You're using an outdated browser. Please upgrade to a modern browser for the best experience.
Please note this is an old version of this entry, which may differ significantly from the current revision.
Subjects:
Immunology
Interferon regulatory factor 8 (IRF8) is a transcription factor of the IRF protein family. IRF8 was originally identified as an essentialfactor for myeloid cell lineage commitment and differentiation. Deletion of Irf8 leads to massive accumulation of CD11b+Gr1+ immature myeloid cells (IMCs), particularly the CD11b+Ly6Chi/+Ly6G− polymorphonuclear myeloid-derived suppressor cell-like cells (PMN-MDSCs). Under pathological conditions such as cancer, Irf8 is silenced by its promoter DNA hypermethylation, resulting in accumulation of PMN-MDSCs and CD11b+ Ly6G+Ly6Clo monocytic MDSCs (M-MDSCs) in mice. IRF8 is often silenced in MDSCs in human cancer patients. MDSCs are heterogeneous populations of immune suppressive cells that suppress T and NK cell activity to promote tumor immune evasion and produce growth factors to exert direct tumor-promoting activity. Emerging experimental data reveals that IRF8 is also expressed in non-hematopoietic cells. Epithelial cell-expressed IRF8 regulates apoptosis and represses Osteopontin (OPN). Human tumor cells may use the IRF8 promoter DNA methylation as a mechanism to repress IRF8 expression to advance cancer through acquiring apoptosis resistance and OPN up-regulation. Elevated OPN engages CD44 to suppress T cell activation and promote tumor cell stemness to advance cancer. IRF8 thus is a transcription factor that regulates both the immune and non-immune components in human health and diseases.
- IRF8
- Function
- Cancer
1. Introduction
Interferon regulatory factor 8 (IRF8), originally termed interferon consensus sequence binding protein (ICSBP), is a member of the IRF transcription factor [1]. IRFs were first identified as a regulator of the type I interferon (IFN-I) response for the activation of IFN-stimulated genes that are essential for immune response to viruses and other pathogens [2,3,4,5], and are now known to be important in turning pathogen associated molecular patterns into chromatin changes and eventually into immune cell activation [6]. IRF8 was first cloned as an IFNγ-inducible nuclear protein-encoding gene that binds to specific IFN-responsive DNA motif in the major histocompatibility complex class I (MHC I) genes [7]. IRF8 has since been determined to be constitutively expressed and IFNγ inducible and plays key roles in the IFN response pathways in immune cell differentiation and function, as well as in non-hematopoietic cell turnover and pathogenesis [8,9,10,11,12,13,14,15,16,17,18,19,20,21].
2. IRF8 Function and Diseases
A 915C> T mutation (R294C) resembles IRF8 KO mice in accumulating immature myeloid cells (IMCs) and causes susceptibility to infection in mice [22,23]. K108E and T80A mutations results in IRF8 loss of function that leads to impaired dendritic cell monocyte development and function in humans or mice [9,24]. A 331C>T [R111* (stop codon)] mutation may cause IRF8 loss of function, resulting in neutrophilia, monocytopenia and decreased CD3+ T cell and CD8+ T cell counts in humans [25]. IRF-8 polymorphisms have been implicated in the development of autoimmune thyroiditis, Behcet’s disease and, increased susceptibility to tuberculosis (TB). The IRF-8 polymorphism, rs17445836, was associated with both development of autoimmune thyroiditis and TB [26,27,28]. Moreover, IRF8 also functions as an apoptosis regulator in hematopoietic tumor cells [29,30,31,32]. IRF8’s intrinsic function in hematopoietic cells thus plays essential roles in myelopoiesis to regulate immune cell activation and immune suppressors under physiological conditions [20,33,34] and in suppression of tumorigenesis [29,30,31,32]. Indeed, IRF8 has been shown to function as a suppressor of acute myeloid leukemia (AML) [35]. However, IRF8 has recently been reported to function as an acute myeloid leukemia (AML)-specific susceptibility factor and promote AML cell proliferation. High IRF8 expression is associated with poorer prognoses in certain AML patients [36]. On the other hand, IRF8 is also expressed in non-hematopoietic cells [8,10,37,38,39,40,41,42,43,44,45]. IRF8 functions as an apoptosis regulator in several types of human tumor cells [38,41,46,47] and mice with IRF8 deletion only in the colon epithelial cells develop significantly more colon tumors than the littermate control mice [41]. Furthermore, IRF8 expression level is positively linked to cancer patient prognosis and response to immunotherapy [18,19]. IRF8 therefore also functions as a tumor suppressor in non-hematopoietic cells.
3. IRF8 Function as a Transcription Factor That Depends on IAD-Interacting Transcription Factors to Exert Its Activity
Analysis and comparison of the IRF8 protein sequences in the National Center for Biotechnology Information with the Blast protein program [48] revealed that IRF8 proteins are highly conserved in mammals, with an 89.44–89.67% similarity in amino acid sequences between human and mouse IRF8 proteins. Human IRF8 gene is located at chromosome 16 with 23,228 bp that encodes 9 exons and 8 introns. The human IRF8 protein is 426 amino acids long with a DNA-binding domain (DBD) and an IRF association domain (IAD) [23]. The DBD binds to unique consensus sequence motifs at promoters of genes, such as IFN-responsive genes, to regulate transcription. The IAD associates with other transcription factors to direct the IRF8 protein complex binding to specific DNA motif to regulate specific gene transcription. IRF8 has weak DNA-binding affinity and its transcription regulatory activity therefore depends on its IAD association with other transcriptional factors to form a transcription complex which renders IRF8 specific and high affinity to bind to the unique DNA motifs at gene promoters [23,34,49,50]. IRF8 functions as either a transcriptional activator or repressor and it is the IAD-associated transcription factor(s) that dictates IRF8 bindingand transcriptional activity [20,23,33,51,52,53,54,55].
IRF8 transcription factor protein complexes selectively binds to certain types of DNA consensus sequence motifs to activate or repress gene transcription [33,56,57,58,59]. Heterodimers of IRF8 in association with IRFs (e.g., IRF1 or IRF2) bind to the IFN-stimulated response element (ISRE: [(A/G)NGAAANNGAAACT]. IRF8-IRF1/IRF2 heterodimers generally bind to promoter region ISRE to repress the transcription of the downstream genes [1]. The IRF8-Ets (Erythroblast Transformation Specific/E-twenty-six) and IRF8-PU1 heterodimers bind to the Ets-RF8 composite element (EICE: GGAANNGAAA) or the IRF8-Ets composite sequence (IECS: GAAANN(N)GGAA) to activate target gene transcription [52,53,56,57,58,60,61,62,63,64,65,66]. In addition, IRF8 has been shown to form a complex with the JUNB/AP-1-BATF3and/or IRF4 and binds to the AP-1-IRFcomposite elements 1 and 2 [AICE1:TTTCNNNNTGA(G/C)T(C/A)A; AICE2: GAAATGA(G/C)T(C/A)A] to activate transcription of genes [11,67,68,69]. For example, IRF8 forms a heterodimer with AP-1 factor BATF and binds to the AICE site to promote gene activation in T helper 17 (Th17) cells, B and dendritic cells [67,69,70]. It has been shown that IRF8 also forms complexes with IRF4, PU.1 and BATF at the ISRE-like sites to activate Il9 and Il21 transcription in Th9 cells [66]. IRF8 can also form a heterodimer with IRF1 or IRF2 [71] that binds to ISRE element to repress transcription of genes induced by IFN and retinoic acid [72,73] or to activate target gene expression [50,70,71]. Although IRF8 binds to IECS to activate gene expression [1,54], IRF8 binding to IECS element at the Asah1 promoter represses acid ceramidase expression [31]. Furthermore, IRF6 interacts with ETV6 to repress Il4 transcription in Th9 cells [66].
4. IRF8 Expression Profiles
The expression of IRF8 was originally detected in hematopoietic cells and its expression has long been thought to be restricted to hematopoietic cells [56]. Although IRF8 is abundantly expressed in hematopoietic cells, particularly in B cells and DCs, it has since been determined that IRF8 is also expressed in epithelial cells in the intestine, colon, ocular lens, skin, lung, liver, and heart [8,10,37,38,39,40,41,42,43,44,45]. Analysis of IRF8 expression from single cell RNA sequencing datasets revealed that IRF8 is indeed expressed in several types of non-hematopoietic cells, including theca cells, melanocytes, enterocytes and intestinal cells in humans.
5. IRF8 Expression and Function in Non-Hematopoietic Cancer Cells
While much research has been dedicated to understanding the role of IRF8 in hematopoietic cells, less research has investigated the role of IRF8 in non-hematopoietic cells. In certain types of cancers, IRF8 functions as a tumor suppressor gene [18,19,42,43,44], and its expression is regulated by DNA methylation or micro-RNA interference [113]. In breast and lung cancer, IRF8 is downregulated as a result of promoter hypermethylation [43,151,152], and a higher IRF8 expression level was associated with a better prognosis [19]. Similarly, in gastric, nasopharyngeal, esophageal, colorectal, lung, prostate, and renal cell carcinomas the expression of IRF8 is downregulated [43,44,153,154,155,156,157]. IFN-γ induced expression of IRF8 was increased when gastric cancer cells were treated with a demethylating agent [153]. In other cancer cell lines with a methylated IRF8 promoter, IFN-γ treatment showed decreased ability to induce IRF8 expression [154]. Taken together, these studies provide evidence for promoter methylation as a mechanism for IRF8 silencing in certain types of human cancer cells. Analysis of human lung tumor specimens at the single cell level validate the early finding that IRF8 is silenced in the tumor cells. Except for certain tumor-infiltrating immune cells, including certain B cells, pDC and myeloid cells, IRF8 is down-regulated in most cell types and is undetectable in human lung cancer cells [158].
Within non-hematopoietic cancer cells, IRF8 plays a protective role against the formation of a metastatic phenotype. In human colon cancer cells, IRF8 protein levels are inversely correlated with the metastatic phenotype [46,47]. IRF8 expression sensitized the metastatic tumor cells to Fas-mediated apoptosis, indicating that loss of IRF8 in cancers could promote earlier metastasis [46]. It has been suggested that colon cancer cells silence IRF8 expression through inhibition of pSTAT1 function at the IRF8 promoter [47]. IRF8 may also function in non-hematopoietic cancers to negatively regulate the expression of MMP3, a matrix metalloproteinase associated with a poor prognosis and metastasis in various solid organ cancers [159,160,161]. In a chemically-induced sarcoma model, an inverse relationship between IRF8 and MMP3 expression was demonstrated; in a mouse model of mammary cancer, loss of MMP3 reduced spontaneous lung metastasis [162]. This suggests that IRF8 may negatively regulate MMP3 expression, protecting against MMP3-induced metastasis in solid organ tumors. While most research supports an anti-metastasis role for IRF8 in non-hematopoietic cancers, one study found that IRF8 promoted EMT-like phenomena, cell motility, and invasion in a human osteosarcoma cell line providing evidence for a possible role in the acquisition of a metastatic-like phenotype [163].
IRF8 appears to impede the progression and formation of cancer by increasing the expression of tumor suppressor genes, such as Caspase 1, p21, p27, and PTEN [44,157,164]. Expression of tumor suppressor genes inhibited lens cell carcinoma growth upon transfection with IRF8 [164]. Similarly, IRF8-induced expression of p27 induced lung cancer cell senescence [44]. Mechanistically, IRF8 was shown to inhibit Akt activity thereby inducing the accumulation of p27 and promoting senescence. While IRF8 increases tumor suppressor genes, one study also showed an IRF8-induced decrease in oncogene (Yap1 and Survivin) expression in renal cell carcinoma cell lines [157]. In this cell line, ectopic expression of IRF8 induced cell cycle G2/M arrest and apoptosis further supporting IRF8’s role in impeding cancer progression.
IRF8 functions as a promoter of Fas-mediated apoptosis. This form of extrinsic apoptosis is key to immune clearance of tumor cells and is targeted in cancer therapy [165,166]. Modification of Fas expression can render immune checkpoint inhibitor (ICI) therapies unsuccessful. Colon cancer cells with low Fas expression exhibited decreased sensitivity to FasL-induced apoptosis (a mechanism utilized by ICIs) [167] and lower Fas expression was correlated with decreased survival in colon cancer patients [168]. One mechanism by which cancer cells may alter the expression of Fas, and thereby reduce sensitivity to apoptosis, is through downregulation of IRF8.
As previously mentioned, IRF8 is downregulated in many cancers. Studies revolving around a loss of IRF8 have elucidated ways in which IRF8 helps facilitate Fas-mediated apoptosis and resulting in tumor immune sensitivity. In soft tissue sarcoma cells, IRF8 was found to be a repressor of FLICE-like protein (FLIP) [169]. Silencing of FLIP and ectopic IRF8 expression each increased susceptibility to FasL-induced apoptosis. This suggests that by repressing FLIP, IRF8 promotes Fas-mediated apoptosis susceptibility. Mechanistically, IRF8 disruption diminishes JAK1 expression and inhibits STAT1 phosphorylation to block IFN-γ induced Fas upregulation in sarcoma cells [170]. IRF8 has also been found to regulate pro-apoptotic and anti-apoptotic factors contributing to Fas-mediated apoptosis. In tumor-induced MDSCs, levels of IRF8 are decreased. IRF8-deficient MDSCs showed an increase in anti-apoptotic Bcl-xL and a decrease in pro-apoptotic Bax, providing a mechanism by which IRF8 downregulation can impede the Fas-mediated apoptosis pathway to evade elimination by T lymphocytes [32]. Another mechanism by which IRF8 can promote Fas-mediated apoptosis is through repression of acid ceramidase (A-CDase). A-CDase upregulation has been implicated in certain cancers such as prostate and breast [171,172,173]. IRF8 induced repression of A-CDase resulted in C16 ceramide accumulation and increased apoptosis sensitivity [31]. This suggests that IRF8 downregulation likely contributes to decreased Fas-mediated apoptosis sensitivity through failure to repress A-CDase. In summary, IRF8 promotes Fas-mediated apoptosis through repression of FLIP and A-CDase and by regulating apoptotic factors such as Bcl-xL and Bax [31,32,169].
IRF8 functions as a link between cancer and the immune system. Poor tumoral homing of DCs and T cells was observed in melanoma-bearing IRF8 KO mice [42]. Additionally, the few DCs that were able to infiltrate the tumor were immature and unable to produce T cell-mediated immune responses. Mechanistically, the decreased tumoral immune infiltration observed in IRF8 KO mice is suggested to occur through the aberrant expression of multiple chemokine receptors and ligands [42]. When IRF8 was induced, immune infiltration was reestablished, and the chemokine expression pattern was reversed. Using a microfluidic device, in which melanoma cells and splenocytes were separated by a microchannel, IRF8 KO splenocytes exhibited a marked decrease in the ability to cross the channel to infiltrate the melanoma cells while melanoma cells exhibited marked propensity to invade the channels suggesting existence of an IRF8-regulated communicating soluble factor [174].
OPN has recently emerged as another immune checkpoint that may compensate PD-L1 function to promote colon tumor immune evasion [175]. IRF8 is expressed in human and mouse gastric and colon epithelial cells [38,41] and binds to the ISRE elements at the Spp1 promoter to repress OPN expression [176]. In human colon and pancreatic cancers, IRF8 expression is down-regulated and OPN is up-regulated [175,176,177,178,179]. scRNA-Seq analysis indicates that OPN is abundantly expressed in tumor cells, MDSCs, and ILCs [175]. Furthermore, scRNA-Seq analysis revealed the tumor-promoting role of SPP1+ cells in cancer [180,181]. OPN protein level is highly elevated in the peripheral blood in human cancer patients and OPN expression is up-regulated in several different types of human cancers [182,183,184,185,186,187,188,189,190]. Silenced IRF8 expression in both tumor cells and tumor-induced myeloid cells by DNA methylation and H3K9me3 deposition at the Irf8 promoter are likely major mechanisms underlying OPN elevation in cancer patients and tumor-bearing mice [41,46,47,154,191]. It was observed that IRF8 KO mice exhibited deficient generation of antigen-specific T cells and this deficiency was due to OPN engagement of CD44 to directly suppress T cell activation [176]. Accordingly, OPN blockade increased activation of tumor-specific T cells and suppressed tumor growth in vivo [175]. OPN is highly expressed in human pancreatic tumor cells and may bind to CD44 on tumor cells via autocrine and paracrine manners to promote pancreatic cancer stemness and progression [177,179]. Therefore, in addition to functioning as a repressor of SPP1 in immune cells such as MDSCs and ILCs, IRF8 not only functions as an intrinsic tumor suppressor [41] but also suppresses tumorigenesis and progression through repressing OPN expression in tumor cells [176].
This entry is adapted from the peer-reviewed paper 10.3390/cells11172630
This entry is offline, you can click here to edit this entry!