Corilagin Counteracts IL-13Rα1 Signaling Pathway in Macrophages to Mitigate Schistosome Egg-Induced Hepatic Fibrosis.

The IL-13Rα1 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-13Rα1 signaling pathway in M2 macrophages in vitro and in vivo. The mRNA and protein expression of IL-13Rα1, PPARγ, KLF4, SOCS1, STAT6, p-STAT6, and TGF-ß 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 PPARγ, KLF4, SOCS1, p-STAT6, and TGF-ß 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-13Rα1 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-13Rα1 signaling pathway and M2 macrophage polarization effectively in vivo. Furthermore, the anti-fibrogenic effect persisted even when IL-13Rα1 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-13Rα1 signaling pathway.

Transcriptional reversion of cardiac myocyte fate during mammalian cardiac regeneration.

RATIONALE: Neonatal mice have the capacity to regenerate their hearts in response to injury, but this potential is lost after the first week of life. The transcriptional changes that underpin mammalian cardiac regeneration have not been fully characterized at the molecular level. OBJECTIVE: The objectives of our study were to determine whether myocytes revert the transcriptional phenotype to a less differentiated state during regeneration and to systematically interrogate the transcriptional data to identify and validate potential regulators of this process. METHODS AND RESULTS: We derived a core transcriptional signature of injury-induced cardiac myocyte (CM) regeneration in mouse by comparing global transcriptional programs in a dynamic model of in vitro and in vivo CM differentiation, in vitro CM explant model, as well as a neonatal heart resection model. The regenerating mouse heart revealed a transcriptional reversion of CM differentiation processes, including reactivation of latent developmental programs similar to those observed during destabilization of a mature CM phenotype in the explant model. We identified potential upstream regulators of the core network, including interleukin 13, which induced CM cell cycle entry and STAT6/STAT3 signaling in vitro. We demonstrate that STAT3/periostin and STAT6 signaling are critical mediators of interleukin 13 signaling in CMs. These downstream signaling molecules are also modulated in the regenerating mouse heart. CONCLUSIONS: Our work reveals new insights into the transcriptional regulation of mammalian cardiac regeneration and provides the founding circuitry for identifying potential regulators for stimulating heart regeneration.

Identification of potent antagonist antibodies against mouse IL-13Rα1 using novel bioassays.

Interleukin-13 (IL-13) is a cytokine implicated in airway diseases such as asthma and idiopathic pulmonary fibrosis. IL-13 signals through a heterodimeric receptor complex consisting of IL-13Rα1 and IL-4Rα, known as the type II IL-4R. IL-4 also signals through this receptor and as such many of the biological effects of IL-13 and IL-4 are similar. Here we describe the development of two sensitive bioassays to determine the potency of antagonists of the mouse type II IL-4R. Both IL-13 and IL-4 dose-dependently induce CCL17 production from J774 mouse monocytic cells and CCL11 production from NIH3T3 mouse fibroblasts in the presence of TNFα. The assays were optimized to minimize TNFα concentration, cell number and incubation time whilst retaining a suitable signal-to-background ratio. Anti-cytokine antibodies or recombinant soluble receptors completely neutralized IL-13 or IL-4 activity in these bioassays. The J774 assay was used to screen a panel of anti-mIL-13Rα1 antibodies for neutralizing activity against this receptor. We report the identification of the first monoclonal antibodies that bind mouse IL-13Rα1 and neutralize both IL-13-induced and IL-4-induced cellular function. These antibodies should prove useful for determining the effects of neutralizing IL-13Rα1 in mouse models of disease. In addition, these bioassays may be used for measuring the bioactivity of mouse IL-13 and IL-4 and for the discovery of additional antagonists of the mouse IL-13Rα1/IL-4Rα complex.

Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system.

We describe protein synthesis, folding and assembly of antibody fragments and full-length aglycosylated antibodies using an Escherichia coli-based open cell-free synthesis (OCFS) system. We use DNA template design and high throughput screening at microliter scale to rapidly optimize production of single-chain Fv (scFv) and Fab antibody fragments that bind to human IL-23 and IL-13α1R, respectively. In addition we demonstrate production of aglycosylated immunoglobulin G (IgG 1) trastuzumab. These antibodies are produced rapidly over several hours in batch mode in standard bioreactors with linear scalable yields of hundreds of milligrams/L over a 1 million-fold change in scales up to pilot scale production. We demonstrate protein expression optimization of translation initiation region (TIR) libraries from gene synthesized linear DNA templates, optimization of the temporal assembly of a Fab from independent heavy chain and light chain plasmids and optimized expression of fully assembled trastuzumab that is equivalent to mammalian expressed material in biophysical and affinity based assays. These results illustrate how the open nature of the cell-free system can be used as a seamless antibody engineering platform from discovery to preclinical development of aglycosylated monoclonal antibodies and antibody fragments as potential therapeutics.

In vitro and in vivo characterisation of anti-murine IL-13 antibodies recognising distinct functional epitopes.

Interleukin-13 (IL-13) sequentially binds to IL-13Ralpha1 and IL-4Ralpha forming a high affinity signalling complex. This receptor complex is expressed on multiple cell types in the airway and signals through signal transducer and activator of transcription factor-6 (STAT-6) to stimulate the production of chemokines, cytokines and mucus. Antibodies have been generated, using the UCB Selected Lymphocyte Antibody Method (UCB SLAM), that block either binding of murine IL-13 (mIL-13) to mIL-13Ralpha1 and mIL-13Ralpha2, or block recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. Monoclonal antibody (mAb) A was shown to bind to mIL-13 with high affinity (K(D) 11 pM) and prevent binding of mIL-13 to mIL-13Ralpha1. MAb B, that also bound mIL-13 with high affinity (K(D) 8 pM), was shown to prevent recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. In vitro, mAbs A and B similarly neutralised mIL-13-stimulated STAT-6 activation and TF-1 cell proliferation. In vivo, mAbs A and B demonstrated equipotent, dose-dependent inhibition of eotaxin generation in mice stimulated by intraperitoneal administration of recombinant mIL-13. In an allergic lung inflammation model in mice, mAbs A and B equipotently inhibited muc5ac mucin mRNA upregulation in lung tissue measured two days after intranasal allergen challenge. These data support the design of therapeutics for the treatment of allergic airway disease that inhibits assembly of the high affinity IL-13 receptor signalling complex, by blocking the binding of IL-13 to IL-13Ralpha1 and IL-13Ralpha2, or the subsequent recruitment of IL-4Ralpha.

Targeting IL-4/IL-13 signaling to alleviate oral allergen-induced diarrhea.

BACKGROUND: Intestinal anaphylaxis (manifested by acute diarrhea) is dependent on IgE and mast cells. OBJECTIVE: We aimed to define the respective roles of IL-4 and IL-13 and their receptors in disease pathogenesis. METHODS: Wild-type mice and mice deficient in IL-4, IL-13, and IL-13 receptor (IL-13R) alpha1 (part of the type 2 IL-4 receptor [IL-4R]) were sensitized with ovalbumin (OVA)/aluminum potassium sulfate and subsequently given repeated intragastric OVA exposures. The IL-4R alpha chain was targeted with anti-IL-4R alpha mAb before or after intragastric OVA exposures. RESULTS: IL4(-/-) (and IL4/IL13(-/-)) mice produced almost no IgE and were highly resistant to OVA-induced diarrhea, whereas allergic diarrhea was only partially impaired in IL13(-/-) and IL13Ralpha1(-/-) mice. IL13Ralpha1-deficient mice had decreased IgE levels, despite having normal baseline IL-4 levels. Intestinal mast cell accumulation and activation also depended mainly on IL-4 and, to a lesser extent, on IL-13. Prophylactic anti-IL-4R alpha mAb treatment, which blocks all IL-4 and IL-13 signaling, suppressed development of allergic diarrhea. However, treatment with anti-IL-4R alpha mAb for 7 days only partially suppressed IgE and did not prevent intestinal diarrhea. CONCLUSION: Endogenously produced IL-13 supplements the ability of IL-4 to induce allergic diarrhea by promoting oral allergen sensitization rather than the effector phase of intestinal anaphylaxis.