Significant and Conflicting Correlation of IL-9 With Prevotella and Bacteroides in Human Colorectal Cancer

Abstract

Background and aim: Gut microbiota (GM) can support colorectal cancer (CRC) progression by modulating immune responses through the production of both immunostimulatory and/or immunosuppressive cytokines. The role of IL-9 is paradigmatic because it can either promote tumor progression in hematological malignancies or inhibit tumorigenesis in solid cancers. Therefore, we investigate the microbiota-immunity axis in healthy and tumor mucosa, focusing on the correlation between cytokine profile and GM signature.

Methods: In this observational study, we collected tumor (CRC) and healthy (CRC-S) mucosa samples from 45 CRC patients, who were undergoing surgery in 2018 at the Careggi University Hospital (Florence, Italy). First, we characterized the tissue infiltrating lymphocyte subset profile and the GM composition. Subsequently, we evaluated the CRC and CRC-S molecular inflammatory response and correlated this profile with GM composition, using Dirichlet multinomial regression.

Results: CRC samples displayed higher percentages of Th17, Th2, and Tregs. Moreover, CRC tissues showed significantly higher levels of MIP-1α, IL-1α, IL-1β, IL-2, IP-10, IL-6, IL-8, IL-17A, IFN-γ, TNF-α, MCP-1, P-selectin, and IL-9. Compared to CRC-S, CRC samples also showed significantly higher levels of the following genera: Fusobacteria, Proteobacteria, Fusobacterium, Ruminococcus2, and Ruminococcus. Finally, the abundance of Prevotella spp. in CRC samples negatively correlated with IL-17A and positively with IL-9. On the contrary, Bacteroides spp. presence negatively correlated with IL-9.

Conclusions: Our data consolidate antitumor immunity impairment and the presence of a distinct microbiota profile in the tumor microenvironment compared with the healthy mucosa counterpart. Relating the CRC cytokine profile with GM composition, we confirm the presence of bidirectional crosstalk between the immune response and the host's commensal microorganisms. Indeed, we document, for the first time, that Prevotella spp. and Bacteroides spp. are, respectively, positively and negatively correlated with IL-9, whose role in CRC development is still under debate.

Keywords: T cells; colorectal cancer; cytokines; gut microbiota; immune response.

Figure 1

T cell subset distribution in the tumor mucosa and surrounding healthy mucosa samples groups. Panel (A) reports the mean percentages (+SD) of T helper subsets with respect to the percentage of CD4⁺ T cells, and panel (B) reports the mean percentages (+SD) of T cytotoxic subsets with respect to the percentage of CD8⁺ T cells. Panel (C) reports the cytofluorimetric analysis of T regulatory cells in the tumor mucosa and surrounding healthy mucosa samples of one representative patient. Statistical analyses were calculated using Wilcoxon signed-rank test. The asterisks (*) represent p-values, *p < 0.01, **p < 0.001. CRC = tumor mucosa; CRC-S = healthy mucosa.

Figure 2

Tissue cytokine levels in 14 CRC patients. The histogram reports the mean (+SEM) cytokine levels (pg/ml) of the evaluated cytokines in CRC-S and CRC of 14 CRC patients. Wilcoxon signed-rank test was performed to test the differences between CRC-S and CRC paired samples. A p-value < 0.05 is considered statistically significant. The asterisks (*) represent p-values, *p < 0.05, **p < 0.01, ***p < 0.001. CRC-S= healthy mucosa; CRC= tumor mucosa.

Figure 3

Rarefaction curves showing the level of saturation of OTUs.

Figure 4

Boxplots showcasing alpha diversity indices (Chao1 index, Shannon index, Evenness, and Breakaway) in CRC and CRC-S samples. Statistical differences were evaluated using paired Wilcoxon signed-rank test for Chao, Shannon, and Evenness indices and using the paired betta analysis implemented in the Breakaway R package. P-values less than 0.05 were considered statistically significant.

Figure 5

Cluster analysis on normalized OTU counts.

Figure 6

Stacked boxplots of microbial composition at phylum level of CRC and CRC-S samples.