Critical Conditions for Studying Interleukin‐11 Signaling In Vitro and Avoiding Experimental Artefacts

Abstract

Interleukin (IL) 11 is a member of the IL6 family of cytokines which require the ubiquitous gp130 receptor to activate canonical (JAK/STAT) and non-canonical (e.g., ERK) signaling pathways. The IL11 cytokine is upregulated in a number of fibro-inflammatory diseases and cancer, where it binds the cognate IL11 receptor alpha subunit (IL11RA) to form a hexameric IL11:IL11RA:gp130 signaling complex. The specific IL11RA receptor is highly expressed on cells of the stromal and parenchymal niche but expressed at low levels on immune cells, highly passaged cells, or transformed cell lines. Consequently, primary cells such as hepatic stellate cells, fibroblasts, and hepatocytes are ideal experimental systems to study IL11 signaling in vitro. In contrast to immortalized cell lines, primary cells better display relevant cellular physiology and pathobiology. This collection of protocols details experimental and culturing conditions for primary cells that preserve meaningful cellular states and physiological responses ex vivo in conventional 2D cell culture systems. Readouts of cellular activity are chosen carefully to capture the non-canonical, post-transcriptional activity of IL11 signaling. Our data suggest that cell type, cell culture conditions, passage number, concentrations of stimuli, timing, and other factors have major implications for studies of IL11 signaling. In vitro experiments with primary cell material need to be planned and executed with great caution. Otherwise, physiologically relevant mechanisms may become dysfunctional and reproducible experimental artefacts can obscure our view of true cytokine biology.

IL11 stimulates ERK/P90RSK to inhibit LKB1/AMPK and activate mTOR initiating a mesenchymal program in stromal, epithelial, and cancer cells

“…All cells were grown and maintained at 37 °C and 5% CO 2 . The culture of primary human hepatic stellate cells (HSCs) and primary human hepatocytes were described previously ( Viswanathan et al., 2021 ). Primary human cardiac fibroblasts (HCFs (52-year-old male), ScienCell) were grown and maintained in complete fibroblasts medium-2 (ScienCell) which contains 5% fetal bovine serum (FBS, ScienCell), 1% fibroblasts growth supplement-2 (FGS-2, ScienCell) and 1% penicillin-streptomycin (P/S, ScienCell).…”

IL11 Activates Pancreatic Stellate Cells and Causes Pancreatic Inflammation, Fibrosis and Atrophy in a Mouse Model of Pancreatitis

“…A critical difference being that the study by Zheng et al used an RSV enhancer-driven SV40 T antigen immortalised PSC-like cell line (HP-1) [ 27 ]. We have shown that high passage primary cells and immortalised cell lines have different/artefactual signalling and phenotypes, as compared to primary cells, when stimulated with IL6 family members [ 28 ]. We suggest that the data shown by Zheng et al are relevant to transformed HP-1 cells but not primary human PSCs.…”

Oxygen–Glucose Deprivation Promoted Fibroblast Senescence and Collagen Expression via IL11

“…IL11 is a member of the IL6 cytokine family, mainly derived from stromal cells such as fibroblasts [ 8 ]. In fibroblasts, IL11 signaling activates JAK/STAT3 and MEK/ERK via IL11RA/IL6ST and possibly AKT [ 21 ].…”

“…IL11 is a member of the IL6 cytokine family, mainly derived from stromal cells such as fibroblasts [ 8 ]. The expression of IL11 promotes tissue fibrosis [ 9 , 10 ].…”