Intestinal microbiota-specific Th17 cells possess regulatory properties and suppress effector T cells via c-MAF and IL-10.

Commensal microbes induce cytokine-producing effector tissue-resident CD4+ T cells, but the function of these T cells in mucosal homeostasis is not well understood. Here, we report that commensal-specific intestinal Th17 cells possess an anti-inflammatory phenotype marked by expression of interleukin (IL)-10 and co-inhibitory receptors. The anti-inflammatory phenotype of gut-resident commensal-specific Th17 cells was driven by the transcription factor c-MAF. IL-10-producing commensal-specific Th17 cells were heterogeneous and derived from a TCF1+ gut-resident progenitor Th17 cell population. Th17 cells acquired IL-10 expression and anti-inflammatory phenotype in the small-intestinal lamina propria. IL-10 production by CD4+ T cells and IL-10 signaling in intestinal macrophages drove IL-10 expression by commensal-specific Th17 cells. Intestinal commensal-specific Th17 cells possessed immunoregulatory functions and curbed effector T cell activity in vitro and in vivo in an IL-10-dependent and c-MAF-dependent manner. Our results suggest that tissue-resident commensal-specific Th17 cells perform regulatory functions in mucosal homeostasis.

c-Maf enforces cytokine production and promotes memory-like responses in mouse and human type 2 innate lymphoid cells.

Group-2 innate lymphoid cells (ILC2s), which are involved in type 2 inflammatory diseases such as allergy, can exhibit immunological memory, but the basis of this ILC2 "trained immunity" has remained unclear. Here, we found that stimulation with IL-33/IL-25 or exposure to the allergen papain induces the expression of the transcription factor c-Maf in mouse ILC2s. Chronic papain exposure results in high production of IL-5 and IL-13 cytokines and lung eosinophil recruitment, effects that are blocked by c-Maf deletion in ILCs. Transcriptomic analysis revealed that knockdown of c-Maf in ILC2s suppresses expression of type 2 cytokine genes, as well as of genes linked to a memory-like phenotype. Consistently, c-Maf was found highly expressed in human adult ILC2s but absent in cord blood and required for cytokine production in isolated human ILC2s. Furthermore, c-Maf-deficient mouse or human ILC2s failed to exhibit strengthened ("trained") responses upon repeated challenge. Thus, the expression of c-Maf is indispensable for optimal type 2 cytokine production and proper memory-like responses in group-2 innate lymphoid cells.

The clinicopathological impact of tumor-associated macrophages in patients with cutaneous malignant melanoma.

BACKGROUND: Tumor-associated macrophages (TAMs) are an immune component of the cutaneous malignant melanoma (CMM) microenvironment and affect tumor growth. TAMs can polarize into different phenotypes, that is, proinflammatory M1 and anti-inflammatory M2 macrophages. However, the role of the macrophage phenotype in CMM remains unclear. METHODS: We examined 88 patients with CMM. Tissue microarrays were constructed, and the density of M1 and M2 macrophages was analyzed by immunohistochemistry. Immune cells coexpressing CD68 and phosphorylated signal transducer and activator of transcription 1 (pSTAT1) were considered M1 macrophages, whereas those coexpressing CD68 and c-macrophage activating factor (c-Maf) were defined as M2 macrophages. These TAMs were counted, and the relationships between the density of M1 and M2 macrophages and clinicopathological factors including prognosis were investigated. RESULTS: The CD68/c-Maf score ranged from 0 to 34 (median: 5.5). The patients were divided based on the median score into the CD68/c-Maf high (≥5.5) and low (<5.5) expression groups. Univariate and multivariate analyses revealed that CD68/c-Maf expression was an independent predictive factor for progression-free survival and an independent prognostic factor for overall survival. CD68/pSTAT1 expression was found in only two patients. CONCLUSION: We suggest that CD68/pSTAT1 coexpression is rarely observed in patients with CMM, and high CD68/c-Maf expression is a predictor of worse prognosis in these patients.

Tumor-infiltrating gamma delta T-cells reveal exhausted subsets with remarkable heterogeneity in colorectal cancer.

The γδT-cells recognize infected or transformed cells. However, unlike αßT-cells, γδT-cells are innate-like immune cells, with no major histocompatibility complex restriction requirements. γδT-cells are the main population of intestinal intraepithelial lymphocytes (IELs) and are associated with the antitumor immune response, particularly in colorectal cancer (CRC). Although CD8+ T-cells exhibit dysfunction and even exhaustion in the tumor microenvironment (TME), which contributes to tumor immune escape, whether the same applies to tumor-infiltrating (TI)-γδT-cells is not completely understood. Here, we sought to investigate the expression pattern of inhibitory receptors and functional state of TI-γδT-cells, and reveal the features of exhausted TI-γδT-cells in the CRC TME. We demonstrated that TI-γδT-cells exhibited exhaustion phenotypes and displayed more severe functional exhaustion than TI-CD8+ T-cells or NK-cells in the TME of CRC. In addition, scRNA-seq analysis of TI-γδT-cells revealed three exhausted subsets with remarkable heterogeneity. The presence of three heterogeneous exhausted γδT-cell (Tex) populations, including Texprog , Textran and Texterm were further confirmed by flow cytometry, on the basis of PD-1 and TIM-3 expression. Finally, we revealed that c-Maf not only contributed to γδT-cell exhaustion via upregulation of inhibitory receptors, but also involved in the exhaustion of CD8+ T and NK-cells. c-Maf may also be an important contributor to γδT-cell exhaustion in CRC patients. These findings indicated that TI-γδT-cells exhibit phenotypic and functional exhaustion in the CRC TME. The revealed features of exhausted TI-γδT-cells may provide help for understanding the mechanisms and the association of γδT-cell exhaustion with tumor development and pathogenesis.

Novel cataract-causing variant c.177dupC in c-MAF regulates the expression of crystallin genes for cell apoptosis via a mitochondria-dependent pathway.

Congenital cataract (CC) is regarded as the most common hereditary ophthalmic disease in children. Mutations in CC-associated genes play important roles in CC formation, which provides the basis for molecular diagnosis and therapy. Among these CC-associated genes, v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (c-MAF) is considered an important transcription factor for eye and lens development. In this study, we recruited a three-generation Chinese Han family with CC. Gene sequencing revealed a novel duplication mutation in c-MAF (NM_005360.5: c.177dup) that caused frameshifting at residue 60 (p. M60fs) of c-MAF. Additionally, in the patient blood samples, the expression levels of related crystallin and noncrystallin genes confirmed that this novel duplication variant impaired the transactivation of c-MAF. Further functional analyses suggested that the c-MAF mutant induces the transcriptional inhibition of CRYAA and CRYGA and subsequently influences ME and G6PD expression levels, ultimately resulting in ROS generation and further leading to cell apoptosis via mitochondria-dependent pathways. In conclusion, we report a novel c-MAF heterozygous mutation that plays a vital role in CC formation in a Chinese family, broadening the genetic spectrum of CC.

Functions and regulation of T cell-derived interleukin-10.

Interleukin (IL)-10 is an essential anti-inflammatory cytokine and functions as a negative regulator of immune responses to microbial antigens. IL-10 is particularly important in maintaining the intestinal microbe-immune homeostasis. Loss of IL-10 promotes the development of inflammatory bowel disease (IBD) as a consequence of an excessive immune response to the gut microbiota. IL-10 also functions more generally to prevent excessive inflammation during the course of infection. Although IL-10 can be produced by virtually all cells of the innate and adaptive immune system, T cells constitute a non-redundant source for IL-10 in many cases. The various roles of T cell-derived IL-10 will be discussed in this review. Given that IL-10 is at the center of maintaining the delicate balance between effective immunity and tissue protection, it is not surprising that IL-10 expression is highly dynamic and tightly regulated. We summarize the environmental signals and molecular pathways that regulate IL-10 expression. While numerous studies have provided us with a deep understanding of IL-10 biology, the majority of findings have been made in murine models, prompting us to highlight gaps in our knowledge about T cell-derived IL-10 in the human system.

T cell-intrinsic miR-155 is required for Th2 and Th17-biased responses in acute and chronic airway inflammation by targeting several different transcription factors.

Asthmatic airway inflammation is divided into two typical endotypes: Th2-mediated eosinophilic and Th1- or Th17-mediated neutrophilic airway inflammation. The miRNA miR-155 has well-documented roles in the regulation of adaptive T-cell responses and innate immunity. However, no specific cell-intrinsic role has yet been elucidated for miR-155 in T cells in the course of Th2-eosinophilic and Th17-neutrophilic airway inflammation using actual in vivo asthma models. Here, using conditional KO (miR155ΔCD4 cKO) mice that have the specific deficiency of miR-155 in T cells, we found that the specific deficiency of miR-155 in T cells resulted in fully suppressed Th2-type eosinophilic airway inflammation following acute allergen exposure, as well as greatly attenuated the Th17-type neutrophilic airway inflammation induced by repeated allergen exposure. Furthermore, miR-155 in T cells appeared to regulate the expression of several different target genes in the functional activation of CD4+ Th2 and Th17 cells. To be more precise, the deficiency of miR-155 in T cells enhanced the expression of c-Maf, SOCS1, Fosl2 and Jarid2 in the course of CD4+ Th2 cell activation, while C/EBPß was highly enhanced in CD4+ Th17 cell activation in the absence of miR-155 expression. Conclusively, our data revealed that miR-155 could promote Th2 and Th17-mediated airway inflammation via the regulation of several different target genes, depending on the context of asthmatic diseases. Therefore, these results provide valuable insights into actual understanding of specific cell-intrinsic role of miR-155 in eosinophilic and neutrophilic airway inflammation for the development of fine-tune therapeutic strategies.

Inhibition of the Otub1/c-Maf axis by the herbal acevaltrate induces myeloma cell apoptosis.

BACKGROUND: The oncogenic transcript factor c-Maf is stabilized by the deubiquitinase Otub1 and promotes myeloma cell proliferation and confers to chemoresistance. Inhibition of the Otub1/c-Maf axis is a promising therapeutic target, but there are no inhibitors reported on this specific axis. METHODS: A luciferase assay was applied to screen potential inhibitors of Otub1/c-Maf. Annexin V staining/flow cytometry was applied to evaluate cell apoptosis. Immunoprecipitation was applied to examine protein ubiquitination and interaction. Xenograft models in nude mice were used to evaluate anti-myeloma activity of AVT. RESULTS: Acevaltrate (AVT), isolated from Valeriana glechomifolia, was identified based on a bioactive screen against the Otub1/c-Maf/luciferase system. AVT disrupts the interaction of Otub1/c-Maf thus inhibiting Otub1 activity and leading to c-Maf polyubiquitination and subsequent degradation in proteasomes. Consistently, AVT inhibits c-Maf transcriptional activity and downregulates the expression of its target genes key for myeloma growth and survival. Moreover, AVT displays potent anti-myeloma activity by triggering myeloma cell apoptosis in vitro and impairing myeloma xenograft growth in vivo but presents no marked toxicity. CONCLUSIONS: The natural product AVT inhibits the Otub1/c-Maf axis and displays potent anti-myeloma activity. Given its great safety and efficacy, AVT could be further developed for MM treatment. Video Abstract.

Molecular switches for regulating the differentiation of inflammatory and IL-10-producing anti-inflammatory T-helper cells.

CD4 T-helper (Th) cells secret a variety of inflammatory cytokines and play critical roles in host defense against invading foreign pathogens. On the other hand, uncontrolled inflammatory responses mediated by Th cells may result in tissue damage and inflammatory disorders including autoimmune and allergic diseases. Thus, the induction of anti-inflammatory cytokine expression becomes an important "brake" to repress and/or terminate aberrant and/or unnecessary immune responses. Interleukin-10 (IL-10) is one of the most important anti-inflammatory cytokines to limit inflammatory Th cells and immunopathology and to maintain tissue homeostasis. Many studies have indicated that Th cells can be a major source of IL-10 under specific conditions both in mouse and human and that extracellular signals and cell intrinsic molecular switches are required to turn on and off Il10 expression in different Th cells. In this review, we will highlight the recent findings that have enhanced our understanding on the mechanisms of IL-10 induction in distinct Th-cell subsets, including Th1, Th2, and Th17 cells, as well as the importance of these IL-10-producing anti-inflammatory Th cells in immunity and inflammation.

Deletion of Cdk5 in Macrophages Ameliorates Anti-Inflammatory Response during Endotoxemia through Induction of C-Maf and Il-10.

Immune response control is critical as excessive cytokine production can be detrimental and damage the host. Interleukin-10 (Il-10), an anti-inflammatory cytokine produced primarily by macrophages, is a key regulator that counteracts and controls excessive inflammatory response. Il-10 expression is regulated through the transcription factor c-Maf. Another regulator of Il-10 production is p35, an activator of the cyclin-dependent kinase 5 (Cdk5), which decreases Il-10 production in macrophages, thus increasing inflammation. However, Cdk5 regulation of c-Maf and the involvement of Il-10 production in macrophages has not yet been investigated. We used in vitro primary bone marrow-derived macrophages (BMDMs) lacking Cdk5, stimulated them with lipopolysaccharid (LPS) and observed increased levels of c-Maf and Il-10. In an in vivo mouse model of LPS-induced endotoxemia, mice lacking Cdk5 in macrophages showed increased levels of c-Maf and elevated levels of Il-10 in lungs as well as in plasma, resulting in ameliorated survival. Taken together, we identified Cdk5 as a potential novel regulator of Il-10 production through c-Maf in macrophages under inflammatory conditions. Our results suggest that inhibition of Cdk5 enhances the c-Maf-Il-10 axis and thus potentiates improvement of anti-inflammatory therapy.

The novel mutation P36R in LRP5L contributes to congenital membranous cataract via inhibition of laminin γ1 and c-MAF.

PURPOSE: The present study investigated a pathogenic mutation and its mechanism on membranous cataract in a congenital membranous cataract family. METHODS: An autosomal dominant four-generation Chinese congenital membranous cataract family was recruited and whole-exome sequencing was performed to screen for sequence variants. Candidate variants were validated using polymerase chain reaction and Sanger sequencing. Wild-type and mutant low-density lipoprotein receptor-related protein 5-like (LRP5L) plasmids were constructed and transfected into human lens epithelial cells (HLE B-3) and human anterior lens capsules. The cell lysates, nuclear and cytoplasmic proteins, and basement membrane components of HLE B-3 cells were harvested. LRP5L and laminin γ1 were knocked down in HLE B-3 cells using specific small-interfering RNA. The protein expression levels of LRP5L, laminin γ1, and c-MAF were detected using immunoblotting and immunofluorescence. RESULTS: We identified a novel suspected pathogenic mutation in LRP5L (c.107C > G, p.P36R) in the congenital membranous cataract family. This mutation was absent in 300 normal controls and 300 age-related cataract patients. Bioinformatics analysis with PolyPhen-2 and SIFT suggested that LRP5L-P36R was pathogenic. LRP5L upregulated laminin γ1 expression in the cytoplasmic proteins of HLE B-3 cells and human anterior lens capsules, and LRP5L-P36R inhibited the effects of LRP5L. LRP5L upregulated c-MAF expression in the nucleus and cytoplasm of HLE B-3 cells, and LRP5L-P36R inhibited c-MAF expression via inhibition of laminin γ1. CONCLUSION: Our study identified a novel gene, LRP5L, associated with congenital membranous cataract, and its mutant LRP5L-P36R contributed to membranous cataract development via inhibition of laminin γ1 and c-MAF.

Long non-coding RNA ANGPTL1-3 promotes multiple myeloma bortezomib resistance by sponging miR-30a-3p to activate c-Maf expression.

Multiple Myeloma (MM) is a malignant hematological disease characterized by monoclonal proliferation of plasma cells in the bone marrow. In recent years, the widespread use of new drugs based on bortezomib (Btz) has significantly improved the remission rate of MM patients. However, drug resistance and disease relapse occur within a few years and MM is still considered to be an incurable disease. The amplification of the long arm of chromosome 1 is one of the most common genetic abnormalities in MM patients. Here, we found that long non-coding RNA ANGPTL1-3 which located in 1q region was overexpressed in MM. Lnc-ANGPTL1-3 expression was correlated with MM International Staging System (ISS) and overall survival. Notably, knockdown of lnc-ANGPTL1-3 increased Btz sensitivity of MM cells. Following exploration revealed that lnc-ANGPTL1-3 competitively interacted with miR-30a-3p to c-Maf, a transcription factor which was reported to be associated with Btz resistance. Taken together, our findings demonstrate that lnc-ANGPTL1-3/miR-30a-3p/c-Maf axis plays a critical role in MM Btz resistance.

Mebendazole elicits potent antimyeloma activity by inhibiting the USP5/c-Maf axis.

c-Maf is a critical oncogenic transcription factor that contributes to myelomagenesis. Our previous studies demonstrated that the deubiquitinase USP5 stabilizes c-Maf and promotes myeloma cell proliferation and survival; therefore, the USP5/c-Maf axis could be a potential target for myeloma therapy. As a concept of principle, the present study established a USP5/c-Maf-based luciferase system that was used to screen an FDA-approved drug library. It was found that mebendazole, a typical anthelmintic drug, preferentially induced apoptosis in c-Maf-expressing myeloma cells. Moreover, oral administration of mebendazole delayed the growth of human myeloma xenografts in nude mice but did not show overt toxicity. Further studies showed that the selective antimyeloma activity of mebendazole was associated with the inhibition of the USP5/c-Maf axis. Mebendazole downregulated USP5 expression and disrupted the interaction between USP5 and c-Maf, thus leading to increased levels of c-Maf ubiquitination and subsequent c-Maf degradation. Mebendazole inhibited c-Maf transcriptional activity, as confirmed by both luciferase assays and expression measurements of c-Maf downstream genes. In summary, this study identified mebendazole as a USP5/c-Maf inhibitor that could be developed as a novel antimyeloma agent.

Transcription factor c-Maf is essential for IL-10 gene expression in B cells.

Interleukin (IL)-10 is an essential anti-inflammatory cytokine that plays important roles as a negative regulator of immune responses to microbial antigens. c-Maf has been suggested as an essential transcriptional factor for IL-10 production in CD4+ T cells and macrophages. However, it remains unclear whether c-Maf participates in IL-10 expression in B cells. In this study, we investigated the role of c-Maf in the transcriptional regulation of IL-10 in regulatory B cells, as well as the underlying molecular mechanism. We found that c-Maf was constitutively expressed in resting B cells. c-Maf expression was upregulated in the presence of LPS and dose-dependently enhanced IL-10 production following binding to the IL-10 promoter. Moreover, a lower expression of c-Maf and decreased production of regulatory B (Breg) cells were detected in mice with collagen-induced arthritis (CIA), which may contribute to the pathological changes. Taken together, these data demonstrate that c-Maf is an indispensable yet constitutive transcription factor for IL-10 gene expression in LPS-activated B cells.

Regulation of c-Maf and αA-Crystallin in Ocular Lens by Fibroblast Growth Factor Signaling.

Fibroblast growth factor (FGF) signaling regulates a multitude of cellular processes, including cell proliferation, survival, migration, and differentiation. In the vertebrate lens, FGF signaling regulates fiber cell differentiation characterized by high expression of crystallin proteins. However, a direct link between FGF signaling and crystallin gene transcriptional machinery remains to be established. Previously, we have shown that the bZIP proto-oncogene c-Maf regulates expression of αA-crystallin (Cryaa) through binding to its promoter and distal enhancer, DCR1, both activated by FGF2 in cell culture. Herein, we identified and characterized a novel FGF2-responsive region in the c-Maf promoter (-272/-70, FRE). Both c-Maf and Cryaa regulatory regions contain arrays of AP-1 and Ets-binding sites. Chromatin immunoprecipitation (ChIP) assays established binding of c-Jun (an AP-1 factor) and Etv5/ERM (an Ets factor) to these regions in lens chromatin. Analysis of temporal and spatial expression of c-Jun, phospho-c-Jun, and Etv5/ERM in wild type and ERK1/2 deficient lenses supports their roles as nuclear effectors of FGF signaling in mouse embryonic lens. Collectively, these studies show that FGF signaling up-regulates expression of αA-crystallin both directly and indirectly via up-regulation of c-Maf. These molecular mechanisms are applicable for other crystallins and genes highly expressed in terminally differentiated lens fibers.

Nephrotic Syndrome in Small Cell Lung Cancer and Induction of C-Mip in Podocytes.

Paraneoplastic nephrotic syndrome is often a complication in patients with cancer, and various histologic lesions have been described in the kidney. We report the case of a 76-year-old woman who presented with a podocytopathy that was found to be associated with a small cell lung carcinoma (SCLC). One cycle of carboplatin-etoposide combination therapy led to resolution of nephrotic syndrome and remission of the lung carcinoma. C-Maf-inducing protein (C-Mip) was overexpressed in both podocytes and cancer cells, but was not found in control kidney and lung tissue samples. C-Mip also was absent in SCLC cells from 30 patients without nephrotic syndrome. Exposing cultured podocytes to a sample of our patient's serum that was collected prior to chemotherapy led to disorganization of the podocyte cytoskeleton and induction of C-Mip expression, which was not observed with control serum or our patient's serum sampled after chemotherapy. These observations suggest that C-Mip may play an important role in SCLC-related podocytopathy and that a circulating factor likely induces its expression in the kidney.

Differential expression patterns of MafB and c-Maf in macrophages in vivo and in vitro.

The large Maf transcription factors c-Maf and MafB are expressed in macrophage-lineage hematopoietic cells, but the expression patterns of MafB and c-Maf in macrophage subtypes and tissue-resident macrophages have not been fully analyzed. First, we analyzed MafB and c-Maf protein expression in tissue-resident macrophages. Mouse lymph nodes, spleens, lungs, and kidneys were subjected to immunohistochemistry using anti-MafB and anti-c-Maf. Both MafB and c-Maf signals were observed in lymph node macrophages. In the splenic macrophages the MafB signal was detected by anti-MafB, but the c-Maf signal was not detected. No expression of c-Maf or MafB was detected in macrophages in the lung and kidney. Flow cytometry analysis revealed a similar pattern of GFP expression in Mafb/GFP knock-in heterozygous mice. To analyze these different expression patterns in greater detail, we examined the expression of MafB and c-Maf by quantitative RT-PCR in different cytokine- or LPS-induced macrophages in vitro. MafB expression was induced by IL-10 or IL-4 with IL-13 and was reduced by LPS or GM-CSF. By contrast, c-Maf expression was induced by IL-10 and reduced by IL-4 with IL-13 or GM-CSF. These results indicate that MafB and c-Maf have different expression patterns in macrophages, suggesting differences in function.

MicroRNAs mediating CNS inflammation: Small regulators with powerful potential.

MicroRNAs (miRNAs) are a family of small non-coding RNAs (~22 nucleotides) that fine-tune protein expression by either silencing mRNA translation or directly targeting gene transcripts for degradation. In the central nervous system (CNS), neuroinflammation plays a critical role in brain injury and neurodegeneration. Increasing evidence supports the involvement of miRNAs as key regulators of neuroinflammation. Altered expression or function of particular miRNAs has been identified in various CNS pathological conditions, including neuroinflammation, neurodegeneration, and autoimmune diseases. Several miRNAs have been shown to play a critical role in the microglia-mediated inflammatory response including miR-155 and miR-146a. In this review, we summarize recent advances in the field of miRNAs associated with CNS inflammation, including our studies of unique inflammatory pathways involving miR-155 and miR-146a. We discuss how specific miRNAs influence microglia activation states in response to inflammatory stimuli, and describe the potential of miRNAs as both biomarkers of inflammation and therapeutic tools for the modulation of microglia behavior.

Regulation of Interleukin-10 Expression.

Interleukin (IL)-10 is an essential anti-inflammatory cytokine that plays important roles as a negative regulator of immune responses to microbial antigens. Loss of IL-10 results in the spontaneous development of inflammatory bowel disease as a consequence of an excessive immune response to the gut microbiota. IL-10 also functions to prevent excessive inflammation during the course of infection. IL-10 can be produced in response to pro-inflammatory signals by virtually all immune cells, including T cells, B cells, macrophages, and dendritic cells. Given its function in maintaining the delicate balance between effective immunity and tissue protection, it is evident that IL-10 expression is highly dynamic and needs to be tightly regulated. The transcriptional regulation of IL-10 production in myeloid cells and T cells is the topic of this review. Drivers of IL-10 expression as well as their downstream signaling pathways and transcription factors will be discussed. We will examine in more detail how various signals in CD4+ T cells converge on common transcriptional circuits, which fine-tune IL-10 expression in a context-dependent manner.

LPS induces the degradation of programmed cell death protein 4 (PDCD4) to release Twist2, activating c-Maf transcription to promote interleukin-10 production.

Programmed cell death protein 4 (PDCD4) is a tumor suppressor and has also been shown to suppress production of the immunomodulatory cytokine IL-10. The precise role of PDCD4 in IL-10 induction in macrophages is still not fully understood. Incubation of macrophages with inhibitors of PI3K and mTOR blocked LPS-stimulated PDCD4 degradation and expression of c-Maf and IL-10 production. PDCD4 and the transcription factor Twist2 were shown to form a complex in untreated cells. LPS disrupted the complex allowing Twist2 to bind to the c-Maf promoter. PI3K and mTOR inhibitors prevented this disruption by stabilizing PDCD4 and thereby decreased Twist2 binding to the c-Maf promoter and induction of c-Maf mRNA. These results indicate a regulatory role for PDCD4 and Twist2 in LPS-induced IL-10 production in macrophages. LPS promotes PDCD4 degradation via a pathway involving PI3K and mTOR, releasing Twist2, which induces IL-10 via c-Maf.