IL-34, IL-36 and IL-38 in colorectal cancer—key immunoregulators of carcinogenesis

Abstract

Colorectal cancer (CRC) is still a big killer nowadays, but the precise underlying mechanism remains to be explored. It is believed that imbalance of host immunity in the local microenvironment plays a critical role in the tumorigenesis of CRC. IL-34 is inversely correlated with overall survival in CRC patients, perhaps via regulating terminal differentiation of a subset of macrophages (M2). It is believed that the recruitment/differentiation of M2 macrophages within the cancer simply represents an increase in number, but the function of these M2 macrophages may be compromised. IL-36s (IL-36α, β and γ) are constitutively expressed in non-cancer colon tissue, but colonic IL-36α, IL-36β and IL-36γ are substantially reduced in the CRC tissues (~ 80%). IL-36α is an independent factor affecting the survival of CRC patients. The level of IL-36α and/or IL-36γ in CRC tissue could potentially be used as biomarkers for predicting the prognosis of CRC at both the later or early stages of CRC. IL-38 is also an anti-inflammatory cytokine. Colonic IL-38 is ~ 95% lower in CRC compared to non-CRC colonic tissue, consistent with the positive correlation between differentiation of CRC, and colonic tumour expression of IL-38. IL-38 is a reliable/sensitive biomarker for distinguishing between CRC and non-cancer colonic tissue. There is a positive correlation between colonic IL-38 in CRC and prognosis and/or overall survival, particularly in advanced CRC, supporting IL-38 probably being a reliable and consistent independent factor in predicting the prognosis of CRC. The findings above may be useful in exploring therapeutic targeting for precision medicine.

Colorectal cancer (CRC) is the third leading cause of cancer-related death, due to a lack of sensitive and reliable biomarkers for early diagnosis (Fuchs et al. 1994). The major reason for delayed diagnosis is that these CDC patients are largely asymptomatic until the obvious clinical symptoms and signs present at advanced stages. Consequently, no effective treatment can be offered to these later-stage CRC patients, apart from offering palliative care. Although the majority of CRC patients are aged over 50 years, of concern is that there is an increasing proportion of patients less than 50 years who are being diagnosed with CRC (US data) (Kasi et al. 2019). The possible explanation for the upsurge of younger aged CRC patients may be due to improved diagnostic screening, as well as intake of highly industrialised processed food and lack of physical activities. Thus, it is critically important to promote CRC cancer screening programmes, including faecal occult blood screening and/or colonoscopy, which could substantially improve early diagnosis and reduce the CRC-associated mortality (Siegel et al. 2014).

Effective standardization of management for CRC patients at the pre- and postoperative levels has increased 5-year survival rate substantially (Welch and Robertson 2016), in addition to advanced surgical techniques and the availability of more-effective targeted therapy. The overall 5-year survival rate for CRC patients varies from 91 to 14%, dependent on the tumour being at a localized stage or a distant metastatic stage (Chen et al. 2020a, b). Thus, early detection of CRC with reliable and sensitive biomarker(s) is critically important for improving the overall survival of CRC, as well as the quality of life of these patients.

Host immunity in cancer and CRC

It is well accepted that host immunity plays an important role in suppressing the development of cancer (Cali et al. 2017). However, the precise underlying mechanism of the host immunity in the oncogenesis of CRC remains to be understood, despite decades of intensive studies (Ponz de Leon et al. 2004). There is a concept that the development of cancer is determined partially by the balance between pro- vs anti-inflammatory responses in the microenvironment, which may either enhance the progression of a tumour non-specifically or promote specific anti-tumour activity (Mager et al. 2016). The important role of host immunity in oncogenesis is elegantly illustrated by the Nobel winners in Medicine in 2018 (Zang 2018), demonstrating that inhibition by cancer cells of the immune checkpoint molecules PD-1 and CTLA-4 can inhibit host anti-cancer immunity during the development of a number of malignancies, which has provided a foundation for precision medicine in the form of a potent therapeutic target (Seidel et al. 2018). Furthermore, carcinogenesis of CRC has been extensively studied (De Robertis et al. 2018), e.g. CRC subtypes and their related genetic mutations (Ponz de Leon and Percesepe 2000), including Th17-mediated cancer immunobiology (De Robertis et al. 2018). Furthermore cytokine expression has been clearly associated with the modulation of CRC (Mager et al. 2016). It is well known that host immunity is critical in the development of cancer(s), for example, the discovery of cancer therapy by inhibition of negative immune regulation (Wei et al. 2018).

Macrophage sub-sets and cancer

Macrophages are the most important leucocytes in the initiation of both innate and acquired immunity. Macrophages are classified as either classical M1 cells, contributing to the elimination of microorganisms and/or foreign materials, whereas M2 macrophages promote immunoregulation/immunosuppression (Ley 2017). However, it is debatable whether macrophages are able to promote the development of cancer or suppress cancer, or more likely both, which may be dependent on their final differentiation, rather than simply grouping them into tumour-associated macrophages (TAMs) as one subset. Therefore, understanding the role of which cytokines may influence patient survival in CRC is critical and could offer key information for the clinicians for the prediction of prognosis, as well as for exploring potential therapeutic targets for precision medicine.

IL-34 and CRC

IL-34, an anti-inflammatory cytokine, regulates host immunoregulatory cells in the body. Dysregulated IL-34 has been detected in a number of auto-immune diseases, e.g. rheumatoid arthritis, psoriasis and in atherosclerosis (Schon 2019). Kobayashi et al. demonstrated that IL-34 expression at both the mRNA and protein levels inversely correlated with overall survival in CRC patients. IL-34 is known to play a key role in the differentiation of macrophages (Boulakirba et al. 2018). More interestingly, the presence of CD163-positive M2 tumour-associated macrophages (IL-34^high/CD163^high M2 cells) inversely correlates with overall survival in CRC (Kobayashi et al. 2019). IL-34 expression by lung cancer cells has been shown to promote chemoresistance and tumorigenesis by immunosuppression within the microenvironment of tumour-associated macrophages via the CSF-1R receptor (Baghdadi et al. 2016). This is supported by the hypothesis that although M1 macrophages may serve as cancer killers, M2 cells serve as cancer promoters, including enhancement of cancer metastasis (Laoui et al. 2011). However, generally, M2 macrophages are thought to be anti-inflammatory macrophages, able to suppress inflammation within the microenvironment. Paradoxically, chronic inflammation is also believed to be a critical factor in the induction and the development of cancer through associated proliferative signalling. A possible explanation may be that the recruitment/differentiation of M2 macrophages within the cancer simply represents an increase in number, but the function of these M2 macrophages may be compromised. Alternatively, the high number of M2 macrophages during the development of cancer may suppress the M1 macrophage population, whose primary purpose is the direct killing of malignant cells in the microenvironment. Such speculation should be clarified in future experiments.

The IL-1 superfamily

The IL-1 superfamily is a huge family, including IL-1α, IL-1β, IL-18, IL-36α, IL-36β, IL-36γ and IL-38 (McEntee et al. 2019). Due to space limitation of the current review, we will focus on two novel cytokines, i.e. the IL-36s and IL-38.

The role of IL-36 in cancer and CRC

There are three subsets in IL-36, named IL-36α, β and γ (formerly IL-1F6, IL-1F8 and IL-1F9). All of these three subsets signal through the IL-1 receptor, i.e. IL-1Rrp2 (IL-1RL2) and IL-1RAcP (Towne et al. 2011), via activating the classic nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPKs), jun N-terminal kinases (JNK) and ERK1/2 kinase cascades (Towne et al. 2004). All of these signalling pathways are key signalling pathways for the development of intestinal cancers (Wagner and Nebreda 2009). The IL-36 isotypes bind to the IL-36 receptor with varying affinities. Such signalling pathways have been confirmed by addition of IL-36 agonists to induce inflammation via activating NF-κB and MAPKs (Towne et al. 2011). The source of IL-36 has been shown to include keratinocytes, monocytes and myeloid dendritic cells (DCs) (Furue et al. 2018). Abnormal production of IL-36 is observed in autoimmune diseases, including psoriasis, SLE and Sjogren’s syndrome (Ding et al. 2018). In addition, IL-36 is closely related to inflammatory bowel disease (Russell et al. 2016), suggesting that IL-36 promotes intestinal inflammation during the development of intestinal diseases. The successful use of IL-36 gene therapy has been reported in a fibrosarcoma animal model (Solahaye-Kahnamouii et al. 2014), demonstrating the impressive potential of IL-36 as a therapeutic target for the management of intestinal abnormalities (Ding et al. 2018).

Notably, a positive correlation has been shown between high colonic IL-36α production in CRC tissues and overall survival of CRC patients (Wang et al. 2014, Chen et al. 2020a). However, non-cancer tissue has been applied as a control for comparison in the study, which compromises the conclusion about the pathogenesis of IL-36α in CRC (Wang et al. 2014). Moreover, there are no studies on IL-36β and IL-36γ in CRC, but there has been an exploration of the relationship between IL-36γ and tertiary lymphoid structure and inflammatory immunity in CRC (Weinstein et al. 2018). In a study by the Sautès-Fridman’s research group, IL-36γ has been shown to promote the development of CRC via inflammation in the tumour microenvironment, in addition to its physiological role in the maintenance of colonic epithelial homeostasis. The study in Bao’s group has drawn together several of these observations to demonstrate that there is constitutive expression of colonic IL-36α, IL-36β and IL-36γ detected in non-cancer colon tissue, mainly distributed in the cytoplasm of colonic epithelial cells, supporting the role of these cytokines in maintaining the homeostasis of intestinal mucosal function (McEntee et al. 2019). Interestingly, colonic IL-36α, IL-36β and IL-36γ production is substantially reduced in the CRC tissues (Chen et al. 2020a), particularly an ~ 80% reduction of IL-36β in CRC tissue. This finding suggests that IL-36β may contribute to the development of CRC. Notably, the differential roles of the IL-36s have been demonstrated by the observation that IL-36α and IL-36γ, but not IL-36β, are upregulated at the molecular and cellular levels in inflammatory bowel disease (IBD) (Nishida et al. 2016), although IL-36α, IL-36β and IL-36γ are all pro-inflammatory cytokines (Ding et al. 2018). This observation further suggests that only IL-36α and IL-36γ, but not IL-36β, contribute to the pathogenesis of intestinal inflammation. The discrepancy in IL-36β production between the findings of Nishida, in intestinal inflammation, where little change was observed (Nishida et al. 2016), and our current findings in CRC, where a substantial reduction in IL-36β was observed, may be due to the different levels of severity of the diseases and/or different pathogenesis.

Furthermore, ROC curve analysis has been applied to determine the specificity and sensitivity of IL-36α, IL-36β and IL-36γ production for the prediction of CRC outcomes in both CRC and non-cancer tissues. It is observed that the area under the curve (AUC) of the ROC curves drawn for IL-36α, IL-36β or IL-36γ production are 0.68, 0.73 or 0.65, respectively. Although IL-36β is slightly better than IL-36α or IL 36γ in terms of specificity and sensitivity for the detection of CRC, these AUC values are not sufficiently high to be used as reliable biomarkers for colorectal cancer. Nevertheless, such data invites speculation that there are differential regulatory roles of the IL-36 isoforms during the tumorigenesis of CRC, which is in line with other reports, demonstrating differential production of IL-36α, IL-36β and IL-36γ also exists in human autoimmunities (Boutet et al. 2016, Fonseca-Camarillo et al. 2018).

Furthermore, the patient’s 5-year survival rate is the golden indicator for determining CRC prognosis (Platell and Semmens 2004). Based on a previous finding, IL-36α^high CRC patients have a better survival than IL-36α^low CRC patients (Chen et al. 2020a). Importantly, multivariate analysis demonstrates that IL-36α is an independent factor affecting the survival of CRC patients. The precise anti-tumour mechanism of the IL-36s remains to be explored, but such data are in line with others, showing IL-36α may exhibit anti-tumour effects in CRC progression [13]. It also is unclear if IL-36α is able to promote host immunity against the development of CRC, which might involve recruiting CD8⁺ cytotoxic tumour-infiltrating T cells (TILs) (Pan et al. 2013). Interestingly, the converse survival result is found for IL36γ expression in CRC, namely the IL-36γ^low CRC patients maintain a better survival rate than the IL-36γ^high patients. It has been reported that IL-36γ is mainly produced by M1 macrophages in the CRC tumour microenvironment (Weinstein et al. 2018), which may contribute to tumoricidal effects (Barros et al. 2013). Notably, the findings in this IL-36 study are consistent with IL-34 in CRC patients, described above.

A better prognostic outcome for CRC patients is detected in the sub-group containing IL-36α^high plus IL-36γ^low patients than the sub-group containing IL-36α^low plus IL-36γ^high group (Chen et al. 2020a). Such data offer some useful information for clinician in dealing with CRC patients to make clinical decisions. In addition, IL-36α and IL-36γ are also good indicators for prognosis for subgroups of CRC patients when stratified by clinicopathological characteristics, especially in more advanced stages of CRC patients. The TNM staging system incorporates the depth of invasion and lymph node metastasis of tumours, which are known to be two of the influential factors determining prognosis of CRC patients (Sobin et al. 2010, Lea et al. 2014). Although there was no significant difference between IL-36γ^high and IL-36γ^low in patients that were T1-3, there was a trend, which may be due to the relatively small number of patients in follow-up, especially the T1-3 patients, who generally had survived well, with very few deaths.

Thus, in summary, the level of IL-36α and/or IL-36γ in CRC tissue could potentially be used as biomarkers for predicting the prognosis of CRC at both the later or early stages of CRC. In addition, the combination of colonic IL-36α plus IL-36γ production seems to be more accurately predictive of the postoperative prognosis of CRC patients. Such data may be useful for both basic research and clinical practice.

The role of IL-38 in cancer and CRC

Since IL-38 shares receptors with IL-36α, β and γ, and may inhibit the activity of these IL-36 isoforms by competing with their signalling pathway (Catalan-Dibene et al. 2018), it is logical to explore the possible role of IL-38 in the development of CRC. IL-38, an anti-inflammatory cytokine (Dinarello et al. 2010), is elevated in the circulation in systemic lupus erythematosus (SLE) patients (Takeuchi et al. 2018). The anti-inflammatory role of IL-38 has been demonstrated by the observation that IL-38 placental expression inversely correlated with the severity of gestational diabetes mellitus (Yu et al. 2017). Paradoxically, it is unclear why there is upregulated circulating IL-38 in SLE patients, since IL-38 is thought to suppress both systemic and local inflammation. The speculation is that increased circulating IL-38 may be a regulatory response to the overwhelming systemic inflammation in the relapsing stage of SLE, but the capacity of IL-38 to mediate its anti-inflammatory action may be compromised in the subsequent signalling pathway and/or at the IL-36 receptor.

In CRC tissue, colonic IL-38 is ~ 95% lower in CRC compared to non-CRC colonic tissue, consistent with the positive correlation between differentiation of CRC and colonic tumour expression of IL-38 (Chen et al. 2020b). These findings invite speculation that colonic IL-38 may be important in maintaining normal intestinal mucosal homeostasis within the microenvironment. In addition, such data also suggest that colonic IL-38 facilitates the suppression of CRC growth and metastasis, perhaps via downregulating gut inflammation, which is in line with the observations of others, where IL-38 has been demonstrated to play an important role in remission and relapse of inflammatory bowel disease (Boutet et al. 2016). It is well known that upregulated local inflammation is likely to promote the development of, and more rapid growth and metastasis of, tumours (Ullman and Itzkowitz 2011). Thus, overall, these data are aligned with the anti-inflammatory role of IL-38, showing disturbed gut immunity is observed in the microenvironment in colorectal cancer (Elinav et al. 2013).

Colonic IL-38 production is more than 2.2-fold higher in CRC located in the left side of the colon, compared to the right, an observation that is consistent with a report showing that the left-sided colon CRC patients have a better prognosis compared to that from the right side (Ghidini et al. 2018). In addition, colonic IL-38 production is correlated positively with differentiation of CRC, as well as the size of CRC, which further supports the potential protective role of IL-38 during the development of CRC. Interestingly, it has been reported that an inverse correlation is observed between the differentiation of lung adenocarcinoma and IL-38 production, potentially explained by the suggestion that IL-38 is related to the production of PD-L1 from lung cancer cells (Takada et al. 2017). Additionally, the discrepancy between CRC and lung carcinoma in relation to IL-38 production at the mucosal surface may also be due to the different races from the different studies (Han Chinese versus Caucasian), and/or different organs, although both are classified as mucosal organs which are regulated by host mucosal-associated lymphoid tissues. It is still unclear what is the precise underlying mechanism of the reduced colonic IL-38 in CRC, compared to normal colonic mucosa, but it might be due to disturbance of colonic mucosal immunity in response(s) to long-term unknown stimuli, maybe in combination, including genetic, environmental and possible infectious factors (Ummarino 2017), in genetically susceptible individuals. Long-term dysregulated colonic mucosal immunity, i.e. compromised IL-38 production, contributes to a higher level of uncontrolled inflammation. Subsequently, chronic intestinal inflammation enhances colonic epithelial cell malignant transformation, and ultimately the development of CRC (Grivennikov 2013), in addition to promoting the growth and metastasis of CRC, which is well supported by the findings that a close correlation exists between chronic inflammatory status and the development of gastrointestinal cancer (Ullman and Itzkowitz 2011).

Subsequently, ROC curves have been utilised to confirm that IL-38 is a reliable and sensitive biomarker for distinguishing between CRC and non-cancer colonic tissue, i.e. AUC reaching 0.89 overall, while for the right-sided colon, it is 0.94; whereas it is 0.89 for the left. Moreover, the evaluation of AUROC of IL-38 for tumour differentiation shows that IL-38 is an excellent, very good or very good prediction biomarker for poorly-, moderately- or well-differentiated CRC (AUC=0.97, 0.89, or 0.89), respectively. The AUROC of IL-38 is supported by the finding from multivariate analysis, indicating that IL-38 is an independent predictor of prognosis and survival.

Overall survival is an objective parameter in determining diagnosis and prognosis. There is a positive correlation between colonic IL-38 in CRC and prognosis and/or overall survival, particularly in advanced CRC (patients with distant metastases, as well as the TNM III-IV classification). Therefore, such findings strongly support IL-38 probably being a reliable and consistent independent factor in predicting the prognosis of CRC.

Interestingly, there is very little published data that demonstrates the relationship between IL-38 and malignancy, except one study of lung adenocarcinoma, which shows an inverse correlation between IL-38 production and poor differentiation of lung adenocarcinoma (Takada et al. 2017), but only in PD-L1 negative lung carcinoma, perhaps as a consequence of the activity of T cell direct action, which can be suppressed by IL-38, which is involved in this sub-group of lung carcinoma patients.

Notably, completely opposite findings are observed between CRC and lung carcinoma. However, no direct comparison study has been performed to determine the underlying mechanism. One possible speculation is that the microenvironment in the gastrointestinal mucosal surface is almost completely different, in comparison to the lung, especially with their pattern of flora, despite both organs being classified as mucosal organs with so-called mucosal-associated lymphoid organs (Marsland et al. 2015). Surprisingly, there is no significant difference of colonic IL-38 in CRC when patients are stratified by TNM, using the Kruskal–Wallis H test, and multivariate analysis, but others have demonstrated a significant correlation between TNM and survival in CRC patients (Rogers et al. 2016). Historically, CRC has usually been diagnosed at a rather later stage due to lack of sensitive diagnostic approaches, which is acknowledged by the authors who have examined IL-38 and CRC, which has resulted in this section of the study being underpowered since there is a relatively small number in the TNM stage I sample size. Similarly, there is no significant difference in IL-38 expression and age or sex. The possible explanation is also due to insufficient numbers of samples in younger CRC patients, whereas no significance in sex might be due to the CRC patients being within groupings mainly over 73 years, who would have lost the benefit of oestrogen in reducing the incidence of CRC in women of fertile age (Lin and Giovannucci 2010).

Conclusions

We have summarised the potential presentation of IL-34, IL-36 and IL-38 expression in colorectal cancer with some clinical linkage. Such findings may be useful in exploring therapeutic targeting for precision medicine. To achieve this goal, the gaps missing between the precise underlying mechanism should be fulfilled. In future research, we should focus on the expression and/or production of these cytokines in CRC and their linkage with the subsets of colorectal cancers, i.e. sporadic colorectal cancer, familial adenomatous polyposis or hereditary non-polyposis colorectal cancer (Ponz de Leon and Percesepe 2000; Ponz de Leon et al. 2004). It has been demonstrated that subsets of colorectal cancers have distinguishing different genetic mutations. More precisely, different genetically manipulated animals as colorectal cancer models, as well as in human clinical samples, will be explored.

Funding information

This work is supported by SJTU 2019, the University of Sydney (SB).

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The authors declare that there is no conflict of interest

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