overexpressing cell clones. In accordance, actin reorganization with formation of stress fibers became evident in KDM2B-overexpressing cells and abolished in the presence of the Rho inhibitor C3 transferase. DU-145 cell migration was significantly enhanced in KDM2B overexpressing cells and abolished in C3-pretreated cells. Conversely, the retardation of cell migration observed in KDM2B knockdown cells was enhanced in C3-pretreated cells. These results establish a clear functional link between the epigenetic factor KDM2B and the regulation of cell adhesion and Rho-GTPases signaling that controls actin reorganization and cell migration.

FGF-2 regulates cell proliferation, migration, and angiogenesis through an NDY1/KDM2B-miR-101-EZH2 pathway.

The histone H3K27 methyltransferase EZH2 plays an important role in oncogenesis, by mechanisms that are incompletely understood. Here, we show that the JmjC domain histone H3 demethylase NDY1 synergizes with EZH2 to silence the EZH2 inhibitor miR-101. NDY1 and EZH2 repress miR-101 by binding its promoter in concert, via a process triggered by upregulation of NDY1. Whereas EZH2 binding depends on NDY1, the latter binds independently of EZH2. However, both are required to repress transcription. NDY1 and EZH2 acting in concert upregulate EZH2 and stabilize the repression of miR-101 and its outcome. NDY1 is induced by FGF-2 via CREB phosphorylation and activation, downstream of DYRK1A, and mediates the FGF-2 and EZH2 effects on cell proliferation, migration, and angiogenesis. The FGF-2-NDY1/EZH2-miR-101-EZH2 axis described here was found to be active in bladder cancer. These data delineate an oncogenic pathway that functionally links FGF-2 with EZH2 via NDY1 and miR-101.

The Epigenetic Factor KDM2B Regulates EMT and Small GTPases in Colon Tumor Cells.

BACKGROUND/AIMS: The epigenetic factor KDM2B is a histone demethylase expressed in various tumors. Recently, we have shown that KDM2B regulates actin cytoskeleton organization, small Rho GTPases signaling, cell-cell adhesion and migration of prostate tumor cells. In the present study, we addressed its role in regulating EMT and small GTPases expression in colon tumor cells. METHODS: We used RT-PCR for the transcriptional analysis of various genes, Western blotting for the assessment of protein expression and immunofluorescence microscopy for visualization of fluorescently labeled proteins. RESULTS: We report here that KDM2B regulates EZH2 and BMI1 in HCT116 colon tumor cells. Knockdown of this epigenetic factor induced potent up-regulation of the protein levels of the epithelial markers E-cadherin and ZO-1, while the mesenchymal marker N-cadherin was downregulated. On the other hand, KDM2B overexpression downregulated the levels of both epithelial markers and upregulated the mesenchymal marker, suggesting control of EMT by KDM2B. In addition, RhoA, RhoB and RhoC protein levels diminished upon KDM2B-knockdown, while all three small GTPases became upregulated in KDM2B-overexpressing HCT116 cell clones. Interestingly, Rac1 GTPase level increased upon KDM2B-knockdown and diminished in KDM2B-overexpressing HCT116 colon tumor- and DU-145 prostate cancer cells. CONCLUSIONS: These results establish a clear functional role of the epigenetic factor KDM2B in the regulation of EMT and small-GTPases expression in colon tumor cells and further support the recently postulated oncogenic role of this histone demethylase in various tumors.

The kinase Akt1 controls macrophage response to lipopolysaccharide by regulating microRNAs.

MicroRNAs regulated by lipopolysaccharide (LPS) target genes that contribute to the inflammatory phenotype. Here, we showed that the protein kinase Akt1, which is activated by LPS, positively regulated miRNAs let-7e and miR-181c but negatively regulated miR-155 and miR-125b. In silico analyses and transfection studies revealed that let-7e repressed Toll-like receptor 4 (TLR4), whereas miR-155 repressed SOCS1, two proteins critical for LPS-driven TLR signaling, which regulate endotoxin sensitivity and tolerance. As a result, Akt1(-/-) macrophages exhibited increased responsiveness to LPS in culture and Akt1(-/-) mice did not develop endotoxin tolerance in vivo. Overexpression of let-7e and suppression of miR-155 in Akt1(-/-) macrophages restored sensitivity and tolerance to LPS in culture and in animals. These results indicate that Akt1 regulates the response of macrophages to LPS by controlling miRNA expression.

TPL2 Kinase Is a Crucial Signaling Factor and Mediator of NKT Effector Cytokine Expression in Immune-Mediated Liver Injury.

Invariant NKT (iNKT) cells represent a subset of innate-like T lymphocytes that function as orchestrators of hepatic inflammation underpinning liver damage. In this study, we demonstrate that TPL2, an MAP3 kinase that has mostly been appreciated for its physiological role in macrophage responses, is a signaling factor in CD3(+)NK1.1(+) iNKT cells and mediator of hepatic inflammation. Genetic ablation of TPL2 in the mouse ameliorates liver injury induced by Con A and impinges on hallmarks of NKT cell activation in the liver without affecting NKT cell development in the thymus. The pivotal role of TPL2 in iNKT cell functions is further endorsed by studies using the iNKT-specific ligand α-galactosylceramide, which causes mild hepatitis in the mouse in a TPL2-dependent manner, including production of the effector cytokines IL-4 and IFN-γ, accumulation of neutrophils and licensing and activation of other immune cell types in the liver. A TPL2 kinase inhibitor mirrors the effects of genetic ablation of TPL2 in vivo and uncovers ERK and Akt as the TPL2-regulated signaling pathways responsible for IL-4 and IFN-γ expression through the activation of the transcription factors JunB and NFAT. Collectively, these findings expand our understanding of the mechanisms of iNKT cell activation and suggest that modulation of TPL2 has the potential to minimize the severity of immune-driven liver diseases.

Loss of miR-200 inhibition of Suz12 leads to polycomb-mediated repression required for the formation and maintenance of cancer stem cells.

In an inducible oncogenesis model, the miR-200 family is inhibited during CSC formation but not transformation, and inhibition of miR-200b increases CSC formation. Interestingly, miR-200b directly targets Suz12, a subunit of a polycomb repressor complex (PRC2). Loss of miR-200 during CSC formation increases Suz12 expression, Suz12 binding, H3-K27 trimethylation, and Polycomb-mediated repression of the E-cadherin gene. miR-200b expression or Suz12 depletion blocks the formation and maintenance of mammospheres, and in combination with chemotherapy suppresses tumor growth and prolongs remission in mouse xenografts. Conversely, ectopic expression of Suz12 in transformed cells is sufficient to generate CSCs. The miR-200b-Suz12-cadherin pathway is important for CSC growth and invasive ability in genetically distinct breast cancer cells, and its transcriptional signature is observed in metastatic breast tumors. The interaction between miR-200 and Suz12 is highly conserved, suggesting that it represents an ancient regulatory mechanism to control the growth and function of stem cells.

The NF-κB1 transcription factor prevents the intrathymic development of CD8 T cells with memory properties.

The role of specific members of the NF-κB family of transcription factors in CD8 T-cell selection and development is largely unknown. Here, we show that mice lacking NF-κB1 develop a unique population of conventional CD8 single-positive (SP) thymocytes with memory T cell-like properties that populate peripheral immune organs. Development of this memory-like population is not due to PLZF(+) thymocytes and instead coincides with changes in CD8 T-cell selection. These include a reduction in the efficiency of negative selection and a dependence on MHC class Ia or Ib expressed by haematopoietic cells. These findings indicate that NF-κB1 regulates multiple events in the thymus that collectively inhibit the excess development of CD8(+) thymocytes with memory cell characteristics.

MicroRNA214 Is Associated With Progression of Ulcerative Colitis, and Inhibition Reduces Development of Colitis and Colitis-Associated Cancer in Mice.

BACKGROUND & AIMS: Persistent activation of the inflammatory response contributes to the development of inflammatory bowel diseases, which increase the risk of colorectal cancer. We aimed to identify microRNAs that regulate inflammation during the development of ulcerative colitis (UC) and progression to colitis-associated colon cancer (CAC). METHODS: We performed a quantitative polymerase chain reaction analysis to measure microRNAs in 401 colon specimens from patients with UC, Crohn's disease, irritable bowel syndrome, sporadic colorectal cancer, or CAC, as well as subjects without these disorders (controls); levels were correlated with clinical features and disease activity of patients. Colitis was induced in mice by administration of dextran sodium sulfate (DSS), and carcinogenesis was induced by addition of azoxymethane; some mice also were given an inhibitor of microRNA214 (miR214). RESULTS: A high-throughput functional screen of the human microRNAome found that miR214 regulated the activity of nuclear factor-κB. Higher levels of miR214 were detected in colon tissues from patients with active UC or CAC than from patients with other disorders or controls and correlated with disease progression. Bioinformatic and genome-wide profile analyses showed that miR214 activates an inflammatory response and is amplified through a feedback loop circuit mediated by phosphatase and tensin homolog (PTEN) and PDZ and LIM domain 2 (PDLIM2). Interleukin-6 induced signal transducer and activator of transcription 3 (STAT3)-mediated transcription of miR214. A miR214 chemical inhibitor blocked this circuit and reduced the severity of DSS-induced colitis in mice, as well as the number and size of tumors that formed in mice given azoxymethane and DSS. In fresh colonic biopsy specimens from patients with active UC, the miR214 inhibitor reduced inflammation by increasing levels of PDLIM2 and PTEN. CONCLUSIONS: Interleukin-6 up-regulates STAT3-mediated transcription of miR214 in colon tissues, which reduces levels of PDLIM2 and PTEN, increases phosphorylation of AKT, and activates nuclear factor-κB. The activity of this circuit correlates with disease activity in patients with UC and progression to colorectal cancer.

The downregulation of GFI1 by the EZH2-NDY1/KDM2B-JARID2 axis and by human cytomegalovirus (HCMV) associated factors allows the activation of the HCMV major IE promoter and the transition to productive infection.

Earlier studies had suggested that epigenetic mechanisms play an important role in the control of human cytomegalovirus (HCMV) infection. Here we show that productive HCMV infection is indeed under the control of histone H3K27 trimethylation. The histone H3K27 methyltransferase EZH2, and its regulators JARID2 and NDY1/KDM2B repress GFI1, a transcriptional repressor of the major immediate-early promoter (MIEP) of HCMV. Knocking down EZH2, NDY1/KDM2B or JARID2 relieves the repression and results in the upregulation of GFI1. During infection, the incoming HCMV rapidly downregulates the GFI1 mRNA and protein in both wild-type cells and in cells in which EZH2, NDY1/KDM2B or JARID2 were knocked down. However, since the pre-infection levels of GFI1 in the latter cells are significantly higher, the virus fails to downregulate it to levels permissive for MIEP activation and viral infection. Following the EZH2-NDY1/KDM2B-JARID2-independent downregulation of GFI1 in the early stages of infection, the virus also initiates an EZH2-NDY1/ΚDM2Β-JARID2-dependent program that represses GFI1 throughout the infection cycle. The EZH2 knockdown also delays histone H3K27 trimethylation in the immediate early region of HCMV, which is accompanied by a drop in H3K4 trimethylation that may contribute to the shEZH2-mediated repression of the major immediate early HCMV promoter. These data show that HCMV uses multiple mechanisms to allow the activation of the HCMV MIEP and to prevent cellular mechanisms from blocking the HCMV replication program.

Regulation of experimental autoimmune encephalomyelitis by TPL-2 kinase.

Tumor progression locus 2 (TPL-2) expression is required for efficient polarization of naive T cells to Th1 effector cells in vitro, as well as for Th1-mediated immune responses. In the present study, we investigated the potential role of TPL-2 in Th17 cells. TPL-2 was found to be dispensable for Th17 cell differentiation in vitro, and for the initial priming of Th17 cells in experimental autoimmune encephalomyelitis (EAE), a Th17 cell-mediated disease model for multiple sclerosis. Nevertheless, TPL-2-deficient mice were protected from EAE, which correlated with reduced immune cell infiltration, demyelination, and axonal damage in the CNS. Adoptive transfer experiments demonstrated that there was no T cell-intrinsic function for TPL-2 in EAE, and that TPL-2 signaling was not required in radiation-sensitive hematopoietic cells. Rather, TPL-2 signaling in radiation-resistant stromal cells promoted the effector phase of the disease. Importantly, using a newly generated mouse strain expressing a kinase-inactive form of TPL-2, we demonstrated that stimulation of EAE was dependent on the catalytic activity of TPL-2 and not its adaptor function to stabilize the associated ubiquitin-binding protein ABIN-2. Our data therefore raise the possibility that small molecule inhibitors of TPL-2 may be beneficial in multiple sclerosis therapy.

TPL2 kinase is a suppressor of lung carcinogenesis.

Lung cancer is a heterogeneous disease at both clinical and molecular levels, posing conceptual and practical bottlenecks in defining key pathways affecting its initiation and progression. Molecules with a central role in lung carcinogenesis are likely to be targeted by multiple deregulated pathways and may have prognostic, predictive, and/or therapeutic value. Here, we report that Tumor Progression Locus 2 (TPL2), a kinase implicated in the regulation of innate and adaptive immune responses, fulfils a role as a suppressor of lung carcinogenesis and is subject to diverse genetic and epigenetic aberrations in lung cancer patients. We show that allelic imbalance at the TPL2 locus, up-regulation of microRNA-370, which targets TPL2 transcripts, and activated RAS (rat sarcoma) signaling may result in down-regulation of TPL2 expression. Low TPL2 levels correlate with reduced lung cancer patient survival and accelerated onset and multiplicity of urethane-induced lung tumors in mice. Mechanistically, TPL2 was found to antagonize oncogene-induced cell transformation and survival through a pathway involving p53 downstream of cJun N-terminal kinase (JNK) and be required for optimal p53 response to genotoxic stress. These results identify multiple oncogenic pathways leading to TPL2 deregulation and highlight its major tumor-suppressing function in the lung.

Toll-like receptor 2-dependent extracellular signal-regulated kinase signaling in Mycobacterium tuberculosis-infected macrophages drives anti-inflammatory responses and inhibits Th1 polarization of responding T cells.

Mycobacterium tuberculosis survives within macrophages and employs immune evasion mechanisms to persist in the host. Protective T helper type 1 (Th1) responses are induced, and the immune response in most individuals is sufficient to restrict M. tuberculosis to latent infection, but most infections are not completely resolved. As T cells and macrophages respond, a balance is established between protective Th1-associated and other proinflammatory cytokines, such as interleukin-12 (IL-12), interferon gamma (IFN-γ), and tumor necrosis factor alpha, and anti-inflammatory cytokines, such as IL-10. The mechanisms by which M. tuberculosis modulates host responses to promote its survival remain unclear. In these studies, we demonstrate that M. tuberculosis induction of IL-10, suppression of IL-12, and inhibition of class II major histocompatibility complex (MHC-II) molecules in infected macrophages are all driven by Toll-like receptor 2 (TLR2)-dependent activation of the extracellular signal-regulated kinases (ERK). Elimination of ERK signaling downstream of TLR2 by pharmacologic inhibition with U0126 or genetic deletion of Tpl2 blocks IL-10 secretion and enhances IL-12 p70 secretion. We demonstrate that M. tuberculosis regulation of these pathways in macrophages affects T cell responses to infected macrophages. Thus, genetic blockade of the ERK pathway in Tpl2(-/-) macrophages enhances Th1 polarization and IFN-γ production by antigen-specific CD4(+) T cells responding to M. tuberculosis infection. These data indicate that M. tuberculosis and its potent TLR2 ligands activate ERK signaling in macrophages to promote anti-inflammatory macrophage responses and blunt Th1 responses against the pathogen.