Cytokine and proinflammatory gene expression in classical Hodgkin lymphoma: its more than NF-κB!
2014 Elsevier Ltd. All rights reserved. The expression of proinflammatory genes including those encoding for cytokines and chemokines throughout the immune system is tightly controlled by the coordinated activity of cell type-specific and activation-dependent transcription factors (TF). Such tight control ensures a concerted cellular immune response and ensures that inflammation is effective but localized. However, deregulated cytokine expression is commonly observed in human lymphoma, and contributes to growth and survival of the malignant cells and to the formation of a supportive tumor microenvironment. The most prominent example for such a deregulation of proinflammatory genes among hematopoietic malignancies is classical Hodgkin lymphoma (cHL) [1]. cHL is a common usually B cellderived malignancy with a characteristic clinical and histopathological presentation. Histopathologically, cHL is characterized by the rare monoor multinucleated Hodgkin-/Reed-Sternberg (HRS) cells which are embedded in an abundant infiltrate of various non-malignant hematopoietic cells, a finding which has attracted the attention of hematologists and pathologists for more than 100 years. In addition, a common clinical feature of cHL patients is their suffering from impressive so-called ‘‘B’’ symptoms including fever and night sweats. It is commonly thought that these characteristics of cHL are mediated by the abundant production of cytokines and chemokines by HRS cells. This includes IL-6, IL-13, IL-21, thymus and activation–regulated chemokine (TARC), macrophage-derived chemokine (MDC), and CCL5/RANTES, to mention some of them. Many of these factors function in an autocrine and/or paracrine manner to stimulate growth and survival of the malignant HRS cells and they modulate the composition of their cellular microenvironment [2]. The assumed critical pathogenic relevance of the crosstalk between HRS and surrounding bystander cells highlights the importance of understanding the molecular basis for the unusually strong upregulation of cytokine and chemokine genes in cHL. Previously, a number of groups including ours have uncovered multiple signaling pathways and TFs linked to the aberrant transcriptional regulation of such genes. This includes the constitutive activation of the IKK/NF-jBand JAK/STAT-pathways and the formation of an aberrant AP-1 TF complex [1,3]. It is well established, that constitutive NF-jB activity contributes to growth and survival of HRS cells as well as to the deregulation of their proinflammatory gene expression. Furthermore, various genomic defects of negative regulators within this signaling pathway, such as deleterious genomic alterations of NFKBIA or TNFAIP3, underline its pathogenic role in cHL [1]. However, deregulation of NF-jB activity is not restricted to cHL but is also found in other human lymphomas, without a similar upregulation of proinflammatory genes, suggesting that other transcriptional regulators are specifically activated in HRS cells that act either alone or in combination with NF-jB to orchestrate their proinflammatory gene expression. 116 S. Kreher et al. / Cytokine 72 (2015) 115–117 In an unbiased attempt to identify such transcriptional regulators we globally mapped DNase I hypersensitive sites (DHSs) in HRS compared to non-Hodgkin cells to identify accessible chromatin regions specific for HRS cells [4]. DHSs indicate the position of cis-regulatory elements bound by TF complexes [5] and have been shown to differ between various cell types [6], providing the opportunity to identify cell type-specific TF binding. Following the definition of a specific active chromatin landscape in HRS cells based on the comparison of the DHS accessibility profiles between HRS and non-Hodgkin cell lines, we therefore conducted a genome-wide search for enriched TF binding motifs within cHL-specific DHSs (Fig. 1). This search revealed in the cHL-specific DHSs a significant enrichment of binding motifs for the inducible transcription factors AP-1, NF-jB, STAT and interferon regulatory factors (IRFs) [4]. Moreover, global footprinting analyses demonstrated that these motifs were specifically occupied in HRS but not in control cells, indicating a chronic activity of the corresponding, normally inducible factors. The important role of AP-1, NF-jB and STAT factors for cHL pathogenesis had already been demonstrated in previously published studies and our studies confirmed this role at the genome-wide level. Given the known important functions of IRFs in the differentiation and transformation of various hematopoietic cell types [7], we set out to examine which of the IRFs were binding to the IRF binding motifs in cHL and examined their expression and functional role in HRS cells. This line of experiments led to the finding of an abundant, largely to HRS cells of cHL restricted expression of IRF5. IRF5 is usually activated upon Toll-like receptor (TLR)-stimulation and regulates the expression of proinflammatory genes such as IL-6, IL-12, TNFa, IP-10, MIP-1a, and CCL5/RANTES. IRF5 plays a key role in the regulation of the proinflammatory cytokine burst after Fig. 1. To identify deregulated transcription factors (TFs) central to classical Hodgkin l global mapping of DNase I hypersensitive sites and analyzed enriched TF binding motifs i regulatory factor (ISRE)-sites among the top enriched motifs. Functionally, high-level IRF5 together with NF-jB resulted in non-Hodgkin cells in activation of essential characteris (lower panel). bacterial or viral infection [7]. Moreover, IRF5 allelic variations have been shown to be associated with autoimmune diseases [7], and IRF5 / mice are resistant to lethal shock by various stimuli due to the incapability to activate proinflammatory cytokines [8]. Given the unique expression of IRF5 in HRS cells we hypothesized that its activity, alone or in combination with TFs such as NF-jB, was the key to the inflammatory phenotype of cHL. Functionally we could confirm this hypothesis. Following inhibition of IRF5 in HRS cell lines we observed a decrease of the HLcharacteristic expression of IL-13, IL-6 and RANTES at the mRNA and/or protein level. Furthermore, reporter assays demonstrated a synergism between IRF5 and NF-jB, exemplarily shown at the RANTES promoter. In complementary gain-of-function experiments we introduced IRF5 and/or NF-jB into non-Hodgkin lymphoma and leukemia cell lines as well as into primary murine splenic B cells. These studies demonstrated a key role of IRF5 in the activation of a whole set of HRS cell-characteristic cytokines and chemokines, including IL-6, IL-13, TNFa, RANTES, and CXCL11. In addition, IRF5-mediated induction of these factors caused an increased attraction of mononuclear cells toward the transfected non-Hodgkin cells. Importantly, full expression of these proinflammatory genes from their endogenous gene loci required IRF5 in combination with NF-jB, since neither IRF5 nor NF-jB alone were able to induce a transcriptional activation comparable to that observed in HRS cells. This finding ties in with the previously demonstrated transcriptional cooperation between IRFs and NF-jB [7]. In addition, we identified the TFs lipopolysaccharide-induced TNF factor (LITAF) and AP-1 as downstream effectors of IRF5 activation, placing IRF5 upstream in the hierarchy of coordinators of proinflammatory gene expression in HRS cells. Apart from proinflammatory genes, IRF5-induced gene expression changes were generally ymphoma (cHL) biology we determined HRS cell-specific accessible chromatin by n these regions (upper panel). By this approach, we identified interferon-stimulated expression was specific to cHL cells and crucial for their survival. Furthermore, IRF5 tics of cHL, including the expression of proinflammatory cytokines and chemokines S. Kreher et al. / Cytokine 72 (2015) 115–117 117 reminiscent to those observed in HRS cells, confirming the key role of IRF5 in cHL (Fig. 1). The inhibition of IRF5 function in HRS cell lines resulted in apoptotic cell death. Apart from a direct effect on cell-intrinsic survival pathways, this may be due to a disruption of cytokineor chemokine-mediated autocrine or paracrine survival pathways. This finding adds a new layer to our knowledge of IRF5 function in lymphoid cells, as until now IRF5 has been linked to the induction of cell death only in lymphoid cells [7]. However, the question now arises of what is the underlying mechanism of IRF5 activation in HRS cells. In antiviral immune responses, IRF5 activation relies on MyD88-dependent signaling following TLR-activation, furthermore, a role of the IKK-related kinases TBK1 and IKKe have been described [7]. More recently, the NF-jB activating kinase IKKb has been shown to activate IRF5 in response to several inflammatory stimuli [9]. This points to a constitutive TLR activation as a possible mechanism for high-level IRF5 expression and activation in HRS cells. How such signals enforce malignant transformation has to be investigated in future studies. Such studies will be also of high relevance for other malignancies with an inflammatory history. Our data link aberrant chromatin activation with specific TF alterations in a hematopoietic malignancy and suggest an exceptional and extensive transcriptional crosstalk between IRF5, the NF-jB signaling pathway and AP-1 family TFs in HRS cells as an explanation for their highly activated phenotype. Uncovering the hierarchical order and coordinating molecular mechanisms of this crosstalk opens therapeutic opportunities for either the inhibition of several of these signaling pathways simultaneously, or the development of specific inhibitors with restricted activity to the respective malignant cells. Only recently, the latter strategy has exemplarily been described as a promising treatment option for multiple myeloma cells [10]. For cHL treatment, a multi-target intervention through concomitant inhibition of IRF5 and NF-jB signaling path