IRG1 restrains M2 macrophage polarization and suppresses intrahepatic cholangiocarcinoma progression via the CCL18/STAT3 pathway

Abstract

Intrahepatic cholangiocarcinoma (ICC) is a highly malignant and aggressive cancer whose incidence and mortality continue to increase, whereas its prognosis remains dismal. Tumor‐associated macrophages (TAMs) promote malignant progression and immune microenvironment remodeling through direct contact and secreted mediators. Targeting TAMs has emerged as a promising strategy for ICC treatment. Here, we revealed the potential regulatory function of immune responsive gene 1 (IRG1) in macrophage polarization. We found that IRG1 expression remained at a low level in M2 macrophages. IRG1 overexpression can restrain macrophages from polarizing to the M2 type, which results in inhibition of the proliferation, invasion, and migration of ICC, whereas IRG1 knockdown exerts the opposite effects. Mechanistically, IRG1 inhibited the tumor‐promoting chemokine CCL18 and thus suppressed ICC progression by regulating STAT3 phosphorylation. The intervention of IRG1 expression in TAMs may serve as a potential therapeutic target for delaying ICC progression.

We found that IRG1 expression remained at a low level in M2 macrophages. IRG1 overexpression can restrain macrophages from polarizing to the M2 type, thus inhibiting the proliferation, invasion, and migration of ICC, while IRG1 knockdown exerted the opposite effects. Mechanistically, IRG1 inhibited the tumor‐promoting chemokine CCL18, thus suppressing ICC progression by regulating STAT3 phosphorylation.

1. INTRODUCTION

Intrahepatic cholangiocarcinoma (ICC) is a notorious tumor whose incidence has increased from 0.44 cases per 100,000 to 1.18 per 100,000. An average annual percentage change of 2.3% has catapulted ICC into an urgent health threat. ¹ , ² ICC is a hot research field due to the rapid progression, unsatisfactory curative effect, and limited survival time of this tumor. Most patients are diagnosed at a late stage, and only 12%–40% of patients are referred to as resectable, which is accompanied by high recurrence and metastasis risks. ³ Systemic therapies, including chemotherapy, precision medicine‐based targeted therapy, and immunotherapy, have some effects but are not curative. ⁴ The search for better treatment methods depends on further in‐depth study of the mechanisms related to the occurrence and development of ICC.

During the complex development of ICC, cancer‐promoting pathways interact with inflammatory signals and remodeling of the tumor microenvironment (TME). ⁵ Tumor‐associated macrophages (TAMs) are a group of highly plastic immune cells in the TME. TAMs tend to exhibit M2 features and have versatile tumor promotion abilities that are involved in tumor initiation, progression, angiogenesis, and metastasis. ⁶ The interplay between TAMs and ICC has been well established. ICC can recruit and activate macrophages via secretion of MCP‐1/CCL2, CSF‐1, and VEGF‐A, and the infiltration of TAMs could be considered a prognostic factor. ⁷ , ⁸ TAMs act as tumor promotors relying on multiple cytokines, matrix proteins, and activating oncogenic pathways. ⁹ , ¹⁰ , ¹¹ In addition, interaction with other stromal cells enables TAMs to exert their immunosuppression and TME remodeling function. ¹² Because TAMs exert tumor‐promoting effects and are highly plastic, decoding the polarization secret of macrophages has become an urgent need for macrophage‐targeted therapy in cancer. ¹³

Immune responsive gene 1 (IRG1), also known as aconitate decarboxylase 1 (ACOD1), enables aconitate decarboxylase activity, encodes itaconate production, ¹⁴ , ¹⁵ and is a noteworthy metabolic remodeling site during macrophage activation. ¹⁶ , ¹⁷ The activation of IRG1 generates a large amount of itaconate. Itaconate blocks the conversion of succinate into fumarate by competitively inhibiting succinate dehydrogenase, which results in interruption of the tricarboxylic acid (TCA) cycle process, and this break of the TCA cycle is followed by the accumulation of succinate and a shift in the energy metabolism mode to glycolysis. ¹⁸ , ¹⁹ IRG1 and its metabolite can exert antibacterial, anti‐inflammatory, and antioxidant effects and thus affect tumor progression through a variety of pathways and mechanisms. ²⁰ , ²¹ , ²² , ²³ Here, we focused on the metabolic reprogramming function of IRG1 in macrophages. The reprogramming of the metabolic state will have an important further impact on the polarization direction and functional state of macrophages. However, little is known about the effect of IRG1 on TAMs in ICC.

In the present study, we first analyzed the expression profile of IRG1 in M1 and M2 macrophages. Moreover, we found that IRG1 can affect the polarization state of macrophages and affect the progression of ICC in vitro and in vivo. Further exploration indicated that IRG1 can inhibit CCL18 secretion by macrophages and thereby suppress the tumorigenic STAT3 pathway.

2. MATERIALS AND METHODS

2.1. Cell lines and cell culture

The human cholangiocarcinoma cell lines HCCC‐9810 and CCLP‐1 were purchased from Shanghai BioLeaf Biotech Co., Ltd., and human monocyte THP‐1 cells were purchased from the National Collection of Authenticated Cell Cultures. The cells were cultured in RPMI 1640 (Gibco™, Thermo Fisher Scientific) medium supplemented with 10% fetal bovine serum (Gibco™, Thermo Fisher Scientific) and 1% penicillin–streptomycin (Gibco™, Thermo Fisher Scientific). For CCL18 treatment experiments, ICC cells were treated with indicated conditioned medium (CM) with or without 20 ng/mL CCL18 (Peprotech) before conducting further functional experiments. The application concentration of CCL18 was determined based on previous research. ²⁴ , ²⁵ , ²⁶ , ²⁷

2.2. Macrophage polarization induction

THP‐1 cells were seeded in six‐well plates at 1 × 10⁶ cells per well. After 48 h of induction with 100 ng/mL PMA, the cells transformed from suspension into adherent cells, namely, resting macrophages (M0); M1‐type macrophages were then induced by further incubation for 24 h with IFN‐γ (PeproTech) (20 ng/mL) and LPS (Sigma) (100 ng/mL), whereas M2‐type macrophages were induced by incubation with IL‐4 (PeproTech) (20 ng/mL) for 24 h.

2.3. Lentivirus infection

Lentiviral vectors for human IRG1 gene overexpression (lenti‐IRG1) and downregulation (lenti‐shIRG1) were obtained from Hanbio. The corresponding empty vectors (lenti‐Con and lenti‐shcon) were used as controls. Transfection was performed following the instructions provided by the manufacturer. Successfully infected cells were selected with 1.5 μg/mL puromycin (Sigma–Aldrich) for 14 days. Selected cells were used for in vitro and in vivo experiments.

2.4. Wound‐healing assay

Intrahepatic cholangiocarcinoma cells were seeded in six‐well plates and cultured with conditioned medium or control medium overnight. The “scratch” wounds were created by straight scraping the cell layer with a pipette tip. Cells were imaged after scratching and cultured with CM for another 48 or 72 h.

2.5. Migration and invasion assay

Migration or invasion assays were performed using 24‐well plates inserted by 8‐μm‐pore‐size transwell filter insert (Corning) with or without precoating with 100 μL diluted Matrigel (300 μg/mL). A total of 4 × 10⁴ ICC cells with serum‐free medium were seeded into the upper chamber, and culture medium containing 10% FBS or conditioned medium was added to the lower chambers. After 48 h, cells on the underside of the membrane were immobilized with methanol and stained with 0.1% crystal violet. The number of penetrated cells was counted under an optical microscope.

2.6. Colony formation assay

A total of 800 cells were well distributed in each well of a six‐well plate and cultured for 14 days. The cells were then fixed with methanol and stained with 0.5% crystal violet for 30 min. The plate was gently washed with PBS and observed. A cell colony was considered to contain at least 30 cells.

2.7. Cell counting kit‐8 (CCK‐8) assay

Cell proliferation ability was tested with a Cell Counting Kit‐8 (CCK‐8) kit (Dojindo) according to the manufacturer's instructions. Briefly, ICC cells were seeded at a density of 1000 per well in a 96‐well plate, and CCK‐8 solution was added each day at the same time for a total of 4 days. The optical density value at 450 nm was determined.

2.8. Western blot

Cell protein was extracted using RIPA buffer (Beyotime) supplemented with a complete EDTA‐free protease inhibitor cocktail. The protein concentration was measured by the Bicinchoninic acid (BCA) method, and 30–40 μg of cell protein was denatured and separated by SDS polyacrylamide gels and then transferred to PVDF membranes (Invitrogen). The membrane was blocked with 5% milk for 1 h and then incubated overnight with primary antibodies at 4°C. Afterward, the membrane was incubated with secondary antibodies at room temperature (RT) for 1 h and visualized by the Odyssey® Imaging System (LI‐COR). Primary antibodies against the following target proteins were used: IRG1 (Cell Signaling Technology) (1:500), CD163 (Cell Signaling Technology) (1:1000), ARG1 (Immunoway) (1:1000), and GAPDH (Sigma–Aldrich) (1:8000).

2.9. Quantitative real‐time PCR

Total RNA was extracted according to the manufacturer's instructions using the AxyPrep Multisource Total RNA Miniprep Kit (Axygen Scientific, Inc.). RNA was reverse‐transcribed into cDNA with a qPCR RT Kit (TOYOBO). Real‐time PCR was performed using THUNDERBIRD SYBR qPCR Mix (TOYOBO). The mRNA level was calculated according to the 2^−ΔΔCt method after normalization to the GAPDH expression level.

2.10. Immunofluorescence staining

Cells were seeded on cover slides in 24‐well plates, incubated overnight with the corresponding CM, and washed with PBS. The cells were fixed in 4% paraformaldehyde for 20 min and gently washed. The cells were then blocked with 1% bovine serum albumin (BSA) for 1 h and incubated with the primary antibodies overnight and then with the secondary antibodies for 1 h at RT. Nuclei were stained with DAPI at RT for 5 min. The slides were backed‐off on glass slides and photographed by laser confocal microscopy.

2.11. Animal models

All experimental procedures involving animals were approved by The Animal Care and Use Committee of Harbin Medical University, China. Male BALB/c nude mice (aged 4–5 weeks) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. For the induction of subcutaneous xenograft tumors, the mice were randomly divided into the indicated groups (n = 6 per group). HCCC‐9810 cells (1 × 10⁷) together with 1 × 10⁶ macrophages were subcutaneously injected into the flanks of mice to form a subcutaneous model. ¹² All mice were well cared for with a free diet and comfortable padding. The tumor size was measured every 3 days with Vernier calipers, and the tumor volume was calculated using the following formula: volume = length × (width)² × 0.5. The mice were sacrificed after 5 weeks, and the tumor volume and weight were assessed by double‐blinded evaluation. For the orthotopic xenograft model, 3 × 10⁶ firefly luciferase‐transfected ICC cells and macrophages (1:1) were mixed and inoculated into the livers of nude mice. After 6 weeks, the tumor size was visualized with the NIGHTOWL LB983 system (Berthold Technologies).

2.12. Statistical analysis

The significance of the differences between different groups was determined by Student's t‐test or one‐way ANOVA. The results are presented as the means ± SEMs. A p‐value <0.05 was considered to indicate statistical significance.

3. RESULTS

3.1. IRG1 is downregulated in M2 macrophages

Many studies have demonstrated significant similarities between TAMs and M2‐type macrophages regarding their phenotypes and functions. Therefore, specific M2‐type macrophages with immunosuppressive and tumor‐promoting effects are often used as models to explore TAMs in vitro and in vivo. By searching the online website Human Protein Atlas (https://www.proteinatlas.org/), throughout human normalized single‐cell RNA (nTPM) from all single‐cell types, IRG1 is a cell‐specific gene that is mainly expressed in mono/macrophages (Figure 1A). ²⁸ However, IRG1 shared limited expression in the macrophage cluster when we searched the online ICC scRNA‐seq database TISCH (http://tisch.comp‐genomics.org/). The expression level of IRG1 is relatively low in the tumor‐associated mono/macrophage cluster of ICC tissue (Figure 1B). ²⁹ This finding aroused our attention and encouraged us to think of the profound meaning underlying the reduction in IRG1 in the context of TAMs. To explore this phenomenon, we induced traditional M0, M1, and M2 macrophages from the THP‐1 cell line as described previously. ³⁰ , ³¹ , ³² Briefly, after PMA‐induced THP‐1 adhesion (M0 macrophages), IL‐4 was used to induce M2 macrophages, whereas IFN‐γ and LPS were applied for M1 polarization. Typical morphological characteristics (Figure 1C) and representative markers of M1 (CD80, CD86, HLA‐DR) and M2 (ARG1, CD163, CD206) macrophages were assessed to demonstrate successful polarization (Figure 1D,E). Next, we explored the IRG1 expression profile of M0, M1, and M2 macrophages at both the mRNA and protein levels. The results indicated that IRG1 remained at a low level during M2 polarization but was more highly expressed in cells polarized to the M1 type compared with M0 (Figure 1F,G). To investigate the expression of IRG1 in ICC specimens, we performed immunohistochemical (IHC) staining of IRG1 and macrophage markers in human ICC and adjacent normal tissue samples. The results indicated that IRG1 tended to be highly expressed in CD86+ M1 macrophages in adjacent normal tissue. However, in tumor tissue with a very low proportion of CD86+ M1 macrophages, the expression level of IRG is relatively low. Especially in poorly differentiated ICC tissue, we can hardly detect the expression of IRG1 (Figure 1H). Therefore, we hypothesize that IRG1 may be a significant regulator of macrophage polarization. Collectively, these data demonstrate that IRG1 preferentially shows lower expression in M2‐like TAMs.

FIGURE 1.

Immune responsive gene 1 (IRG1) is downregulated in M2 macrophages. (A) Summary of IRG1 RNA and protein expression in different cell type based on date generated within the Human Protein Atlas Project. (B) Overview of IRG1 (ACOD1) expression in different cell clusters based on scRNA‐seq database TISCH. (C) Typical morphology of M0, M1, and M2 macrophages induced in vitro. (D) mRNA levels of the M1 markers CD80, CD86, and HLA‐DR in M0 and M1 macrophages. (E) mRNA levels of the M2 markers CD163, CD206, and ARG1 in M0 and M2 macrophages. (F) IRG1 mRNA levels in M0, M1, and M2 macrophages. (G) IRG1 protein levels in M0, M1, and M2 macrophages and quantification of the relative band densities. (H) Immunohistochemical (IHC) staining of CD68, CD86, and IRG1 in intrahepatic cholangiocarcinoma (ICC) and adjacent normal tissue specimens. All bar graphs show the means ± SEMs of three independent experiments performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001.

3.2. IRG1 attenuates M2 polarization of macrophages

Because the polarization state is the key determiner of macrophage function, we determined whether IRG1 could have an impact on M2‐like macrophage polarization. We transfected IRG1‐overexpression and ‐knockdown lentiviruses into THP‐1 cells, and the transfection efficiency was confirmed at the mRNA and protein levels after the infected cells had been induced to polarize into M2 macrophages using PMA and IL‐4 as described previously (Figure 2A–D). To determine the relationship between IRG1 expression and macrophage polarization, we assessed M2‐specific markers, and as demonstrated by qPCR and Western blotting, IRG1 knockdown and IRG1 overexpression slightly increased and markedly decreased M2 marker expression, respectively, under the same M2 polarization induction conditions (Figure 2E–H). Consistent results were obtained with immunofluorescence images, which showed that IRG1 overexpression induced fewer ARG1‐positive M2 macrophages, whereas IRG1 knockdown exerted the opposite effect (Figure 2I,J). Meanwhile, we also analyzed the M0 and M1 marker expression levels under IL‐4 induction. The expression level of IRG1 did not affect the expression of M0 marker. Interestingly, IRG1 could slightly promote M1 polarization (Figure S1A–C). Taken together, these results suggest that IRG1 overexpression impairs the polarization of THP‐1 cells to M2‐like macrophages.

FIGURE 2.

Immune responsive gene 1 (IRG1) attenuates M2 polarization of macrophages. (A, B) The IRG1 overexpression efficiency was measured by qPCR (A) and Western blot analysis (B), and the relative protein density was determined. (C, D) qPCR and Western blot analysis of the IRG1 knockdown efficiency. (E, F) M2 marker mRNA and protein expression levels in the IRG1‐overexpression and control groups and quantification of the protein density. (G, H) M2 marker mRNA and protein expression levels in the IRG1 knockdown and control groups and relative band density. (I) Immunofluorescence labelling of ARG1 after M2 polarization induction in the IRG1‐overexpression and control groups. (J) Immunofluorescence labelling of ARG1 after M2 polarization induction in the IRG1‐knockdown and control groups. All bar graphs show the means ± SEMs of three independent experiments performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001.

3.3. Macrophage‐expressed IRG1 inhibits ICC cell progression

Tumor‐associated macrophages have been well recognized for their tumor‐promoting effects. According to the above results, we postulated that IRG1 might promote tumor progression by modulating macrophage polarization. To validate this hypothesis, the progressiveness of ICC cells (both CCLP‐1 and HCCC‐9810 cells) was analyzed after culturing with CM from IRG1‐knockdown (shIRG1 CM) or IRG1‐overexpressing M2 macrophages (IRG1 CM). We found that compared with the blank group without adding CM, the proliferation, migration, and invasion abilities of ICC cells were enhanced after culturing with CM from control M2‐like macrophages (Con CM). However, the promotion ability was attenuated by CM from IRG1‐overexpressing M2‐like macrophages. Specifically, transwell and wound‐healing assays indicated that ICC cells from the IRG1‐overexpression CM group had less invasion and migration potential than those from the control M2 CM group. Opposite results were obtained with the IRG1‐knockdown group (Figure 3A–D). In addition, cell counting and colony formation assays revealed that IRG1 overexpression attenuated the proliferation ability of HCCC‐9810 and CCLP‐1 cells compared with that of control M2 macrophages (Figure 3E–H). Based on these classical experiments, we can conclude that the IRG1 expression profile affects ICC aggressiveness by regulating macrophage polarization.

FIGURE 3.

Macrophage‐expressed immune responsive gene 1 (IRG1) inhibits intrahepatic cholangiocarcinoma (ICC) progression. (A, B) Transwell experiments verified the migration and invasion of ICC cells cultured with conditioned medium (CM) from IRG1‐overexpressing (A) or IRG1‐knockdown (B) M2 macrophages. (C, D) Wound‐healing images of ICC cells cultured with CM from IRG1‐overexpressing (C) or IRG1‐knockdown (D) M2 macrophages. (E, F) ICC cell proliferation in the different CM‐supplemented groups was assessed by the CCK‐8 assay. (G, H) Representative images and colony number quantification of the colony formation experiment after ICC cells were cultured with CM from IRG1‐overexpressing (G) or IRG1‐knockdown (H) M2 macrophages. All bar graphs show the means ± SEMs of three independent experiments performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001.

3.4. Macrophage‐expressed IRG1 suppresses ICC oncogenesis in vivo

Considering the encouraging results described above, we used a mouse xenograft model to investigate the influence of IRG1 on ICC growth in vivo. We adopted a continuous injection method to simulate the internal environment as previously described (Figure 4A). ¹² After subcutaneous injection with mixed macrophages and ICC cells, macrophages were injected into the tumor centre every 3 days until macroscopic tumors grew to a proper volume. As expected, compared with the control M2 macrophage group, the size and weight of tumors were significantly smaller in the IRG1‐overexpression group (Figure 4B,C), whereas the IRG1‐knockdown group (Figure 4E,F) showed more malignant tumor development. Tumor growth curves also indicated that IRG1‐overexpressing macrophages significantly slowed the growth rate of tumors compared with that of the control group, whereas IRG1 knockdown had the opposite effect (Figure 4D,G). To further determine the effect of IRG1 in vivo, an orthotopic xenograft model was constructed. Firefly luciferase‐labeled ICC cells with either IRG1‐overexpressing or IRG1‐knockdown M2 macrophages were coinjected intrahepatically (Figure 4H). As expected, the bioluminescence results also indicated that IRG1‐overexpressing macrophages exerted a higher inhibitory effect on ICC growth than IRG1‐knockdown macrophages (Figure 4I,J). Collectively, IRG1 overexpression in macrophages is a stimulating force for ICC inhibition in vivo.

FIGURE 4.

Macrophage‐expressed immune responsive gene 1 (IRG1) suppresses intrahepatic cholangiocarcinoma (ICC) oncogenesis in vivo. (A) Schematic of the ICC mouse subcutaneous xenograft model. (B) Images of tumors derived after subcutaneous injection of HCCC‐9810 cells alone or mixed with IRG1‐overexpressing M2 macrophages. (C) The weights of the tumors were quantified. (D) The tumor growth curve in the Con and IRG1 groups was measured every 3 days. (E) Images of tumors derived after subcutaneous injection of HCCC‐9810 cells alone or mixed with IRG1‐knockdown M2 macrophages. (F) The weights of the tumors were quantified. (G) The tumor growth curve of the shcon and shIRG1 groups was measured every 3 days. (H) Schematic of the ICC mouse orthotopic xenograft model. (I, J) Representative images of the orthotopic xenograft model and volumes of orthotopic tumors. All bar graphs show the means ± SEMs of three independent experiments performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001.

3.5. CCL18 contributes to the tumor‐promoting effect of IRG1

Secreted cytokines are the most classical and straightforward connection between macrophages and tumor cells. We hypothesized that IRG1 might regulate the secretion of protumor factors in response to cues from potential IRG1‐related cytokines. To validate this hypothesis, we selected several TAM‐related cytokines and validated their connection with IRG1 in the STRING database (Figure 5A). ³³ As shown in the network diagram, IRG1 is related to a cluster of cytokines, among which IL‐6, IL‐10, CCL18, MMP9, and IL‐17a are typical factors secreted by TAMs. We first validated the expression level of these candidate cytokines at the mRNA level. ³⁴ , ³⁵ , ³⁶ We found that tumor‐promoting cytokines, including IL‐10, CCL18, MMP9, and IL‐17a, were present in IRG1‐overexpressing M2 macrophages (Figure 5B). Among them, a notable change in the CCL18 levels drew our attention because it is a marker cytokine of M2 macrophages whose function has not been well established in ICC. CCL18 from TAMs has been proven to promote cancer progression, including breast cancer, ovarian cancer, and pancreatic ductal adenocarcinoma. ³⁷ , ³⁸ , ³⁹ However, whether CCL18 plays a role in ICC remains a mystery. Further ELISA confirmed that the CCL18 levels were significantly decreased in IRG1‐overexpressing M2 macrophage CM compared with the control group (Figure 5C). The inhibition of tumor malignancy in the IRG1‐overexpressing CM group was reversed by exogenous addition of CCL18 (Figure 5D–G). The above results indicate that IRG1 in M2 macrophages exerts a tumor promotion effect via CCL18 secretion.

FIGURE 5.

CCL18 contributes to the tumor‐promoting effect of immune responsive gene 1 (IRG1). (A) Protein–protein interaction network of IRG1 and several M2‐related cytokines. (B) qPCR validation of potential cytokines. (C) Levels of secreted CCL18 in the conditioned medium (CM) of the Con and IRG1‐overexpression groups. (D) Migration and invasion ability of CCLP‐1 and HCCC‐9810 cells after exogenous addition of CCL18. (E) Representative images of the wound‐healing assay after intrahepatic cholangiocarcinoma (ICC) cells were cultured with IRG1 CM containing CCL18. (F) The growth curves of ICC cells were tracked by the CCK‐8 assay. (G) Colony formation images of ICC cells after treatment with CCL18 and colony number counts. All bar graphs show the means ± SEMs of three independent experiments performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001.

3.6. IRG1 regulates STAT3 phosphorylation in ICC cells via CCL18

CCL18 exerts a variety of tumor‐promoting effects by activating downstream pathways. Reportedly, CCL18 is closely related to the PI3K/AKT, NF‐kB, and JAK2/STAT3 signaling pathways. ³⁹ , ⁴⁰ , ⁴¹ Previous research has proven that tumor‐associated neutrophils (TANs) and TAMs interact to promote ICC progression by activating STAT3. ¹² Therefore, we measured the STAT3 phosphorylation level and found that p‐STAT3 was upregulated in response to M2 macrophage CM. IRG1‐overexpression CM weakened p‐STAT3 activation, and the effect could be restored by exogenous addition of CCL18 (Figure 6A,B). Collectively, our results suggest that the p‐STAT3 activation status in ICC is a consequence of IRG1‐regulated CCL18 secretion (Figure 6C).

FIGURE 6.

Immune responsive gene 1 (IRG1) regulates STAT3 phosphorylation in intrahepatic cholangiocarcinoma (ICC) cells via CCL18. (A) Western blot analysis of the STAT3 and p‐STAT3 levels after CCLP‐1 and HCCC‐9810 cells were cultured with conditioned medium (CM) with or without CCL18 addition. (B) Relative p‐STAT3 protein density quantification. (C) Schematic depicting the contribution of IRG1 in M2 macrophages to ICC progression.

4. DISCUSSION

ICC exhibits striking malignancy accompanied by very low therapy efficiency and poor prognosis. Previous studies have well established that abundant infiltration of TAMs is often accompanied by worse tumor behavior. ⁴² However, macrophages are confirmed to play paradoxical roles in different disease patterns. In tumor disease, macrophages are believed to be educated by the TME and exert immunosuppressive and tumor‐promoting effects. ³⁵ However, in many inflammation‐related diseases, such as IBD or rheumatoid arthritis, macrophages tend to exhibit a proinflammatory M1 type. ⁴³ , ⁴⁴ These phenomena remind us that macrophages are highly plastic and can be educated either by disease heterogeneity or manpower. We have thoroughly unveiled many important mechanisms of macrophage polarization, particularly its distinct characteristics among general M1 and M2 types. ⁴⁵ It is vital to determine an operable method to regulate the polarization of macrophages. The secret may lie in their internal metabolic modification in light of the distinct metabolic pathways between M1 and M2 macrophages. ⁴⁶ M1 macrophages are fond of glycolytic metabolism, whereas M2 macrophages are characterized by increased mitochondrial respiration. ⁴⁶ , ⁴⁷ Whether the TCA cycle can be unaffected is crucial for the polarization state of macrophages. ¹⁹ Breaking or blocking any node of this cycle will prompt metabolic reprogramming. Therefore, macrophages can adopt different survival modes and show corresponding functional differences. ⁴⁸ IRG1, which encodes itaconate, is considered one break point of the TCA cycle. ⁴⁹ Itaconate can inhibit the activity of succinate dehydrogenase in mitochondria and thereby inhibits the mitochondrial TCA cycle. IRG1 drives metabolite reprogramming of macrophages, and the fate of macrophages is thus obviously changed. ⁵⁰ , ⁵¹

In this study, we demonstrated that macrophages are an essential driving force in ICC progression. IRG1 functions as a polarization regulator in macrophages and thereby influences the progression of ICC. More specifically, IRG1 expression in M2‐like TAMs is markedly lower than that in M1 macrophages. The overexpression of IRG1 inhibited the M2 polarization of macrophages, and IRG1 knockdown exerted the opposite effect. As such, IRG1‐overexpressing macrophages can inhibit the malignant progression of ICC cells both in vitro and in vivo. Further investigation indicated that IRG1 may affect the secretion of cytokines from macrophages. Among these cytokines, CCL18 exhibited a significant reduction in the context of IRG1 overexpression. Further investigation revealed that the tumor promotion effect of CCL18 was related to increased STAT3 phosphorylation levels.

Macrophages exposed to inflammatory stimuli including LPS undergo metabolic reprogramming to increase IRG1‐dependent itaconate. ⁵² Meanwhile, Runtsch et al. proved that the IRG1‐coding protein itaconate could inhibit JAK1/STAT6‐mediated macrophage M2 polarization. ⁵⁰ This endows IRG1 with therapeutic potential for inflammation‐related diseases, but little is known in cancer. Recently, researchers have focused on the immune regulation effect of IRG1. Deletion of IRG1 in mice reversed the immunosuppressive function of TAMs and enhanced the function of antitumor CD8+ T cells. ⁵³ , ⁵⁴ , ⁵⁵ Besides, some studies focused on the effects of tumor cell‐derived IRG1. Tumor cell‐derived IRG1/itaconate can promote hepatocyte apoptosis‐related hepatocarcinogenesis. ⁵⁶ Conversely, IRG1/itaconate controls metabolic homeostasis and suppresses ER‐positive breast cancer growth. ⁵⁷ From the perspective of our study, we revealed the role of IRG1 in tumor progression from the perspective of intratumoral macrophage regulation. Focusing on macrophage–tumor cell interaction, we believe that targeting IRG1 to reshape macrophage can have a positive therapeutic effect.

Mechanically, we found that IRG1 affects the secretion of chemokine CCL18. CCL18 is an important factor for TAM‐mediated immunosuppressive and protumor function. ⁵⁸ CCL18 has been proven to recruit CD4+ T cells and to be directly involved in tumor invasion, migration, epithelial‐to‐mesenchymal transition, and angiogenesis. ³⁴ , ⁵⁹ , ⁶⁰ , ⁶¹ Existing research based on 14 pairs of ICC tumors and nontumor liver tissues indicates that infiltration of CCL18+ macrophages is often associated with an immunosuppressive microenvironment. ⁶² The specific regulatory mechanism of CCL18 in ICC has not been revealed yet. Here we found that the secretion of TAM‐specific CCL18 may be regulated by IRG1. Further exploration reveals that promoting STAT3 phosphorylation may be the mechanism by which CCL18 exerts its cancer‐promoting effect. Besides, IRG1 can inhibit the production of various cancer‐promoting cytokines except for CCL18 (IL‐10, MMP9, and IL‐17a), which has positive therapeutic significance for ICC.

The optimal strategy for macrophage‐targeted therapy is reversing the tumor‐promoting polarization of macrophages and restoring their antigen‐presenting and phagocytosis function. Existing therapeutic strategies targeting macrophages can be broadly divided into the following categories: eliminating macrophages or inhibiting the recruitment of macrophages, inhibiting or reversing the cancer‐promoting polarization of macrophages, blocking the functional approaches of macrophages, and targeting immunosuppressive molecules. ⁶³ , ⁶⁴ , ⁶⁵ , ⁶⁶ , ⁶⁷ , ⁶⁸ , ⁶⁹ Chemokine inhibitors, CSF1R inhibitors, and anti‐CD47/SIRPα antibodies are considered promising TAM‐targeted therapies. ⁷⁰ Chimeric antigen receptor (CAR) macrophages targeting HER2 have also successfully decreased tumor burden by promoting M1 polarization and boosting anti‐tumor T cell activity. ⁷¹ Utilizing existing biotechnology based on macrophage modification, the potential clinical value of itaconate could be revisited. Perhaps IRG1 could be constructed as a CAR component that helps macrophages resist tumor‐promoting polarization and exert a comprehensive antitumour effect. In addition, nano‐targeted drug delivery systems (nano‐TDDSs) have emerged as a promising strategy for macrophage remodeling. Derivatives of itaconate, such as dimethyl itaconic acid (DI), 4‐octyl itaconic acid (4‐OI), and 4‐ethyl itaconic acid (4‐EI), delivered by nano‐TDDSs may be worth studying. ⁷²

In conclusion, our study revealed that IRG1 plays an important role in inhibiting macrophage polarization toward the M2 phenotype. Furthermore, IRG1 may decrease CCL18 secretion, which would inhibit STAT3 phosphorylation in ICC and thus exert its tumor‐suppressing effect. Targeting IRG1 or itaconate may have immense potential in macrophage‐targeted therapy.

AUTHOR CONTRIBUTIONS

Menghua Zhou: Conceptualization; data curation; formal analysis; investigation; methodology; validation; visualization; writing – original draft; writing – review and editing. Hongjun Yu: Investigation; methodology; resources; writing – review and editing. Miaoyu Bai: Investigation; methodology; resources; writing – review and editing. Shounan Lu: Methodology; resources; writing – review and editing. Chaoqun Wang: Methodology; resources; writing – review and editing. Shanjia Ke: Methodology; resources; writing – review and editing. Jingjing Huang: Resources; writing – review and editing. Zihao Li: Resources; writing – review and editing. Yanan Xu: Resources; writing – review and editing. Bing Yin: Resources; writing – review and editing. Xinglong Li: Resources; writing – review and editing. Zhigang Feng: Resources; writing – review and editing. Yao Fu: Resources; writing – review and editing. Hongchi Jiang: Project administration; resources; supervision; writing – review and editing. Yong Ma: Conceptualization; funding acquisition; methodology; project administration; supervision; writing – review and editing.

FUNDING INFORMATION

This work was jointly supported by the Harbin Medical University Postgraduate Innovation and Practical Research Project (YJSCX2020‐28HYD), Scientific Foundation of the First Affiliated Hospital of Harbin Medical University (2019 L01, HYD2020JQ0007, HYD2020JQ0011), Heilongjiang Postdoctoral Foundation (LBH‐Z11066, LBH‐Z12201 and LBH‐Q17097), China Postdoctoral Science Foundation (2012 M510990, 2012 M520769 and 2013 T60387), Chen Xiaoping Foundation for the Development of Science and Technology of Hubei Province (CXPJJH122002‐025), Natural Science Foundation of Heilongjiang Province of China (LC2018037), and National Natural Scientific Foundation of China (81100305, 81470876, 82370643 and 81270527).

CONFLICT OF INTEREST STATEMENT

The authors have no conflict of interest.

ETHICS STATEMENT

Approval of the research protocol by an Institutional Reviewer Board: All procedures followed were in accordance with the ethical standards of the institutional review board of the First Affiliated Hospital of Harbin Medical University.

Informed Consent: All clinical samples used in this study were obtained from the First Affiliated Hospital of Harbin Medical University, and written informed consent was obtained from the patients and/or their immediate relatives using recognized guidelines.

Registry and the Registration No. of the study/trial: N/A.

Animal Studies: All animal experimental protocols were approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University (No. IRB‐AF/SC‐04/01.0), and the experimental procedures were conducted in strict accordance with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Supporting information

Figure S1.

Zhou M, Yu H, Bai M, et al. IRG1 restrains M2 macrophage polarization and suppresses intrahepatic cholangiocarcinoma progression via the CCL18/STAT3 pathway. Cancer Sci. 2024;115:777‐790. doi: 10.1111/cas.16068

DATA AVAILABILITY STATEMENT

Data are available on request from the authors.