Mechanism of enteral nutrition in regulating Tfh/Tfr immune imbalance in colorectal cancer via the TLR4 signaling pathway

Abstract

This study aims to investigate the regulatory effects and underlying mechanisms of enteral nutrition (EN) on colorectal cancer (CRC). An inflammatory colorectal tumor model was established in mice using the azoxymethane-dextran sulfate sodium method. The mice were divided into groups including a model group and groups treated with EN or EN plus the Toll-like receptor 4 (TLR4) activator lipopolysaccharide. The T follicular helper/T follicular regulatory (Tfh/Tfr) cell ratios in splenic tissues were analyzed by flow cytometry. Protein expression related to the TLR4 pathway and cytokines in colonic tissues was assessed by Western blotting and quantitative real-time polymerase chain reaction. Histopathological changes were evaluated using hematoxylin-eosin staining. The proportion of Tfh cells to Tfr cells is significantly imbalanced in CRC, and this imbalance was corrected following EN treatment. This correction of Tfh/Tfr imbalance was found to inhibit tumor growth, suggesting that the regulatory effect of EN on CRC may be mediated through the modulation of the Tfh/Tfr cell balance. At the molecular level, we discovered that EN was found to suppress the activation of the TLR4 signaling pathway. Furthermore, the addition of lipopolysaccharide abolished the effects of EN, indicating that the regulation of the Tfh/Tfr cell ratio by EN is mediated through the TLR4 signaling pathway. This study provides evidence that EN can inhibit the progression of CRC, potentially by regulating Tfh/Tfr immune imbalance via the TLR4 signaling pathway. These findings highlight the potential of EN as a therapeutic strategy for CRC treatment.

Introduction

In the modern era, dietary patterns and eating habits have undergone dramatic changes. To accommodate the fast pace of work and life, people’s eating habits have become increasingly irregular, placing a significant burden on the digestive system.⁽¹⁾ Over time, such habits elevate the risk of developing malignant tumors in the digestive system. Malignant tumor cells invade the normal intestinal mucosa, consuming the body’s nutrients through unchecked proliferation. Furthermore, tumor cells induce swelling of the gastrointestinal mucosa, thereby reducing the patient’s appetite. This combination of nutrient depletion and reduced food intake gradually leads to declining physiological function, malnutrition, exacerbated negative nitrogen balance, and ultimately cachexia—a common and often fatal complication in patients with digestive system tumors.⁽²⁾ With the continuous advancement of evidence-based medicine, clinical approaches to treating malignant tumors of the digestive system have evolved toward more diversified and comprehensive strategies. Enteral nutrition (EN) refers to the delivery of essential nutrients and energy to patients through oral or nasogastric feeding, to improve their physical condition and promote the recovery of gastrointestinal function.⁽³⁾ Currently, EN has been widely used as a supportive therapy in the treatment of various cancers.^(4,5) However, most studies have focused on its role in postoperative recovery, whereas research on its anti-tumor effects remains limited. This study aims to investigate the regulatory effects of EN on colorectal cancer (CRC) and explore its underlying molecular mechanisms.

T follicular helper (Tfh) cells are a subset of CD4⁺T cells characterized by the specific expression of C-X-C chemokine receptor type 5 (CXCR5). These cells express key molecules such as Programmed Death-1 (PD-1), B-cell lymphoma 6 (BCL-6), and secrete the cytokine IL-21, which acts on B lymphocytes to promote their proliferation, activation, and germinal centers (GCs) formation. Tfh cells play a critical role in adaptive immunity.⁽⁶⁾ T follicular regulatory (Tfr) cells are another subset of CD4⁺T cells that possess dual features of regulatory T cells (Treg) and Tfh cells. Tfr cells express Treg-specific the transcription factor Foxp3 and B lymphocyte-induced maturation protein 1 (Blimp-1) and secrete IL-10. They also express Tfh-associated molecules, including PD-1, CXCR5, and BCL-6. The primary function of Tfr cells is to inhibit Tfh cells, suppressing Tfh and B cell activity in GCs, thereby preventing excessive humoral immune responses.^(7–9) Previous studies have shown that the imbalance in the numbers and functions of Tfh and Tfr cells is associated with various diseases, including autoimmune disorders, rheumatic diseases, and tumors. A dynamic balance between Tfh and Tfr cells is essential for maintaining immune system homeostasis.⁽¹⁰⁾ Although several studies have reported the roles of Tfh and Tfr cells in the progression of various human tumors,^(11,12) data on their involvement in CRC progression remain limited.

Toll-like receptors (TLRs) are a class of pattern recognition receptors expressed on the surface of immune cells. TLRs activate both innate and adaptive immune responses.⁽¹³⁾ The TLR signaling pathway consists of myeloid differentiation primary response 88 (MyD88)-dependent and MyD88-independent pathways, which ultimately activate the NF-κB pathway, inducing the release of pro-inflammatory cytokines and promoting lymphocyte proliferation and activation. Studies have shown that CRC patients with low Toll-like receptor 4 (TLR4) signaling expression have a significantly prolonged survival time,⁽¹⁴⁾ and that activation of TLR4 signaling can promote epithelial-mesenchymal transition and metastasis in CRC.^(15–16) Therefore, it is hypothesized that EN may correct the Tfh/Tfr immune imbalance in CRC by modulating TLR4 signaling, thereby intervening in tumor progression.

Materials and Methods

Animal models

Thirty healthy male Kunming mice (9–12 weeks old, weighing 40–45 ‍g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). After adaptive feeding for 7 days in a temperature- and humidity-controlled environment, the mice were randomly divided into five groups: Control group, Peptisorb-fed (FMT, Peptisorb-feeding treatment) group, model group, model + FMT group, and model + FMT + lipopolysaccharide (LPS) group. Throughout the experiment, all mice had ad libitum access to food and water. On the first day of modeling, all model groups were intraperitoneally injected with azoxymethane (AOM) at a concentration of 10 ‍mg/kg. Subsequently, 2.5% dextran sulfate sodium (DSS) solution was administered in the drinking water during weeks 2, 5, and 8. Tumor formation was assessed following the completion of DSS treatment. After successful modeling, the model + FMT and model + FMT + LPS groups received the EN formula Peptisorb [a short-peptide EN solution manufactured by Nutricia Pharmaceutical (Wuxi) Co., Ltd., Wuxi, China] as the sole nutrient source for 7 consecutive days, with no standard chow provided. The model + FMT + LPS group received the same EN but was additionally administered LPS intraperitoneally at a dose of 60 ‍μg/ml (equivalent to 60 ‍mg/kg body weight). The FMT group was fed only Peptisorb without undergoing azoxymethane-dextran sulfate sodium (AOM-DSS) treatment. The control group had free access to drinking water and standard chow.

Histopathological examination of colonic tissue

Colonic tissues were sectioned into paraffin slices and stained with hematoxylin-eosin (HE). Mucosal damage was observed under a light microscope, with evaluations based on two parameters: inflammatory cell infiltration and tissue damage. The average score of these two parameters was calculated to assess the extent of pathological changes.

Detection of CD4⁺CXCR5⁺Foxp3⁺ Tfr and CD4⁺CXCR5⁺PD-1⁺ Tfh cells in spleen

Spleen tissues were ground and incubated with antibodies in the dark for 15 ‍min. A total of 2 ‍ml of 1× Lysing Buffer (Beyotime, Shanghai, China) was added to each tube, mixed thoroughly, and incubated in the dark for 10 ‍min. After centrifugation, the samples were washed once with 2 ‍ml phosphate-buffered saline (PBS). Subsequently, 2 ‍ml of 1× Fixation Buffer (prepared by diluting with PBS at a 1:19 ratio, Beyotime) was added, and the samples were incubated in the dark at 4°C for 30 ‍min. After centrifugation, 2 ‍ml of 1× Permeabilization Buffer (prepared by diluting with PBS at a 1:4 ratio, Beyotime) was used to wash the samples, followed by incubation with 2 ‍ml of 1× Permeabilization Buffer at room temperature in the dark for 30 ‍min. After centrifugation and washing with PBS, 10 ‍μl of PerCP-Cy5.5-FOXP3 antibody was added to each sample, mixed thoroughly, and incubated in the dark at room temperature for 30 ‍min. Samples were then washed with 2 ‍ml PBS, resuspended in 500 ‍μl PBS, and analyzed using flow cytometry.

Quantitative real-time polymerase chain reaction (RT-qPCR)

RT-qPCR was performed according to the manufacturer’s instructions. Briefly, colon tissue samples were rapidly frozen in liquid nitrogen, and total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA). cDNA was synthesized with the PrimeScript RT reagent Kit (Takara, Kusatsu, Japan). PCR amplification was conducted using SYBR Green PCR Master Mix (Solarbio, Beijing, China) on an ABI 7500 Fast Real-time PCR system under the following conditions: 95°C for 10 ‍min; followed by 40 cycles of 95°C for 15 ‍s and 60°C for 60 ‍s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal reference for mRNA levels. The relative expression of target mRNA was calculated using the 2^−ΔΔCT method. Primer sequences used in this study are listed in Table 1.

Table 1.

Primer Sequences for RT-qPCR

Gene	Forward primer (5'-3')	Reverse primer (5'-3')
Foxp3	5'-AGCTTAGAGAAGACAGACCCAT-3'	5'-TCCAGCTTGGCAAGACTCCT-3'
IL-10	5'-GCTCCAAGACCAAGGTGTCTA-3'	5'-GGGGGATGACAGTAGGGGAA-3'
TGF-β	5'-CAGCTCCTCATCGTGTTGGT-3'	5'-CAACTTCTTCTCCCCGCCAT-3'
IL-21	5'-GGCTCTCGTTCCCACAGATG-3'	5'-TGCACTTCCACAGGCTTTCA-3'
TLR4	5'-GTCAGCAAACGCCTTCTTCC-3'	5'-CTTCAAGGGGTTGAAGCTCAG-3'
MYD88	5'-GTCCCTGCCCTGAAGATGAC-3'	5'-GAAGATGCAAACCTCGCTGC-3'
GAPDH	5'-CCCTTAAGAGGGATGCTGCC-3'	5'-ACTGTGCCGTTGAATTTGCC-3'

Detection of protein expression in colonic tumors

Protein expression in colonic tumors was analyzed using Western blotting. Colonic mucosal tissues were rinsed twice with ice-cold PBS and then lysed in RIPA buffer containing 1% protease and 1% phosphatase inhibitors for 10 ‍min. The tissues were homogenized on ice using an ultrasonic homogenizer, and the lysates were centrifuged at 13,000 × g for 15 ‍min at 4°C. The supernatant was collected, and protein concentration was determined using a BCA protein assay kit (Beyotime) at a wavelength of 570 ‍nm. Proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk prepared in TBST solution and incubated overnight at 4°C with the following primary antibodies: Foxp3 (1:1,000; Abcam, Cambridge, UK), CTLA-4 (1:800, Abcam), IL-10 (1:500, CST, Danvers, MA), TGF-β (1:500, CST), and IL-21 (1:500, CST). After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:10,000) at room temperature for 60 ‍min. Protein bands were visualized using ECL chemiluminescent substrate and exposed for imaging. The intensity of target protein bands was analyzed using image analysis software, and the relative expression of the target proteins was calculated by normalizing the gray value of the target protein bands to the internal control bands. The results represent the relative expression levels of the target proteins in the samples.

Statistical analysis

Statistical analyses were performed using SPSS ver. 13.0. Measurement data were expressed as mean ± SD ($\stackrel{\_}{\mathrm{x}}$ ± s). Comparisons between groups were conducted using the t test, while comparisons among multiple groups were performed using one-way ANOVA followed by Tukey’s post-hoc test. A p value of less than 0.05 was considered statistically significant.

Results

Significant changes in Tfh/Tfr in the model group

An inflammatory colorectal tumor model was established in mice using the AOM-DSS method. HE staining revealed that the intestinal mucosal structure in the normal control group was intact, with continuous epithelial tissue and orderly arranged glands. In contrast, the model group exhibited disrupted intestinal mucosal layers, irregular morphology, disorganized arrangement, the formation of GCs, and a significant infiltration of inflammatory cells (Fig. 1A). To verify the changes in the Tfh/Tfr ratio in the model animals, splenic cells from the model group were analyzed by flow cytometry. The results showed a significant increase in Tfh cells and a marked decrease in Tfr cells in the model group compared with the control group, leading to a notable alteration in the Tfh/Tfr ratio (Fig. 1B and E). Western blotting analysis of colonic tissues revealed that, compared to the control group, the expression of TLR4, a key protein in the TLR4 signaling pathway, was significantly upregulated in the model group, accompanied by an evident increasing trend in MYD88 expression (Fig. 1C and D).

Fig. 1.

Significant changes in Tfh/Tfr in the model group. (A) HE staining of intestinal tissues from model animals. (B) Flow cytometry analysis of splenic cells in mice. (C) Western blotting detection of key proteins in colonic tissues from mice. (D) Statistical analysis of protein band intensities in Western blotting. (E) Statistical analysis of flow cytometry results. (***p<0.001, **p<0.01 vs Control)

Regulation of Tfh/Tfr ratio by EN

To investigate the regulatory effect of EN on the Tfh/Tfr ratio in model animals, mice were assigned to the following groups: Control + PBS, Control + FMT, Model + PBS, and Model + FMT. After treatment, spleen tissues were collected for flow cytometry analysis. The results showed that FMT did not affect the Tfh/Tfr ratio under physiological conditions. Compared to the Control group, the Model group exhibited a significant increase in Tfh cells, a notable decrease in Tfr cells, and a marked alteration in the Tfh/Tfr ratio. Notably, in the Model + FMT group, Tfh cell numbers were significantly reduced, Tfr cell numbers were increased, and the Tfh/Tfr ratio was lowered to a level comparable to that of the Control group (Fig. 2A–C). Next, to explore the impact of FMT on tissue inflammation, we used RT-qPCR to assess the expression of inflammatory cytokines in intestinal tissues. The results indicated that under physiological conditions, FMT had no effect on the expression of inflammatory cytokines in the intestinal tissues. Compared to the Control group, the Model group showed significantly reduced mRNA transcription levels of Foxp3, IL-10, and TGF-β, while IL-21 mRNA levels were significantly upregulated. However, in the Model + FMT group, the transcription levels of Foxp3, IL-10, and TGF-β were significantly restored, and IL-21 levels were notably downregulated compared with the Model + PBS group (Fig. 2D–G). These findings suggest that FMT significantly regulates the Tfh/Tfr ratio in the Model group and may suppress intestinal inflammation.

Fig. 2.

Regulation of the Tfh/Tfr ratio by EN. (A) Flow cytometry analysis of splenic Tfh and Tfr cells in each group of mice. (B, C) Statistical analysis of flow cytometry results showing Tfh/Tfr ratio changes. (D) Relative mRNA expression levels of Foxp3 in intestinal tissues. (E) Relative mRNA expression levels of IL-21 in intestinal tissues. (F) Relative mRNA expression levels of IL-10 in intestinal tissues. (G) Relative mRNA expression levels of TGF-β in intestinal tissues. (***p<0.001 vs Control + PBS, ^###p<0.01 vs model + PBS)

Regulation of the Tfh/Tfr ratio in CRC by EN via the TLR4 signaling pathway

Previous studies have confirmed that the TLR4 signaling pathway is significantly activated in this CRC model. Based on these findings, we further investigated the regulatory mechanism of FMT on Tfh/Tfr ratios in CRC using the TLR4 signaling pathway activator LPS. Experimental animals were divided into three groups: Model + PBS, Model + FMT, and Model + FMT + LPS. After treatment, splenic tissues were collected for flow cytometry analysis. The results showed that, compared to the Model + PBS group, the number of Tfh cells was significantly reduced, while Tfr cell numbers were increased in the Model + FMT group, resulting in a decreased Tfh/Tfr ratio. However, following the addition of LPS, Tfh cell numbers increased, Tfr cell numbers decreased, and the Tfh/Tfr ratio was elevated compared to the Model + FMT group (Fig. 3A and B). These findings indicate that LPS counteracts the regulatory effect of FMT on the Tfh/Tfr ratio. Subsequently, the expression levels of inflammatory cytokines in intestinal tissues were assessed by RT-qPCR. The results demonstrated that, compared to the Model + FMT group, the addition of LPS significantly downregulated the mRNA transcription levels of Foxp3, IL-10, and TGF-β, while significantly upregulating IL-21 mRNA transcription levels (Fig. 3C). Western blotting analysis showed trends consistent with the RT-qPCR results, indicating that the expression levels of these proteins were also altered following LPS treatment (Fig. 3D and E).

Fig. 3.

Regulation of the Tfh/Tfr ratio in CRC by EN via the TLR4 signaling pathway. (A) Flow cytometry analysis of Tfh and Tfr cells in spleen tissues from each group. (B) Statistical analysis of Tfh/Tfr ratios from flow cytometry results. (C) Relative mRNA expression levels of Foxp3, IL-21, IL-10, and TGF-β in intestinal tissues. (D) Western blotting analysis of Foxp3, IL-21, IL-10, and TGF-β protein expression levels in intestinal tissues. (E) Statistical analysis of protein expression levels from Western blotting results. (***p<0.001 vs model + PBS, Control + PBS, ^###p<0.01 vs model + FMT, model + PBS, ^&&&p<0.01 vs model + FMT)

Study on the molecular mechanism of EN regulating the TLR4 signaling pathway

To investigate the effect of FMT on the colorectal tumor phenotype, we measured the tumor diameters in the Model + PBS, Model + FMT, and Model + FMT + LPS groups. The results showed that the tumor diameters in both the Model + FMT and Model + FMT + LPS groups were significantly reduced compared to the Model + PBS group. However, compared with the Model + FMT group, the tumor diameters in the Model + FMT + LPS group were significantly increased. These findings suggest that FMT can inhibit tumor growth, an effect that is counteracted by LPS (Fig. 4A and B). Next, RT-qPCR and Western blotting were used to assess the transcriptional and protein expression levels of the key TLR4 signaling pathway components, TLR4 and MyD88. The results demonstrated that, compared to the Control group, the transcriptional and protein expression levels of TLR4 and MyD88 were significantly upregulated in the Model + PBS group. In the Model + FMT group, the expression levels of these factors were significantly downregulated compared with the Model + PBS group. However, in the Model + FMT + LPS group, the expression levels of TLR4 and MyD88 were significantly increased relative to the Model + FMT group (Fig. 4C–E).

Fig. 4.

Study on the molecular mechanism of EN regulating the TLR4 signaling pathway. (A) Images of colorectal tumors in each group of mice. (B) Statistical analysis of tumor weight in each group. (C) Relative mRNA expression levels of TLR4 and MyD88 in colorectal tissues. (D) Western blotting analysis of TLR4 and MyD88 protein expression levels in colorectal tissues. (E) Statistical analysis of protein expression levels from Western blotting results. (***p<0.001 vs model, Control + PBS, ^###p<0.01 vs model + FMT, model + PBS, ^&&&p<0.01 vs model + FMT)

Discussion

CRC is one of the most common malignant tumors of the digestive system. According to the latest estimates from the International Agency for Research on Cancer, CRC ranks third in incidence and second in mortality globally.⁽¹⁷⁾ Although the overall mortality rate of CRC has declined in recent years, the mortality rate among individuals under 50 years old has continued to rise, indicating a trend of younger CRC patients. Most patients are diagnosed at an advanced stage, and certain subtypes of CRC still lack effective treatment options.^(18–20) Early-stage CRC can be cured through surgical resection. However, for advanced or metastatic CRC, chemotherapy or targeted therapies are typically required to enhance therapeutic efficacy. Unfortunately, the clinical outcomes of these treatments are inconsistent and often cause significant harm to normal physiological functions, underscoring the urgent need for novel therapeutic strategies.

EN and parenteral nutrition (PN) are two commonly used postoperative nutritional support methods. Both provide patients with essential nutrients, such as calories and nitrogen, to promote the restoration of positive nitrogen balance, alleviate stress responses, replenish nutrients, and reduce hypermetabolic reactions.^(21–24) Recent studies suggest that PN can increase the hepatic burden postoperatively, particularly in patients undergoing gastrointestinal surgeries for conditions like gastric cancer, thereby impeding gastrointestinal recovery and, in severe cases, leading to complications such as gut-derived infections.⁽²⁵⁾ In contrast, EN not only preserves the function and integrity of the gastrointestinal mucosal barrier but also aligns with patients’ physiological needs after surgery, helping to correct surgical trauma. EN regulates metabolism, improves nutritional status and immunity, and suppresses inflammatory responses, thereby providing a solid foundation for postoperative recovery.⁽²⁶⁾ Moreover, EN directly supplies energy to the intestinal tract, modulates immune cells and lymphatic tissues, and facilitates gastrointestinal function recovery, thereby significantly reducing the risk of complications like postoperative intestinal obstruction and enhancing surgical safety.⁽²⁷⁾ This study demonstrates that EN also exhibits tumor-inhibitory effects.

The dynamic balance between Tfh and Tfr cells is a critical node regulating humoral immunity and inflammatory responses. Recent reports indicate that follicular T cells are key mediators in the immunopathogenesis of CRC and may exert either anti-tumor or pro-tumor effects through different mechanisms.⁽²⁸⁾ In this study, a significant imbalance in the Tfh/Tfr ratio was observed in CRC model animals, characterized by an increase in Tfh cells and secretion of the cytokine IL-21, alongside a relative deficiency in the number or function of immunosuppressive Tfr cells and a decrease in inhibitory cytokines such as IL-10 and TGF-β. This imbalance may lead to dysregulated immune surveillance and exacerbated chronic inflammation, thereby promoting CRC progression. However, EN treatment restored this ratio, suggesting that the regulatory effects of EN on CRC may be mediated through modulation of the Tfh/Tfr balance.

Dysregulation of TLRs is closely associated with the development of inflammatory diseases, cancer, and autoimmune disorders.^(29,30) TLR4, a core member of the TLR family, has been reported to play a regulatory role in the onset and progression of CRC. Studies have found that TLR4 expression is abnormally elevated in CRC patients, while its expression is low under normal conditions.⁽³¹⁾ Targeted inhibition of TLR4 signaling can attenuate inflammatory responses in CRC.^(32,33) Consistent with previous reports, the expression of TLR4 and MYD88 was significantly upregulated in model mice, suggesting that TLR4 may be activated via a MYD88-dependent pathway. Further results indicated that EN inhibited the activation of the TLR4 signaling pathway. However, the addition of the TLR4 activator LPS counteracted the effects of EN, demonstrating that the regulation of the Tfh/Tfr ratio by EN is mediated through the TLR4 signaling pathway.

In conclusion, this study provides evidence that EN can inhibit the progression of CRC, and its mechanism may involve the regulation of Tfh/Tfr immune imbalance through the TLR4 signaling pathway. However, future studies are needed to further elucidate the mechanisms by which EN suppresses CRC progression and to evaluate, in clinical CRC patients, the effects of EN formulations on TLR4 pathway activity in tumor tissue, follicular T cell infiltration, and patient prognosis.

Author Contributions

Conceptualization, MZ; methodology, LK and TC; formal analysis, MZ and LK; investigation, CD; data curation, LK and TC; writing – original draft preparation, MZ; writing – review and editing, CD. All authors have read and agreed to the published version of the manuscript.

Funding

Not applicable.

Ethics Statement

All animal experiments were complied with the ARRIVE guidelines and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Drum Tower Hospital Group Suqian Hospital (IACUC-2023-NJ513).

Data Available Statement

Data is available from the corresponding author on request.

Conflict of Interest

No potential conflicts of interest were disclosed.