Regulation of interferon lambda-1 (IFNL1/IFN-λ1/IL-29) expression in human colon epithelial cells

Highlights

• First characterization of IFN-λ1 gene regulation in colon epithelial cells.

• ZEB1 and BLIMP-1 inhibit IFN-λ1 gene expression in colon epithelial cells.

• ZEB1 specifically represses the type-III IFN family (IFN-λ1) over type-I IFNs.

• NF-κB family members differentially regulate IFN-λ1 in colon epithelial cells.

Abstract

The efficient regulation of intestinal immune responses is critical to colon health. Viruses, for example noraviruses, are key pathogens of the intestine. The lambda interferons (comprising three ligands: IFNL1, L2 and L3 – the so-called “Type III” interferons) constitute the most recently discovered IFN family and are known to be important in intestinal anti-viral defense. A fourth family member, IFNL4, was recently described. Expression of the IFN-lambda receptor is restricted to epithelial and immune cells; together, these ligands and their receptor represent an important anti-viral and immunoregulatory component of the immune/epithelial inteface. We investigated control of IFNL1 expression in human colon epithelial cells. We used the TLR3 agonist poly I:C to drive expression of IFNL1 in SW480 cells, and small interfering RNA (siRNA) to knockdown target transcription factors. We identified ZEB1 and BLIMP-1 as transcription factors that strongly inhibited IFNL1 expression in SW480 cells. Interestingly, while BLIMP-1 inhibited both type-III and type-I interferons (IFN-β), the inhibitory action of ZEB1 was specific for IFNL1. We also defined the NF-κB family member, p65 as a key activator of IFNL1 and NF-κB p50 as a key inhibitor. Finally, we demonstrated that siRNA targeting of ZEB1 or NF-κB p50 resulted in a significant elevation of secreted IFN-λ1 protein and expression of the anti-viral gene OAS1, while knockdown of p65 inhibited these events. Our data provide insight to the regulation of IFNL1 expression in the human colon and suggest novel therapeutic approaches to elevate IFNλ-1 protein where required.

Introduction

The type III IFNs were first described in 2003 in two independent studies, describing three highly related cytokines known as: IFN-λ1, IFN-λ2 and IFN-λ3 (IL-29, IL-28A and IL-28B, respectively) [1], [2]. These have since been found to have critical roles in coordinating anti-viral and other inflammatory responses. Type III IFNs are members of the IL10-IFN family, which also includes the IL-10-like cytokines (IL-10, IL-19, IL-20, IL-22, IL-24 and IL-26), the type I IFNs (IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω), and the type II IFN, IFN-γ. The gene structures of the IFN-λs (IFNL1, IFNL2, and IFNL3; formerly termed IL29, IL28A, IL28B, respectively) have been reviewed in detail [3], [4] and resemble those of the IL-10-like cytokines rather than those of the intron-less type I IFN genes, cytokines with which type III IFNs share many functional properties. The three IFN-λ proteins are encoded on human chromosome 19q13 and display a high degree of homology (IFN-λ2 and -λ3 being nearly 96% identical at the protein level [1], [3]). Recently, a fourth family member, IFNL4, was described. Interestingly, this gene encodes a functional protein only in primates and is disrupted in individuals of a particular genotype [5].

Expression of the IFNL genes has been found to be responsive to TLR signaling resulting from both viral and bacterial ligands and is most pronounced in plasmacytoid dendridic cells (pDCs) and epithelial cells [3], [4], [6]. Other cell-types, such as monocytes and fibroblasts, also express them [1], [2], [3], [4], [6]. All IFN-λ ligands signal through a single heterodimeric receptor complex consisting of the IL-28Rα signaling chain and the accessory chain IL-10Rβ [1], [3]. While several cell types can be induced to produce the IFN-λ ligands, expression of the type III IFN receptor is restricted to epithelial cells and leukocytes, including pDCs and T cells; therefore IFN-λ ligands are selectively active on these cell types [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]. The primary outcome of type III IFN signaling is similar to that of type I IFNs. pSTAT1 homodimer and pSTAT1/2 heterodimer signal transduction leads to the activation of target-gene promoter GAS and ISRE elements, respectively [1], resulting in the expression of interferon-stimulated genes (ISGs) encoding anti-viral proteins such as 2′5′-oligoadenylate synthetase 1 and 2 (OAS1 and 2), Mx-1 and 2 proteins, and IFN-inducible dsRNA-activated protein kinase [4]. While the outcome of type I and type III IFN expression may appear redundant, differences in receptor distribution confer selectivity of effect. Type III IFN expression and response is most prominent at mucosal surfaces such as in the lungs and gastro-intestinal tract, while type I IFN expression and response is most prominent in liver, spleen and kidneys [14]. This selective receptor expression confers tight regulation of the response to type III IFNs to epithelial cells at mucosal surfaces and to the pDCs that can survey those surfaces, pointing to an important regulatory function for type III IFNs at the interfaces between the body and the outside environment.

Due to functional similarities between the type I and type III IFNs, promoter regulation studies have drawn comparisons between type III and type I IFNs. Initial work focused on proximal control elements contained within the first 600 bp 5′ of the transcription start site (TSS); NF-κB and IRF sites were found to be occupied within the first 300 bases of the IFN-λ1 promoter following Newcastle Disease Virus infection, for example [17]. The effect of NF-κB and IRF on IFNL promoter function was also investigated by Osterlund et al., whose overexpression studies suggested that IFNL1 was regulated by IRF3, while IFNL2 and IFNL3 were more responsive to IRF7 [18]. These authors drew comparison with type I IFN regulation; IFNB is regulated by IRF-3 while IFNA is more dependent on IRF-7 [19], [20], [21]. A more recent study of IFNL1 activation in response to bacteria found that activation was dependent upon distal NF-κB sites at −1137 and −1183 bp of the promoter in monocyte-derived dendritic cells (MDDC) [22], demonstrating that the regulation of IFNL1 can be both stimulation and cell-type specific.

We recently presented the first characterization of IFNL1 transcriptional regulation in human bronchial epithelial cells. We studied both the endogenous gene and luciferase reporter constructs extending to 4 kb 5′ of the transcription start site, and revealed a hitherto undescribed role for ZEB1 as a powerful and selective IFNL1 transcriptional repressor [23]. BLIMP-1 (previously characterized as an IFN-β repressor) was also shown to repress IFNL1. In addition, we further defined roles for NF-κB and IRF; NF-κB c-REL/p52 and c-REL/p65 heterodimers, and IRF1, were activators while the NF-κB-p50 homodimer was repressive. Thus, we demonstrated that while there is overlapping regulation of type I and type III IFNs in these cells, ZEB1 (also known as AREB6 and TCF8 [24]) represents a key point of differential regulation between IFNL1 and the type I IFNs, suggesting possible specific modulation of the type III IFNs.

The type III IFNs show potentially important mucosal epithelia specificity and anti-viral properties within the gastro-intestinal tract. Mice lacking functional receptors for type III IFNs in their intestinal epithelia were impaired in their ability to control oral infection by rotavirus, while mice lacking functional receptors for type I IFN signaling were not. Treatment of mice with IFN-λ repressed rotavirus replication in the gut while type I IFN did not [25]. In addition, using intestinal cell lines and murine and human colonic tissue, IFN-λ1 and IFN-λ2 were demonstrated to be strong inhibitors of HCMV protein expression, thereby causing an increased expression of anti-viral proteins such as Mx-1 and OAS1. This study also demonstrated that increased IFN-λ1 led to induction of the neutrophil chemoattractant IL-8, which further associates type III IFNs with inflammatory responses [26] and confirms an earlier similar observation in human PBMC [8].

Mucosal epithelial cells such as those of the colon not only provide a physical barrier against pathogens such as viruses but also serve as regulators of innate and adaptive immune responses, secreting cytokinesin response to viral infection and so triggering cellular infiltration and polarization of T-cells [3]. Expression and subsequent secretion of IFN-λ1 is thought to be a critical factor in maintaining the epithelial/immune cell interface particularly through its ability to inhibit Th2 polarization [3], [9], [11], [12]. Thus it was of interest for us in this study to determine how IFN-λ1 expression is regulated in colon epithelial cells exposed to viral insult. In the present study we identified the key transcription factors acting on endogenous IFNL1 expression in colon epithelia responding to the viral-mimic poly I:C.