Unique IL-13R?2/STAT3 mediated IL-13 regulation detected in lung conventional dendritic cells, 24?h post viral vector vaccination.
ABSTRACT: This study demonstrates that 24?h following viral vector-based vaccination IL-13R?2 functions as a master sensor on conventional dendritic cells (cDCs), abetted by high protein stability coupled with minimal mRNA expression, to rapidly regulate DC mediated IL-13 responses at the lung mucosae, unlike IL-13R?1. Under low IL-13, IL-13R?2 performs as a primary signalling receptor, whilst under high IL-13, acts to sequester IL-13 to maintain homeostasis, both in a STAT3-dependent manner. Likewise, we show that viral vector-derived IL-13 levels at the vaccination site can induce differential STAT3/STAT6 paradigms in lung cDC, that can get regulated collaboratively or independently by TGF-?1 and IFN-?. Specifically, low IL-13 responses associated with recombinant Fowlpox virus (rFPV) is regulated by early IL-13R?2, correlated with STAT3/TGF-?1 expression. Whilst, high IL-13 responses, associated with recombinant Modified Vaccinia Ankara (rMVA) is regulated in an IL-13R?1/STAT6 dependent manner associated with IFN-?R expression bias. Different viral vaccine vectors have previously been shown to induce unique adaptive immune outcomes. Taken together current observations suggest that IL-13R?2-driven STAT3/STAT6 equilibrium at the cDC level may play an important role in governing the efficacy of vector-based vaccines. These new insights have high potential to be exploited to improve recombinant viral vector-based vaccine design, according to the pathogen of interest and/or therapies against IL-13 associated disease conditions.
Project description:Interleukin (IL)-32?, a newly identified IL-32 isoform, has been reported to exert pro-inflammatory effects through the association with protein kinase C delta (PKC?). In this study, we further examined the effects of IL-32? on IL-13 and IL-13R?2 expression and the related mechanism in THP-1 cells. Upon stimulating IL-32?-expressing and non-expressing cells with phorbol 12-myristate 13-acetate (PMA), the previous microarray analysis showed that IL-13R?2 and IL-13 mRNA expression were significantly decreased by IL-32?. The protein expression of these factors was also confirmed to be down-regulated. The nuclear translocation of transcription factors STAT3 and STAT6, which are necessary for IL-13R?2 and IL-13 promoter activities, was suppressed by IL-32?. Additionally, a direct association was found between IL-32?, PKC?, and signal transducer and activator of transcription 3 (STAT3), but not STAT6, revealing that IL-32? might act mainly through STAT3 and indirectly affect STAT6. Moreover, the interaction of IL-32? with STAT3 requires PKC?, since blocking PKC? activity eliminated the interaction and consequently limited the inhibitory effect of IL-32? on STAT3 activity. Interfering with STAT3 or STAT6 binding by decoy oligodeoxynucleotides (ODNs) identified that IL-32? had additive effects with the STAT3 decoy ODN to suppress IL-13 and IL-13R?2 mRNA expression. Taken together, our data demonstrate the intracellular interaction of IL-32?, PKC?, and STAT3 to regulate IL-13 and IL-13R?2 synthesis, supporting the role of IL-32? as an inflammatory modulator.
Project description:This study demonstrates that the fate of a vaccine is influenced by the cytokines produced by the innate lymphoid cells (ILC) recruited to the vaccination site, and it is vaccine route and adjuvant dependent. Intranasal virus vaccination induced ST2/IL-33R+ ILC2 in lung, while intramuscular vaccination induced exclusively IL-25R+ ILC2 in muscle. Interestingly, a larger proportion of IL-13+ ILC2s were detected in muscle following i.m. viral vector vaccination compared to lung post i.n. delivery. These observations revealed that ILC2 were the main source of IL-13 at the vaccination site (24?h post vaccination) responsible for inducing T cells of varying avidities. Moreover, recombinant fowlpox viral vector-based vaccines expressing adjuvants that transiently block IL-13 signalling at the vaccination site using different mechanisms (IL-4R antagonist or IL-13R?2 adjuvants), revealed that the level of IL-13 present in the milieu also significantly influenced IFN-?, IL-22 or IL-17A expression by ILC1/ILC3. Specifically, an early IL-13 and IFN-? co-dependency at the ILC level may also be associated with shaping the downstream antibody responses, supporting the notion that differentially regulating IL-13 signalling via STAT6 or IL-13R?2 pathways can modify ILC function and the resulting adaptive T- and B-cell immune outcomes reported previously. Moreover, unlike chronic inflammatory or experimentally induced conditions, viral vector vaccination induced uniquely different ILC profiles (i.e., expression of CD127 only on ILC2 not ILC1/ILC3; expression of IFN-? in both NKP46+ and NKp46- ILCs). Collectively, our data highlight that tailoring a vaccine vector/adjuvant to modulate the ILC cytokine profile according to the target pathogen, may help design more efficacious vaccines in the future.
Project description:Monocytes/macrophages are innate immune cells that play a crucial role in the resolution of inflammation. In the presence of the Th2 cytokines interleukin-4 (IL-4) and interleukin-13 (IL-13), they display an anti-inflammatory profile and this activation pathway is known as alternative activation. In this study we compare and differentiate pathways mediated by IL-4 and IL-13 activation of human monocytes/macrophages. Here we report differential regulation of IL-4 and IL-13 signaling in monocytes/macrophages starting from IL-4/IL-13 cytokine receptors to Jak/Stat-mediated signaling pathways that ultimately control expression of several inflammatory genes. Our data demonstrate that although the receptor-associated tyrosine kinases Jak2 and Tyk2 are activated after the recruitment of IL-13 to its receptor (containing IL-4R? and IL-13R?1), IL-4 stimulates Jak1 activation. We further show that Jak2 is upstream of Stat3 activation and Tyk2 controls Stat1 and Stat6 activation in response to IL-13 stimulation. In contrast, Jak1 regulates Stat3 and Stat6 activation in IL-4-induced monocytes. Our results further reveal that although IL-13 utilizes both IL-4R?/Jak2/Stat3 and IL-13R?1/Tyk2/Stat1/Stat6 signaling pathways, IL-4 can use only the IL-4R?/Jak1/Stat3/Stat6 cascade to regulate the expression of some critical inflammatory genes, including 15-lipoxygenase, monoamine oxidase A (MAO-A), and the scavenger receptor CD36. Moreover, we demonstrate here that IL-13 and IL-4 can uniquely affect the expression of particular genes such as dual-specificity phosphatase 1 and tissue inhibitor of metalloprotease-3 and do so through different Jaks. As evidence of differential regulation of gene function by IL-4 and IL-13, we further report that MAO-A-mediated reactive oxygen species generation is influenced by different Jaks. Collectively, these results have major implications for understanding the mechanism and function of alternatively activated monocytes/macrophages by IL-4 and IL-13 and add novel insights into the pathogenesis and potential treatment of various inflammatory diseases.
Project description:Colorectal cancer (CRC) is one of the most common causes of cancer-related death worldwide due to the distant metastases. Compelling evidence has reported that epithelial-mesenchymal transition (EMT) is involved in promoting cancer invasion and metastasis. However, the precise molecular events that initiate this complex EMT process remain poorly understood. Here, we showed that the pleiotropic cytokine interleukin-13 (IL-13) could induce an aggressive phenotype displaying EMT by enhancing the expression of EMT-promoting factor ZEB1. Importantly, STAT6 signaling inhibitor and STAT6 knockdown significantly reversed IL-13-induced EMT and ZEB1 induction in CRC cells, whereas ectopic STAT6 expression in STAT6null CRC cell line markedly promoted EMT in the present of IL-13. ChIP-PCR and Luciferase assays revealed that activated STAT6 directly bound to the promoter of ZEB1. Otherwise, we found IL-13 also up-regulated the stem cell markers (nanog, CD44, CD133 and CD166) and promoted cell migration and invasion through STAT6 pathway. We also found that siRNA-mediated knockdown of IL-13R?1 could reverse IL-13-induced ZEB1 and EMT changes by preventing STAT6 signaling. Finally, we demonstrated positive correlation between IL-13R?1 and ZEB1 at mRNA levels in human CRC samples. Taken together, our findings first demonstrated that IL-13/IL-13R?1/STAT6/ZEB1 pathway plays a critical role in promoting EMT and aggressiveness of CRC.
Project description:Recently we reported that IL-4 and IL-13 signaling in murine early thymic progenitors (ETPs) expressing the heteroreceptor (HR) comprising IL-4 receptor ? (IL-4R?) and IL-13 receptor ? 1 (IL-13R?1) activate STAT6 and inhibit ETP maturation potential toward T cells. In this study, we asked whether IL-4 and IL-13 signaling through the HR mobilizes other STAT molecules to shape ETP fate decision. The findings indicate that HR+ ETPs undergoing cytokine signaling display increased STAT1, but not STAT3, phosphorylation in addition to STAT6 activation. In parallel, the ETPs had a STAT1-dependent heightened expression of IRF-8, a transcription factor essential for development of CD8?+ dendritic cells (DCs). Interestingly, STAT1 phosphorylation and IRF-8 upregulation, which are independent of STAT6 activation, guided ETP maturation toward myeloid cells with a CD8?+ DC phenotype. Furthermore, these CD8?+ DCs display a thymic resident phenotype, as they did not express SIRP?, a molecule presumed to be involved in cell migration. These findings suggest that IL-4 and IL-13 cytokine-induced HR signaling provides a double-edged sword that simultaneously blocks T cell lineage potential but advances myeloid maturation that could impact T cell selection and central tolerance.
Project description:Macrophages coexpress both the interleukin (IL)-2R? chain (?(c)) and IL-13R?1. These receptor chains can heterodimerize with IL-4R? to form type I or type II IL-4 receptor complexes, respectively. We used macrophages derived from Il2rg and Il13ra1 knockout (KO) mice to evaluate the requirements for these receptor chains for induction of the alternative macrophage activation (AMA) pathway by IL-4 and IL-13. Absence of ?(c) significantly decreased activation of STAT6 by IL-4 but not IL-13. However, although activation of STAT6 by IL-4 was markedly reduced in ?(c) KO macrophages, it was not abolished, indicating that IL-4 can still signal through type II IL-4 receptors via the IL-13R?1 chain. IL-13 failed to activate STAT6 in macrophages derived from Il13ra1 KO mice; however, these cells remained fully responsive to IL-4. The inability of IL-13 but not IL-4 to signal in Il13ra1(-/-) macrophages correlated with the inability of IL-13 but not IL-4 to induce expression of genes such as Arg1, Retnla and Ccl11 that are characteristically expressed by alternatively activated macrophages. In addition, IL-13 but not IL-4 failed to induce membrane fusion and giant cell formation by Il13ra1 KO macrophages. These findings demonstrate that the IL-13R?1 chain is essential for induction of the AMA pathway by IL-13 but not IL-4.
Project description:Early life immune responses are deficient in Th1 lymphocytes that compromise neonatal vaccination. We found that IL-4 and IL-13 engage a developmentally expressed IL-4R?/IL-13R?1 heteroreceptor to endow IFN regulatory factor 1 (IRF-1) with apoptotic functions, which redirect murine neonatal Th1 reactivation to cell death. IL-4/IL-13-induced STAT6 phosphorylation serves to enhance IRF-1 transcription and promotes its egress from the nucleus. In the cytoplasm, IRF-1 can no longer serve as an anti-viral transcription factor but, instead, colocalizes with Bim and instigates the mitochondrial, or intrinsic, death pathway. The new pivotal function of IRF-1 in the death of neonatal Th1 cells stems from the ability of its gene to bind STAT6 for enhanced transcription and the proficiency of its protein to precipitate Bim-driven apoptosis. This cytokine-induced, IRF-1-mediated developmental death network weakens neonatal Th1 responses during early life vaccination and increases susceptibility to viral infection.
Project description:The IL-13R?1 signaling pathway and M2 macrophages play crucial roles in schistosome egg-induced hepatic fibrosis via the expression of pro-fibrotic molecules. This study aims to investigate the inhibitory effect and mechanism of action of corilagin on schistosome egg-induced hepatic fibrosis via the IL-13R?1 signaling pathway in M2 macrophages in vitro and in vivo. The mRNA and protein expression of IL-13R?1, PPAR?, KLF4, SOCS1, STAT6, p-STAT6, and TGF-? was measured in vitro with corilagin treatment after IL-13 stimulation and in vivo corilagin treatment after effectively killing the adult schistosomes in schistosome-infected mice. Histological analysis of liver tissue was assessed for the degree of hepatic fibrosis. The results revealed that corilagin significantly reduced the expression of PPAR?, KLF4, SOCS1, p-STAT6, and TGF-? compared with model group and praziquantel administration (p < 0.01 or p < 0.05) in vivo and in vitro, which indicated a strong inhibitory effect of corilagin on IL-13R?1 signaling pathway. As well, the inhibitory effect of corilagin showed a significant dose-dependence (p < 0.05). The area of fibrosis and distribution of M2 macrophages in mouse liver tissue were reduced significantly and dose-dependently with corilagin treatment compared to model group or praziquantel administration (p < 0.01 or p < 0.05), indicating that corilagin suppressed IL-13R?1 signaling pathway and M2 macrophage polarization effectively in vivo. Furthermore, the anti-fibrogenic effect persisted even when IL-13R?1 was up- or down-regulated in vitro. In conclusion, corilagin can suppress schistosome egg-induced hepatic fibrosis via inhibition of M2 macrophage polarization in the IL-13R?1 signaling pathway.
Project description:In this study, we examined the role IL-13 receptor alpha 1 (IL-13R?1) plays in macrophage differentiation and function. The findings indicate that IL-13R?1 is expressed on the M2 but not on the M1 subset of macrophages and specifically heterodimerizes with the IL-4R? chain to form a type II receptor, which controls the differentiation and function of these cells. Indeed, BM cells from IL-13R?1(+/+) and IL-13R?1(-/-) mice yield equivalent numbers of macrophages when cultured under M2 polarizing conditions. However, IL-13R?1(-/-) BM cells yield a much higher number of macrophages than IL-13R?1(+/+) BM cells when the differentiation is carried out under M1-polarizing conditions. Further analyses indicated that macrophages that express IL-13R?1 also display surface markers associated with an M2 phenotype. In addition, the IL-13R?1(+) macrophages were highly efficient in phagocytizing zymosan bioparticles both in vitro and in vivo, and supported differentiation of naïve T cells to a Th2 phenotype. Finally, when stimulated by IL-13, a cytokine that uses the heteroreceptor, the cells were able to phosphorylate STAT6 efficiently. These previously unrecognized findings indicate that IL-13R?1 serves as a marker for M2 macrophages and the resulting heteroreceptor influences both their differentiation and function.
Project description:There continues to be a major need for more effective inflammatory bowel disease (IBD) therapies. IL-13R?2 is a decoy receptor that binds the cytokine IL-13 with high affinity and diminishes its STAT6-mediated effector functions. Previously, we found that IL-13R?2 was necessary for IBD in mice deficient in the anti-inflammatory cytokine IL-10. Here, we tested for the first time a therapeutic antibody specifically targeting IL-13R?2. We also used the antibody and Il13ra2-/- mice to dissect the role of IL-13R?2 in IBD pathogenesis and recovery. Il13ra2-/- mice were modestly protected from induction of dextran sodium sulfate (DSS)-induced colitis. Following a 7-day recovery period, Il13ra2-/- mice or wild-type mice administered the IL-13R?2-neutralizing antibody had significantly improved colon health compared to control mice. Neutralizing IL-13R?2 to increase IL-13 bioavailability promoted resolution of IBD even if neutralization occurred only during recovery. To link our observations in mice to a large human cohort, we conducted a phenome-wide association study of a more active variant of IL-13 (R130Q) that has reduced affinity for IL-13R?2. Human subjects carrying R130Q reported a lower risk for Crohn's disease. Our findings endorse moving anti-IL-13R?2 into preclinical drug development with the goal of accelerating recovery and maintaining remission in Crohn's disease patients.