DC-derived IL-27 suppresses anti-tumor immunity via the modulation of gut microbiota

doi:10.21203/rs.3.rs-1452354/v1

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Research Article

DC-derived IL-27 suppresses anti-tumor immunity via the modulation of gut microbiota

https://doi.org/10.21203/rs.3.rs-1452354/v1

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Background

Chronic inflammation has been shown to be crucial in promoting tumor initiation and development in many cancer types, including hepatocellular carcinoma (HCC). It is characterized by the persistent expression of inflammatory cytokines surrounding the tissues. IL-27 is a heterodimeric cytokine that consists of EBI3 and p28, which can be expressed by many antigen presenting cells, including monocytes, macrophages, and dendritic cells (DCs). The role of IL-27 in tumor development is controversial. Gut microbiota plays an important role in immune system development and homeostasis, while how it can be regulated to impact anti-tumor immune response is poorly understood.

Results

We demonstrated a tumor-promoting role of DC-derived IL-27 via the modulation of gut microbiota in murine models. IL-27p28 DC-conditional knockout (Itgax-IL-27p28^f/f) mice exhibited reduced tumor growth compared with WT controls. T cell anti-tumor response was enhanced, and inhibitory receptor expression was decreased. Interestingly, when WT mice received fecal transplant from Itgax-IL-27p28^f/f mice, tumor growth was significantly inhibited. Their gut microbiota exhibited distinct features and gavage of bifidobacterium, a bacteria strain that was greatly increased in Itgax-IL-27p28^f/f mice, reduced tumor growth. Furthermore, MHC class II expression on tumor necrosis factor and inducible nitric-oxide-synthase-producing DCs (TIP-DCs) was increased in Itgax-IL-27p28^f/f mice, and their activation could be inhibited by the gut microbiota modulated by IL-27. Their function of activating CD8⁺ T cells was also enhanced in the absence of DC-derived IL-27.

Conclusion

DC-derived IL-27 could promote tumor development and suppress anti-tumor immune response by inhibiting the function of TIP-DCs via the modulation of gut microbiota. This indirect effect of IL-27 on adaptive immune response provides insights into possible organ-specific functions of IL-27 and targeting IL-27 in tumor immunotherapy.

IL-27p28

T cells

anti-tumor immunity

TIP-DCs

gut microbiota

Tumor development is considered as an evolution of complex interactions between the tissue/tumor cells and tumor microenvironment¹. Many factors affect tumor initiation and development, such as aging, cancer-inducing substances, infectious agents, radiation and diet². Chronic inflammation has been shown to be crucial in promoting tumor initiation and development in many cancer types, including HCC³. It is characterized by the persistent expression of inflammatory cytokines surrounding the tissues. The expression and signaling dysregulation of IL-6 family cytokines, consisted of IL-6, IL-11, IL-31, ciliary neurotrophic factor (CNTF), cardiotrophin1 (CT1) and cardiotrophin-like cytokine factor 1(CLCF1), leukemia inhibitory factor (LIF) and oncostatin M⁴, in the tumor microenvironment were frequently associated with cancer progression and poor clinical outcomes^5–9. On the other hand, IL-12 is considered as one of the most potent cytokines in mediating anti-tumor responses¹⁰ by inducing Th1 responses and enhancing IFN-γ production, as well as promoting NK function. IL-27 was initially discovered as a member of IL-12 family¹¹, and later regarded as a member of IL-6 family by phylogenetic analysis¹². Its function in tumor progression and anti-tumor immune response is still not clear.

IL-27 is a heterodimeric cytokine that consists of EBI3 and p28, which can be expressed by many antigen presenting cells, including monocytes, macrophages and DCs¹¹. By binding to WSX-1 and gp130, IL-27 transduces intracellular signaling via STAT1 and STAT3 activation¹³. IL-27 was initially shown to be a pro-inflammatory cytokine that could promote Th1 responses by activating naïve CD4⁺ T cells and NK cells to produce IFN-γ^13–15. The pro-inflammatory function of IL-27 was later challenged by evidence that IL-27 signaling could be critical in regulating pro-inflammatory cytokine production and suppressing T cell hyperactivity during infections^16,17. Subsequently, many studies have demonstrated the anti-inflammatory functions of IL-27 in various animal models, such as Toxoplasma gondii infection, experimental autoimmune encephalomyelitis (EAE), and chronic inflammatory bowel disease (IBD)^18–20. Furthermore, IL-27 is also found to promote the expression of inhibitory receptors on T cells, such as Tim-3, LAG-3, and PD-1^21–23, indicating a possible mechanism for its immune suppressive function.

The role of IL-27 in tumor development is controversial. IL-27 has been shown to promote anti-tumor immune response by enhancing Th1 and CTL responses in TBJ neuroblastoma and colon carcinoma^24,25. Other studies also indicate that IL-27 could promote NK or NKT cell activation, which results in reduced tumor growth^26,27. However, IL-27 signaling could drive Tim-3 and IL-10 expression and T cell dysfunction in B16 melanoma²⁸. Moreover, IL-27 is shown to confer a pro-tumorigenic activity by suppressing T cell function via CD39²³. Therefore, the function of IL-27 in anti-tumor immune response is still not clear and need further investigation.

The gut microbiota plays an important role in immune system development^29,30 and homeostasis^31,32. It has been shown that maternal microbiota could shape early postnatal innate immune development³³. The lack of Bifidobacterium is associated with systemic and intestinal inflammation and immune dysregulation during the first months of life³⁴. Many studies have demonstrated the link between gut microbiota and tumor development³⁵. Studies using germ-free animals revealed tumor-promoting effects of the microbiota in variant cancers, including colorectal cancer³⁶ and gastric cancer³⁷. Similar conclusion was made by using antibiotics that deplete intestinal microbiota^38,39. Anti-tumor effects of some commensal bacteria strains have also been suggested. Commensal Bifidobacterium could promote anti-tumor immune responses and facilitate anti-PD-L1 treatment efficacy in murine B16 melanoma model⁴⁰. The immune modulatory function of gut microbiota during anti-PD1 therapy was also confirmed in human melanoma patients⁴¹. The role of microbiota in tumor development and therapy has been studied in many tumor types, such as sarcomas, non-small cell lung cancer, renal cell carcinoma and lymphoma^42–44. Due to the anatomical and functional relationship between liver and gut, gut microbiota has been suggested to impact HCC development^45,46. It has been shown that fecal microbial diversity is increased in early HCC patients⁴⁷, and patients with primary HCC exhibit increased proteobacteria, a pro-inflammatory bacteria strain, in their fecal microbiota⁴⁸. Nevertheless, how gut microbiota can be regulated and how it can impact anti-tumor immune response are poorly understood.

DC-derived IL-27 is especially important in regulating anti-tumor immune response as DCs are essential in modulating adaptive immunity. In this study, we investigated the role of DC-derived IL-27p28 in tumor development in murine HCC models by using Itgax-IL-27p28^f/f mice. We found that tumor development was significantly reduced in the Itgax-IL-27p28^f/f mice and Th1/CTL responses were promoted in the absence of DC-derived IL-27p28. This enhanced anti-tumor response was dependent on both CD4⁺ and CD8⁺ T cells. Moreover, DC-derived IL-27 may modulate gut microbiota to affect the anti-tumor adaptive immune responses. Further studies showed that the activation of TIP-DCs could be inhibited by the gut microbiota modulated by IL-27, which resulted in suppressed T cell proliferation and function. Our results demonstrated the role of DC-derived IL-27p28 in tumor development and its function in regulating adaptive immune response via modulating gut microbiota.

Cell line

The murine HCC cell line hepa1-6 was obtained from the American Type Culture Collection (Manassas, VA) and cultured in DMEM with 10% fetal bovine serum (Gibco, Gaithersburg, MD). The murine HCC cell line hepa1-6 has recently been authenticated by mouse cell line authentication service provided by Applied Biological Materials (Richmond, BC, Canada).

Mice and murine HCC models

C57BL/6 Itgax-IL-27p28f/f mice and C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-I mice) were provided by Dr. Zhinan Yin (Jinan University, Guangzhou, China). All mice were housed in specific-pathogen-free facilities and in accordance with the National Animal Care and Use Committee. All animal experiments were approved by the Institutional Laboratory Animal Care and Use Committee of Soochow University (Suzhou, China).

Two murine HCC models were established in the study. To generate the orthotopic HCC model by hydrodynamic injection, 1 × 10⁶ hepa1-6 cells in 2 ml PBS were injected via tail vein within 8 to 10 s. Mice were sacrificed 3 weeks later, and tumor nodules were counted. The DEN-induced spontaneous HCC model was generated by injecting 14-day-old C57BL/6 mice with 25 mg/kg DEN (Sigma-Aldrich, St Louis, MO). Eight months later, mice were sacrificed, and the livers were removed. Tumor size was measured, and the number of tumor nodules was counted.

Flow cytometry

For cell-surface staining, cells were blocked with CD16/32 FcR-block (BioLegend, San Diego, CA) for 15 min, and stained with fluorescent dye-conjugated mAb for 30 min at 4°C. For intracellular cytokine staining, cells were stimulated for 4–6 h with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of brefeldin A (10 µg/ml; BD Bio- sciences, San Jose, CA). Cells were stained for surface markers for 30 min, then fixed with 4% paraformaldehyde, permeabilized with 1% saponin (Sigma-Aldrich, St Louis, MO), and stained for cytokines for 30 min at 4°C. The antibodies used for FACS staining were FITC-anti-mouse-NK1.1 (PK136), PE/CF594-anti-mouse-CD3 (145-2C11), PE/CF594-anti-mouse-CD45 (30-F11), PerCP/Cy5.5-anti-mouse-CD4 (RM4–5), PerCP/Cy5.5-anti- mouse-NK1.1 (PK136), PE/CY7-anti-mouse-CD19 (1D3), APC/H7-anti-mouse-CD4 (GK1.5), and ACP/Cy7-anti-mouse-CD11b (M1/70) were purchased from BD Bioscience (San Jose, CA). The FITC-anti-mouse-CD8 (53–6.7), FITC-anti-mouse-I-A/I-E (M5/114.15.2), FITC- anti-mouse-CD4 (GK1.5), PE-anti-mouse-Ly6G (1A8), PE-anti- mouse-CD4 (RM4-5), PE-anti-mouse-NK1.1 (PK136), PE-anti-mouse-IFN-γ (XMG1.2), PE-Rat-IgG2b, kappa Isotype (RTK4530), PE/Dazzle594-anti-mouse-CD45 (30-F11), PerCP/Cy5.5-anti-mouse-TCRγδ (GL3), PerCP/Cy5.5- anti-mouse-IL-17A (TC11-18H10.1), PerCP/Cy5.5-anti-mouse-CD45.1 (A20), APC-anti-mouse-CD8 (53–6.7), APC-anti-mouse-CD4 (RM4-5), APC-anti-mouse-F4/80 (BM8), Alexa fluor 700-anti-mouse-CD45 (30-F11), APC-anti-mouse- IFN-γ (XMG1.2), PE/Cy7-anti-mouse-TNF-α (MP6-XT22), and PE/Cy7- anti-mouse-Ly6C(HK1.4), PE/Cy7-anti-mouse-CD44 (IM7), APC/Cy7-anti-mouse-CD11b (M1/70), APC/Cy7-anti-mouse-CD62L (MEL-14), Brilliant Violet421-anti-mouse CD8a (53 − 6.7), Brilliant Violet510-anti-mouse CD3ε (145-2C11), Brilliant Violet510-anti-mouse CD11c (N418) were purchased from BioLegend (San Diego, CA).

Neutralization of IL-27 in vivo.

To neutralize IL-27 in vivo, mice were injected i.p. with anti-IL-27p28 50ug (R&D Systems, Minneapolis, MN) per mouse immediately after the tumor cell inoculation on day 0 and one week later. Monoclonal goat IgG (Bio X Cell, Lebanon, NH) were used as isotype control.

Fecal transplantation

Fecal pellets from WT and Itgax-IL-27p28^f/f mice were collected, and each fecal pellet was resuspended in 1ml PBS. The suspension from each fecal pellet was used for oral gavage of one recipient mouse at 200ul per gavage. Mice were gavaged with WT or Itgax-IL-27p28^f/f mice fecal suspension or PBS as control every other day for one week prior to tumor inoculation and once a week on day 7 and day 14 post tumor implantation.

Bacterial gavage

Bifidobacterium species including B. bifidum, B. longum, B. lactis and B. breve were purchased from China General MicroBiological Culture Collection Center (BeiJing, China). Bacteria were cultured in an anaerobic chamber according to manufacturer's instructions. To confirm the compositions, bacteria were harvested and washed with PBS and centrifuged at 3000 rpm for 30 minutes. The pellet was sent for sequencing (GENEWIZ, Suzhou, China). For bacteria transplantation, a cocktail of Bifidobacterium species was resuspended in PBS at 5x10⁹ CFU/ml. Each mouse was given 200µl of Bifidobacterium (1x10⁹ CFU/mouse) by oral gavage with the same schedule as fecal transplant.

Fecal sample processing, sequencing, and analysis

Mice feces were collected into autoclaved EP tube and stored at − 80°C. Extracted DNA samples were amplified, DNA libraries were constructed, and the sequencing 16S rRNA was performed on Illumina MiSeq platform (GENEWIZ, Beijing, China). Sequences were grouped into operational taxonomic units (OTUs) using the clustering program VSEARCH (1.9.6) against the Silva 132 database pre-clustered at 97% sequence identity. The Ribosomal Database Program (RDP) classifier was used to assign taxonomic category to all OTUs at confidence threshold of 0.8. The RDP classifier uses the Silva 132 database which has taxonomic categories predicted to the species level. Base on the OUT analysis results obtained, Shannon index and Chao1 index, coordinates analysis (PCoA) were conducted by R package to display microbiome space between samples. A heatmap of the identified key variables was completed by the Heat map builder.

Bifidobacterium bacteria were harvested and centrifuged to collect the pellet. Bacterial 16S rRNA were amplified and sequenced (GENEWIZ, Beijing, China). Briefly, 16S rRNA was amplified from extracted DNA using broad-range bacterial primers 27F and 1492R. Amplified products from samples were verified by gel electrophoresis using the PCR reaction mixture in agarose gels. The 16Sr RNA was sequenced by Sanger Sequencing. The sequencing data was blasted in NCBI database for confirmation.

Activation of T cells with OVA peptide-pulsed TIP-DCs

TIP-DCs (CD45 + CD11b + Ly6C+) were sorted using a FACSAria III flow cytometer (BD Biosciences, San Jose, CA) and pulsed with 10 µg/ml OVA peptide for 2 hr at 37° in a total volume of 1 ml, then washed four times in RPMI-1640. In addition, CD8⁺ T cells from OT-I mice were separated by using mouse CD8 isolation cocktail kits, respectively (Stem cell, Vancouver, BC). CD8⁺ T cells in the suspension were then isolated and co-cultured with TIP-DC pulsed with or without OVA peptides (10 ug/ml, Sigma-Aldrich, St. Louis, MO) in a 96-well at a ratio of 3:1. After three days, production of IFN-γ and TNF-α by CD8 + T cells was analyzed by flow cytometry.

T cell proliferation assays

CD8⁺ T cells from OT-I mice were separated by using mouse CD8⁺ isolation cocktail kits, respectively (Stem cell, Vancouver, BC). Sorted CD8⁺ T cells were labeled with CFSE (ThermoFisher, Grand Island, NY) according to the manufacturer's instructions. Briefly, cells were resuspended in 50ml PBS and centrifuged for 5 minutes at 300g. Cells were then resuspended in 10 ml of 5 µM CellTrace CFSE staining solution and incubated in a 37°C water bath for 20 minutes. Then 40 ml serum-free RPMI-1640 medium was added, and cells were incubated for 5 minutes. After incubation, cells were centrifuged for 5 minutes at 300g and resuspended in serum-free RPMI-1640 medium. CFSE-labeled CD8⁺ T cells were seeded in anti-CD3 (5ug/ml, Biolegend, San Diego, CA) and anti-CD28 (2ug/ml, Biolegend, San Diego, CA) pre-coated 96-well plate and cultured in the presence of various concentrations of IL-27. After three day, CFSE expression on CD8⁺ T cells was analyzed by flow cytometry. In some experiments, CFSE-labeled CD8⁺ T cells were co-cultured with TIP-DCs pulsed with OVA peptides in 96-well plates at a ratio of 3:1. After three days, CFSE expression on CD8⁺ T cells was analyzed by flow cytometry.

Statistical analysis

Multiple comparisons were performed using one-way ANOVA with Tukey’s post-hoc test when comparing among all groups or Dunnett’s post-hoc test when comparing to control only. Comparisons between two groups were performed using Students’ unpaired t-test. All statistical analyses were performed with Graphpad Prism version 7 (GraphPad Software, San Diego, CA). Data were shown as mean ± SEM, and p < 0 .05 was considered statistically significant. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.

DC-derived IL-27 promoted tumor development in murine HCC models

To investigate the role of DC-derived IL-27 in tumor development, orthotopic murine HCC model was established as previously described by hydrodynamic injection (HGT) of hepa1-6 cells into wild-type (WT) or IL-27p28 DC conditional knockout (Itgax-IL-27p28^f/f) mice. Tumor growth was significantly reduced in the liver of Itgax-IL-27p28^f/f mice compared with the WT mice (Fig. 1a-b). To further confirm the result, we chemically induced HCC with diethyl nitrosamine (DEN) in both WT and Itgax-IL-27p28^f/f mice (Fig. 1c-d). Smaller and fewer tumor nodules were observed in Itgax-IL-27p28^f/f mice than in WT mice, indicating that DC-derived IL-27p28 promoted tumor development in murine HCC models. Furthermore, we neutralized IL-27 by using anti-IL-27p28 (Anti-p28) antibody in orthotopic HCC model (Fig. 1e-f). As expected, mice treated with anti-p28 antibodies developed fewer tumor nodules compared with the isotype control-treated mice. Taken together, these results demonstrated that DC-derived IL-27 promoted tumor growth in murine HCC models.

DC-derived IL-27 inhibited T cell activation in the tumor microenvironment

To elucidate the anti-tumor immune response regulated by DC-derived IL-27, we examined the percentages and numbers of T cells and NK cells in the spleen and liver of WT and Itgax-IL-27p28^f/f tumor bearing mice in orthotopic HCC model (Fig. 2a-d). While there was no significant difference in the spleen, both frequencies and numbers of T cells were significantly increased in the liver of Itgax-IL-27p28^f/f mice compared with WT mice. Among the T cells, both CD4⁺ T and CD8⁺ T cells were increased. The effector CD4⁺ T cells also exhibited higher percentages in the spleen, and both higher percentages and numbers in the liver of Itgax-IL-27p28^f/f mice (Fig. 2e-h). The percent and number of effector CD8⁺ T cells were increased in both spleen and liver of Itgax-IL-27p28^f/f mice compared with the WT (Fig. 2e-h), suggesting that DC-derived IL-27 may suppress T cell function, especially at the tumor site.

To further assess the anti-tumor function of the immune effector cells, we examined the cytokine production by CD4⁺ T, CD8⁺ T and NK cells using flow cytometry (Fig. 2i-l). The percentages of IFN-γ- and TNF-α-producing CD8⁺ T cells were significantly increased in the spleen of Itgax-IL-27p28^f/f mice compared with the WT mice. Moreover, the percentages and numbers of IFN-γ- and TNF-α-producing CD4⁺ T cells and CD8⁺ T cells were greatly increased in the liver of Itgax-IL-27p28^f/f mice compared with the WT mice. The percent and number of TNF-α-producing NK cells were also increased in the liver of Itgax-IL-27p28^f/f mice. We further confirmed the tumor-promoting role of DC-derived IL-27 in a melanoma lung metastasis model (Fig. S1a). There was an increase in CD8⁺T cell percentages and both effector CD4⁺T and CD8⁺T cell percentages were increased in the lung (Fig. S1b-c). Thus, in the absence of DC-derived IL-27, both CD4⁺ T and CD8⁺ T cells had enhanced immune activation phenotypes and increased IFN-γ and TNF-α production especially in the tumor microenvironment.

DC-derived IL-27 promoted the expression of inhibitory receptors on both CD4 ⁺ and CD8⁺ T cells in tumor-bearing mice

Immune checkpoint inhibitors are usually highly expressed on T cells in the tumor microenvironment. It has been shown that IL-27 signaling is essential for inducing T-cell immunoglobulin and mucin-domain containing-3 (Tim3)⁺ exhausted T cells²¹. The expression of co-inhibitory receptors on CD4⁺ and CD8⁺ T cells is also found to be driven by IL-27 by a study using single-cell mass cytometry²⁸. We then examined the expression of programmed cell death protein 1 (PD-1) and Tim-3 on CD4⁺ T cells and CD8⁺ T cells in naïve and tumor-bearing WT and Itgax-IL-27p28^f/f mice. There was no PD-1 or Tim-3 expression on CD4⁺ and CD8⁺ T cells in spleen and liver of naïve WT and Itgax-IL-27p28^f/f mice (Fig. 3a). As expected, PD-1 and Tim3 were expressed at a relatively higher level on both CD4⁺ and CD8⁺ T cells in tumor-bearing liver than in spleen of WT mice (Fig. 3b-g). Interestingly, this increased PD-1 and Tim3 expression on T cells in the tumor microenvironment was completely abolished in the absence of DC-derived IL-27 in the Itgax-IL-27p28^f/f mice (Fig. 3b-g). This was also observed in the melanoma lung metastasis model (Fig. S1d). These results demonstrated that DC-derived IL-27 is essential in the upregulation of inhibitory receptor expression on T cells in the tumor microenvironment.

DC-derived IL-27 promoted tumor development through modulating gut microbiota

Many studies have demonstrated the important roles of gut microbiota in viral infections, autoimmune diseases, and tumor development^49–51. In addition, there could be bidirectional crosstalk occurring between the liver and gut⁵². To address whether gut microbiota could play a role in IL-27-mediated immune suppression and tumor development, we collected feces from WT and Itgax-IL-27p28^f/f mice and transplanted into Itgax-IL-27p28^f/f and WT mice respectively by gavage with PBS as control (Fig. 4a-b). Interestingly, tumor growth in WT mice was significantly inhibited after receiving Itgax-IL-27p28^f/f feces, while the tumor growth in Itgax-IL-27p28^f/f mice was not affected by WT fecal transfer. These results suggest that the tumor-suppressive immune mechanism in Itgax-IL-27p28^f/f mice can be mediated and transferred through gut microbiota.

To identify specific bacteria stains involved in promoting anti-tumor immune responses, we examined the fecal bacterial content of WT and Itgax-IL-27p28^f/f mice with or without fecal transplant using 16s ribosomal RNA (rRNA) miSeq Illumina platform (Fig. 4c-h). The shared bacteria genera were shown in the Veen diagram, which indeed demonstrated a distinct difference in gut bacteria compositions between WT and Itgax-IL-27p28^f/f mice (Fig. 4c). Principal coordinate analysis revealed that samples from WT mice receiving Itgax-IL-27p28^f/f fecal material became more like samples obtained from Itgax-IL-27p28^f/f mice, suggesting the gut microbiota modulation via gavage was successfully performed (Fig. 4d). In contrast, Itgax-IL-27p28^f/f mice receiving WT fecal material did not significantly change in gut bacterial composition, which is consistent with the results of tumor growth, suggesting the gut bacteria in Itgax-IL-27p28^f/f mice may be dominant over those in WT mice. The result analyzed by unweighted pair group method with arithmetic mean (UPGMA) further confirmed the finding that WT mice receiving Itgax-IL-27p28^f/f fecal material became similar to Itgax-IL-27p28^f/f mice in their gut bacterial composition, whereas WT mice feces transplant did not significantly modify the gut microbiota of the Itgax-IL-27p28^f/f mice (Fig. 4e). The indices for community diversity and richness were calculated and described as Shannon index and Chao1 index respectively (Fig. 4f). The diversity and richness of gut bacterial community were decreased in Itgax-IL-27p28^f/f mice. Receiving fecal material from Itgax-IL-27p28^f/f mice decreased the community richness and diversity in WT mice, while receiving fecal material from WT mice increased the community richness and diversity in Itgax-IL-27p28^f/f mice, especially as shown in Chao1 index. Taken together, these results demonstrated that the gut bacterial compositions are significantly different between WT and Itgax-IL-27p28^f/f mice, and the tumor-promoting immune mechanism in the absence of DC-derived IL-27 could be mediated by gut microbiota.

Since the diversity and richness of the bacterial community could be modified in the Itgax-IL-27p28^f/f mice receiving WT fecal materials, but the tumor growth was not affected, the tumor growth phenotypes are more likely to be related to the bacterial composition than the diversity and richness. To further identify the difference in bacterial composition, we analyzed for differentially abundant taxa across all permutations between various groups of mice (Fig. 4g). Taxonomically, nine different bacterial phyla including Actinobacteria, Bacteroidetes, Cyanobacteria, Deferribacteres, Firmicutes, Proteobacteria, Saccharibacteria, Tenericutes, and Verrucomicrobia were identified. We further narrowed down to genus-level taxa that have been associated with anti-tumor effects. Bifidobacterium, Parasutterella and Akkermansia, which belong to Actinobacteria, Proteobacteria and Verrucomicrobia respectively and have been associated with anti-tumor functions, were increased in all groups of Itgax-IL-27p28^f/f mice, Itgax-IL-27p28^f/f mice receiving WT feces, and WT mice receiving Itgax-IL-27p28^f/f feces compared to WT mice (Fig. 4h-i). Among them, Bifidobacterial has been well studied for its role in activating host immune responses⁴⁰. To establish its role in our murine HCC model, we inoculated a cocktail of Bifidobacterium species, including B.bifidum, B.longum, B.breve and B.lactis to tumor-bearing mice by gavage (Fig. 4j). Treatment with Bifidobacterium significantly inhibited tumor growth compared with the control group. To exclude any possible influence of tumor growth on gut microbiota, we also examined the fecal bacterial content of naïve WT and Itgax-IL-27p28^f/f mice which exhibited similar differences, suggesting the changes in microbiota were induced by intrinsic expression of DC-derived IL-27 (Fig. S2). Taken together, these results demonstrated that DC-derived IL-27 could promote tumor growth and suppress anti-tumor immune responses through the modulation of gut microbiota.

Ly6C ⁺ MHCII ⁺ (TIP)-DCs could be modulated by gut microbiota in the absence of DC-derived IL-27 to promote T cell functions

Gut microbiota has been shown to modulate DC phenotypes and functions. Depletion of gut microbiota using antibiotic cocktail could reduce Ly6C⁺MHC II⁺ cells⁵³. Bifidobacterium could increase the MHC II⁺ DCs in the tumor microenvironment to promote anti-tumor immune response⁴⁰. Thus, we hypothesis that DC-derived IL-27 may regulate DC function via modulation of gut microbiota. To address this, we first examined the major myeloid cell subsets in spleen and liver of WT and Itgax-IL-27p28^f/f mice including DCs, myeloid-derived suppressor cells (MDSCs), and macrophages (Fig. 5a-d). There was a slight decrease of DCs in the spleen of Itgax-IL-27p28^f/f mice compared to WT mice, while no difference was observed in tumor-bearing liver. The expressions of CD80, CD86 or MHC II were similar between WT and Itgax-IL-27p28^f/f mice on DCs, MDSCs, and macrophages both in spleen and liver (Fig. 5b, 5d-f). However, both percentages of CD11b⁺Ly6G^-Ly6C⁺MHC II⁺ DC population out of CD11b⁺Ly6G^-Ly6C⁺ population or total leukocyte were significantly increased in the liver of Itgax-IL-27p28^f/f mice compared with WT mice (Fig. 5g). This DC population has been previously reported as tumor necrosis factor and inducible nitric-oxide-synthase-producing DCs (TIP-DC)³⁴. TIP-DCs have high level expression of MHCII and CD86 and play a role in immune responses against bacterial infection and tumor by enhancing T cell priming ^54,55. To confirm that TIP-DC could be modulated by gut microbiota, we examined this population in WT and Itgax-IL-27p28^f/f mice receiving fecal transplant (Fig. 5h). The level of MHC class II expression on the CD11b⁺Ly6G^-Ly6C⁺ population from tumor-bearing liver was higher in Itgax-IL-27p28^f/f mice than in WT mice, and its expression was significantly increased in WT mice receiving Itgax-IL-27p28^f/f fecal materials compared to PBS control, suggesting that MHC class II expression on TIP-DCs could be modulated by gut microbiota.

We then investigated whether TIP-DCs from WT or Itgax-IL-27p28^f/f mice were different in their functions of priming T cells. To exclude the direct effect of IL-27 on T cells, we treated native CD8⁺ T cells activated with anti-CD3/anti-CD28 with or without recombinant IL-27 (Fig. 6a). No difference was observed in T cell proliferation in the presence or absence of IL-27. We then isolated TIP-DCs from WT or Itgax-IL-27p28^f/f tumor-bearing mice by flow sorting and pulsed them with OVA antigen (Fig. 6b). CD8⁺ T cells from OT-1 mice were co-cultured with OVA-pulsed TIP-DCs for three days and analyzed for proliferation and TNF-α and IFN-γ production. The proliferation of the CD8⁺ T cells measured by CFSE labelling was enhanced by TIP-DCs from Itgax-IL-27p28^f/f mice compared with the WT control (Fig. 6c). CD8⁺ T cells stimulated by TIP-DCs from Itgax-IL-27p28^f/f mice produced significantly higher level of TNF-α and IFN-γ compared with the WT control (Fig. 6d). These results demonstrated that in the absence of DC-derived IL-27, TIP-DCs’ function of priming T cells was significantly promoted. Taken together, these results suggest that TIP-DCs could be modulated by gut microbiota in the absence of DC-derived IL-27 to promote anti-tumor T cell functions.

The role of IL-27 in tumor development has been controversial. In the current study, we investigated the function of DC-derive IL-27 in murine HCC models and found that it promoted HCC tumor development and suppressed anti-tumor immune response. Interestingly, the tumor growth phenotype of Itgax-IL-27p28^f/f mice could be transferred via gut microbiota, and some bacteria strains were identified in the gut of Itgax-IL-27p28^f/f mice that could inhibit tumor development and were significantly reduced in the WT mice. Furthermore, the percentage and MHC class II expression of TIP-DCs were increased, and their T cell-activating function was enhanced in Itgax-IL-27p28^f/f mice compared with the WT mice. The phenotypes of the TIP-DCs could be modulated by gut microbiota. Therefore, our results have uncovered a novel mechanism of DC-derived IL-27 regulating anti-tumor immune response via modulation of gut microbiota and provided new insights to the function of DC-derived cytokines in adaptive immune responses.

Both tumor-suppressive and tumor-promoting functions of IL-27 have been described in the previous studies. IL-27 has been shown to activate STAT1 signaling and promote IFN-γ production¹⁶. Many studies demonstrated that IL-27 could inhibit tumor growth and promote anti-tumor immunity by upregulating Th1 and CTL responses in tumor models such as neuroblastoma, colon carcinoma, and B16 melanoma^24,25,27. On the other hand, IL-27 has been shown to act as a negative regulator of T cell responses in IL-27ra^f/f mice challenged with viral, bacterial or parasitic pathogens^16,56,57. Moreover, IL-27 could promote tumor progression by inducing an immune-suppressive phenotype in tumor-associated macrophage and promoting co-inhibitory receptor Tim-3 expression on T cells^21,23. Recently, the single-cell mass cytometry results also revealed that co-inhibitory receptor expression on T cells is driven by IL-27²⁸. Currently, SRF388, an IL-27 neutralizing antibody, is currently being evaluated in a Phase I study in patients with advanced solid tumors, including HCC (#NCT04374877). In fact, IL-27 is expressed at a relatively higher level in liver than in other organs (http://gepia2.cancer-pku.cn/#general). Its expression has also been shown to be increased in patients infected with hepatitis B virus^58–60, which is one of the important inducing factors in liver carcinogenesis⁶¹. The levels of IL-27 in pre-diagnostic sera were significantly associated with increased risk of HCC development⁶², suggesting IL-27 could play an important role in liver chronic inflammation and carcinogenesis. We found that DC-derived IL-27 could promote tumor development and suppress anti-tumor adaptive immune response. A previous study using the same IL-27p28 conditional knockout mice showed tumor-suppressive role of DC-derived IL-27 in a subcutaneous melanoma model²⁷. However, DC-derived IL-27 promoted melanoma lung metastasis and suppressed anti-tumor immune response in the lung (Fig. S1), suggesting the role of IL-27 regulating anti-tumor immune response could be organ-specific. The direct or indirect effects of IL-27 on the immune responses may be proportionally different depending on the tissue-specific environment.

In murine HCC models, the activation and cytokine production by both CD4⁺ and CD8⁺ T cells were enhanced in the absence of DC-derived IL-27. Many previous studies have demonstrated an important role of IL-27 in T cell activation and differentiation promoting Th1 phenotype and CTL self-renewal^16,63−65. The immune-suppressive function of IL-27 we observed in HCC models could be due to the indirect effect of IL-27 on T cells that is dominant in the liver tumor microenvironment. It has been shown that IL-27 could induce co-inhibitory receptor expression on T cells, such as PD-1 and Tim-3, in many tumor models^21,28,66. We also demonstrated that CD4⁺ and CD8⁺ T cells expressed high level of PD-1 in tumor-bearing livers in WT mice while the expression was greatly diminished in Itgax-IL-27p28^f/f mice. However, the mechanism of IL-27 inducing inhibitory receptor expression on T cells needs further investigation.

The link between gut microbiota and tumor development has been demonstrated in many tumor types, including melanoma, prostate cancer and lymphoma^35,40,42,67. Due to the close anatomical and functional relationship between liver and gut, gut microbiota has been shown to play a role in HCC development^45,46. Interestingly, we found the tumor-suppressive phenotype of Itgax-IL-27p28^f/f mice could be transferred to WT mice by fecal transplantation. However, Itgax-IL-27p28^f/f mice receiving WT fecal materials did not exhibit significant changes in their gut microbiota composition and tumor development, suggesting the bacterial strains that were present in the gut of Itgax-IL-27p28^f/f mice might be dominant when coexisting with those from the WT mice. The diversity and richness were decreased in Itgax-IL-27p28^f/f mice compared with the WT mice. Tumor growth could be inhibited with lower microbiota diversity and richness in mice that are treated with antibiotic cocktail (ABX)⁴¹. Many studies also suggest that microbiota diversity could be used to predict cancer progression^68–70. However, we found Itgax-IL-27p28^f/f mice that received WT fecal materials increased the diversity and richness compared to PBS-treated Itgax-IL-27p28^f/f mice but had similar tumor growth. Thus, diversity and richness may not be the only indicator and the composition of the gut microbiota could be more critical in modulating tumor development. Bifidobacterium and Akkermansia were increased in the gut of Itgax-IL-27p28^f/f mice compared with WT mice. Both are found to play important roles in tumor development and treatment response^{40,44,71,72,65}. However, our results do not rule out the possible contributions of other commensal bacteria species affected by IL-27, and how IL-27 regulates gut microbiota needs further investigation.

We discovered that the population of CD11b⁺Ly6G^-Ly6C⁺MHC II⁺ cells were significantly increased in Itgax-IL-27p28^f/f mice. Moreover, the MHC II expression on these cells could be modulated by fecal transplantation which is consisted with previous finding showing that the gut microbiota could affect MHC II expression on CD11b⁺Ly6C⁺ cells⁴⁰. This population has been given a name of TIP-DC^54,35. TIP-DCs have been shown to play an important role in protecting the host from bacteria infection^35,73, viral infection⁷⁴ and tumor growth⁷⁵. In TIP-DC mediated anti-tumor responses, they are essential in CD8⁺T cells activation⁷⁵ and enhanced cytotoxicity⁵⁴. By co-culture experiments, we also demonstrated that TIP-DCs from Itgax-IL-27p28^f/f mice are more competent in promoting CD8⁺ T cell proliferation and cytokine production, suggesting that they could be the link between modified gut microbiota and enhanced adaptive anti-tumor immune response.

Our results demonstrated that DC-derived IL-27 could promote tumor growth and suppress anti-tumor immune response in murine HCC through the modulation of gut microbiota. This indirect effect of IL-27 on adaptive immune response provides insights into possible organ-specific functions of IL-27 and targeting IL-27 in tumor immunotherapy.

HCC: Hepatocellular carcinoma

DC: Dendritic cell

Itgax-IL-27p28^f/f: IL-27p28 DC-conditional knockout

TIP-DC: Tumor necrosis factor and inducible nitric-oxide-synthase-producing DC.

CNTF: Ciliary neurotrophic factor

CT1: Cardiotrophin1

CLCF1: Cardiotrophin-like cytokine factor 1

LIF: Leukemia inhibitory factor

EAE: Experimental autoimmune encephalomyelitis

IBD: Inflammatory bowel disease

OT-I mice: C57BL/6-Tg(TcraTcrb)1100Mjb/J mice

OTUs: Operational taxonomic units

WT: Wild-type

HGT: Hydrodynamic injection

Anti-p28: Anti-IL-27p28

Tim3: T-cell immunoglobulin and mucin-domain containing-3

PD-1: Programmed cell death protein 1

rRNA: Ribosomal RNA

UPGMA: Unweighted pair group method with arithmetic mean

ABX: Antibiotic cocktail

Ethics approval and consent to participate:

Not applicable.

Consent for publication:

Not applicable.

Availability of data and materials:

RNA-Seq data that support the findings of this study are available from the corresponding author upon request. The authors declare that the data supporting the findings of this study are available within the article and provided as Source Data file.

Competing interests:

The authors declare that they have no competing interests.

Funding:

This work has been supported by China Postdoctoral Science Foundation (2019M661938), Jiangsu Planned Projects for Postdoctoral Research Funds (2019K098), Natural Science Foundation of Jiangsu Higher Education Institutes (20KJD320001), Translational Research Grant of NCRCH (2021ZKQC01), Life Science Institute Grand Challenge Fund, and the start-up grant of National University of Singapore.

Author contributions:

Y.L, Y.S, and H.L designed the research. Y.L, Y.S, Z.J and H.G performed the experiments and analyzed the data. L.L, Y.Z, Y.M and HY.T assisted in the experiments. Z.Y provided Itgax-IL-27p28^f/f mice and OT-I mice. D.W and H.L supervised the study. Y.L, Y.S, and H.L wrote the manuscript.

Acknowledgements:

Not applicable.

Authors’ information:

Not applicable.

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No competing interests reported.

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DC-derived IL-27 suppresses anti-tumor immunity via the modulation of gut microbiota

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Abstract

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Background

Materials And Methods

Cell line

Mice and murine HCC models

Flow cytometry

Fecal transplantation

Bacterial gavage

Fecal sample processing, sequencing, and analysis

Activation of T cells with OVA peptide-pulsed TIP-DCs

T cell proliferation assays

Statistical analysis

Results

DC-derived IL-27 promoted tumor development in murine HCC models

DC-derived IL-27 inhibited T cell activation in the tumor microenvironment

DC-derived IL-27 promoted tumor development through modulating gut microbiota

Discussion

Conclusions

Abbreviations

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