Production of recombinant human interleukin-38 and its inhibitory effect on the expression of proinflammatory cytokines in THP-1 cells

Abstract

Interleukin (IL)-38 is the latest member of the IL-1 cytokine family. However, as a result of lacking efficient method to generate relatively large quantity of IL-38, its precise functions are poorly understood. In the present study, the cloning, expression, purification, and activity analysis of recombinant human IL-38 was described. Human IL-38 cDNA was cloned into the prokaryotic expression vector pET-44. The recombinant IL-38 containing a C-hexahistidine tag was expressed in Escherichia coli BL21 (DE3) which induced by isopropyl-β-D-thiogalactoside. The expressed fusion protein was purified by Ni-NTA affinity chromatography. IL-38 protein was largely found in the soluble fraction. The purified IL-38 appeared a single band on SDS-PAGE, the yield of IL-38 was 4 mg from 1 L of bacterial culture, and the purity was more than 98% with low endotoxin level (<0.1 EU/μg). Western blotting confirmed the identity of the purified protein. Activity analysis showed that IL-38 can inhibit effectively the expression of proinflammatory cytokines, such as tumor necrosis factor-α, IL-1β, IL-17, and monocyte chemoattractant protein-1 in lipopolysaccharide-activated THP-1 cells. The production and characterization of biologically active IL-38 will be beneficial for its potential role in clinical applications.

Biology of IL‐38 and its role in disease

“…Another study demonstrated that knockdown of IL‐38 with siRNA in peripheral blood mononuclear cells resulted in increased production of IL‐6, APRIL and CCL2 in response to TLR ligands, supporting the hypothesis that IL‐38 acts as an antagonist . THP‐1 cells stimulated with LPS produced lower concentrations of TNF and IL‐1β in the presence of recombinant IL‐38 . However, it has also been reported that IL‐38 can increase proinflammatory cytokine production in response to LPS or IL‐1β .…”

“…34 THP-1 cells stimulated with LPS produced lower concentrations of TNF and IL-1β in the presence of recombinant IL-38. 35 However, it has also been reported that IL-38 can increase proinflammatory cytokine production in response to LPS or IL-1β. 21,22 These studies that increased IL-6 in these higher concentrations.…”

IL-36, IL-37, and IL-38 Cytokines in Skin and Joint Inflammation: A Comprehensive Review of Their Therapeutic Potential

“…On the one hand, since IL-36γ expression is enhanced by TLR4 activation, the IL-38-mediated inhibition of TLR signaling indirectly decreases the release of the agonist IL-36γ [39]. For instance, IL-38 reduces TLR4-mediated inflammation by significantly decreasing IL-6 and IL-23 produced by THP1 cells or primary M1 macrophages upon LPS stimulation [95,96]. Since blocking the TLR4 pathway in an animal model of DITRA syndrome significantly limits the auto-inflammatory response [97], the IL-38-mediated targeting of the TLR signaling in inflammatory skin conditions is encouraging.…”

IL-38: A New Player in Inflammatory Autoimmune Disorders

“…Three candidate receptors, IL-36R, IL-1R1, and IL-1 receptor accessory protein-like1 (IL-1RAPL1) have been proposed for IL-38. IL-38 binds to these receptors, prevents the binding of agonistic ligands and inhibits subsequent signaling pathways, thus exerting anti-inflammatory effects through PBMCs, a human leukemia monocytic cell line (THP-1s), Mφs, and dendritic cells (DCs) to inhibit the activation and function of Th1s and Th17s and promote Treg expansion [14,15,16]. However, the primary pathway is still under dispute.…”