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The Generation of an Engineered Interleukin-10 Protein With Improved Stability and Biological Function.

ABSTRACT: Interleukin-10 (IL-10) is an immunoregulatory cytokine that plays a pivotal role in modulating inflammation. IL-10 has inhibitory effects on proinflammatory cytokine production and function in vitro and in vivo; as such, IL-10 is viewed as a potential treatment for various inflammatory diseases. However, a significant drawback of using IL-10 in clinical application is the fact that the biologically active form of IL-10 is an unstable homodimer, which has a short half-life and is easily degraded in vivo. Consequently, IL-10 therapy using recombinant native IL-10 has had only limited success in the treatment of human disease. To improve the therapeutic potential of IL-10, we have generated a novel form of IL-10, which consists of two IL-10 monomer subunits linked in a head to tail fashion by a flexible linker. We show that the linker length per se did not affect the expression and biological activity of the stable IL-10 molecule, which was more active than natural IL-10, both in vitro and in vivo. We confirmed that the new form of IL-10 had a much-improved temperature- and pH-dependent biological stability compared to natural IL-10. The IL-10 dimer protein binds to the IL-10 receptor similarly to the natural IL-10 protein, as shown by antibody blocking and through the genetic modifications of one monomer in the IL-10 dimer specifically at the IL-10 receptor binding site. Finally, we showed that stable IL-10 is more effective at suppressing LPS-induced-inflammation in vivo compared to the natural IL-10. In conclusion, we have developed a new stable dimer version of the IL-10 protein with improved stability and efficacy to suppress inflammation. We propose that this novel stable IL-10 dimer could serve as the basis for the development of targeted anti-inflammatory drugs.

SUBMITTER: Minshawi F

PROVIDER: S-EPMC7431522 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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The Generation of an Engineered Interleukin-10 Protein With Improved Stability and Biological Function.

Minshawi Faisal F Lanvermann Sebastian S McKenzie Edward E Jeffery Rebecca R Couper Kevin K Papoutsopoulou Stamatia S Roers Axel A Muller Werner W

Frontiers in immunology 20200811

Interleukin-10 (IL-10) is an immunoregulatory cytokine that plays a pivotal role in modulating inflammation. IL-10 has inhibitory effects on proinflammatory cytokine production and function <i>in vitro</i> and <i>in vivo</i>; as such, IL-10 is viewed as a potential treatment for various inflammatory diseases. However, a significant drawback of using IL-10 in clinical application is the fact that the biologically active form of IL-10 is an unstable homodimer, which has a short half-life and is ea ...[more]

PMID: 32849644

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Project description:Background and aimsThe impairment of intestinal epithelial barrier is the main etiologic factor of inflammatory bowel disease. The proper intestinal epithelial proliferation and differentiation is crucial for maintaining intestinal integrity. Metformin is a common anti-diabetic drug. The objective is to evaluate the protective effects of metformin on ileal epithelial barrier integrity using interleukin-10 deficient (IL10KO) mice.MethodsWild-type and IL10KO mice were fed with/without metformin for 6 weeks and then ileum was collected for analyses. The mediatory role of AMP-activated protein kinase (AMPK) was further examined by gain and loss of function study in vitro.ResultsCompared to wild-type mice, IL10KO mice had increased proliferation, reduced goblet cell and Paneth cell lineage differentiation in the ileum tissue, which was accompanied with increased crypt expansion. Metformin supplementation mitigated intestinal cell proliferation, restored villus/crypt ratio, increased goblet cell and Paneth cell differentiation and improved barrier function. In addition, metformin supplementation in IL10KO mice suppressed macrophage pro-inflammatory activity as indicated by reduced M1 macrophage abundance and decreased pro-inflammatory cytokine IL-1?, TNF-? and IFN-? expressions. As a target of metformin, AMPK phosphorylation was enhanced in mice treated with metformin, regardless of mouse genotypes. In correlation, the mRNA level of differentiation regulator including bmp4, bmpr2 and math1 were also increased in IL10KO mice supplemented with metformin, which likely explains the enhanced epithelial differentiation in IL10KO mice with metformin. Consistently, in Caco-2 cells, metformin promoted claudin-3 and E-cadherin assembly and mitigated TNF-?-induced fragmentation of tight junction proteins. Gain and loss of function assay also demonstrated AMPK was correlated with epithelial differentiation and proliferation.ConclusionsMetformin supplementation promotes secretory cell lineage differentiation, suppresses inflammation and improves epithelial barrier function in IL10KO mice likely through activation of AMPK, showing its beneficial effects on gut epithelial.

Dataset Information

The Generation of an Engineered Interleukin-10 Protein With Improved Stability and Biological Function.

Publications

The Generation of an Engineered Interleukin-10 Protein With Improved Stability and Biological Function.

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