Inflammation drives colorectal cancer development, and colorectal cancer risk is influenced by dietary factors, including dietary fiber. Hyperactive WNT signaling occurs in colorectal cancer and may regulate inflammation. This study investigated (i) relationships between the inflammatory potential of diet, assessed using the Energy-adjusted Dietary Inflammatory Index (E-DII), and markers of WNT signaling, and (ii) whether DII status modulated the response to supplementation with two types of dietary fiber. Seventy-five healthy participants were supplemented with resistant starch and/or polydextrose (PD) or placebo for 50 days. Rectal biopsies were collected before and after intervention and used to assess WNT pathway gene expression and crypt cell proliferation. E-DII scores were calculated from food frequency questionnaire data. High-sensitivity C-reactive protein (hsCRP) and fecal calprotectin concentrations were quantified. hsCRP concentration was significantly greater in participants with higher E-DII scores [least square means (LSM) 4.7 vs. 2.4 mg/L, P = 0.03]. Baseline E-DII score correlated with FOSL1 (β = 0.503, P = 0.003) and WNT11 (β = 0.472, P = 0.006) expression, after adjusting for age, gender, body mass index, endoscopy procedure, and smoking status. WNT11 expression was more than 2-fold greater in individuals with higher E-DII scores (LSM 0.131 vs. 0.059, P = 0.002). Baseline E-DII modulated the effects of PD supplementation on FOSL1 expression (P = 0.04). More proinflammatory diets were associated with altered WNT signaling and appeared to modulate the effects of PD supplementation on expression of FOSL1. This is the first study to investigate relationships between the E-DII and molecular markers of WNT signaling in rectal tissue of healthy individuals.

Prevention Relevance: Our finding that more inflammatory dietary components may impact large bowel health through effects on a well-recognized pathway involved in cancer development will strengthen the evidence base for dietary advice to help prevent bowel cancer.

Approximately half of colorectal cancer cases are attributable to “modifiable” lifestyle factors, e.g., obesity and diet (1, 2). For example, there is “probable” evidence that higher consumption of foods containing dietary fiber lowers colorectal cancer risk (3). However, because foods and nutrients are not consumed in isolation, it is important to assess diet healthfulness holistically when investigating relationships with disease-related outcomes (4).

Inflammation modulates colorectal cancer risk (5–8), and individuals with inflammatory bowel disease (IBD) are at increased risk of colorectal cancer (9). The Dietary Inflammatory Index (DII) quantifies the inflammatory potential of the whole diet (10) and comprises 45 food parameters, including 36 anti-inflammatory components, e.g., dietary fiber (10). The DII has been validated in various cohorts and shown to correlate with the expression of inflammatory markers, e.g., C-reactive protein (CRP), IL6, and IL-10 (11–15). Furthermore, more proinflammatory DII scores are associated with greater risk of all-cause mortality (16) and of cancers (17) including colorectal cancer (18). A systematic review and meta-analysis of nine studies revealed that individuals in the highest DII category of exposure had 40% increased risk of colorectal cancer compared with those in the lowest category, translating to a 7% increase in colorectal cancer risk for each one-point increase in DII score (18). The underlying mechanisms linking DII and colorectal cancer risk are not fully understood, but are likely to include effects of the inflammation-related components of the diet on insulin sensitivity, the gut microbiome, local inflammation (which promotes cell proliferation and mutagenesis; ref. 19), and the production of reactive oxygen species (7, 18), as well as modulation of molecular pathways, e.g., WNT signaling.

The WNT signaling pathway regulates cellular processes such as proliferation that contribute to the maintenance of homeostasis and tissue self-renewal in the large intestine (20). Aberrant WNT signaling in colorectal cancer includes abnormal expression of β-catenin and adenomatous polyposis coli (APC; ref. 21). Furthermore, WNT genes, e.g., WNT11, are upregulated in colonic tissue from patients with ulcerative colitis (UC) (22). Recent evidence suggests that WNT signaling may influence the inflammatory state via cross-talk with pathways including NF-κB and MAPK (23). WNT signaling may also regulate the activity of inflammatory pathways, e.g., β-catenin inhibits NF-κB signaling (24), and the expression of inflammatory cytokines and chemokines, e.g., WNT5A induces IL1 and IL6 (25–27). In addition, inflammatory cytokines regulate mucosal WNT signaling via Protein Kinase B (AKT) signaling (28).

The WNT pathway plays an important role in the link between diet, adiposity, and physical activity, and gastrointestinal cancers including colorectal cancer (29, 30) and several dietary factors modulate WNT pathway activity (31, 32). We have shown that higher adherence to the World Cancer Research Fund (WCRF) Cancer Prevention Recommendations, which include anti-inflammatory components of the DII such as dietary fiber, was associated with altered expression of WNT pathway components (33). Adherence to the subrecommendation on dietary fiber intake was associated with significantly lower rectal expression of β-catenin and of WNT11 (33). Higher dietary fiber intake protects against colorectal cancer (3), and short-chain fatty acids (SCFA) produced by dietary fiber fermentation, primarily butyrate, are chemoprotective and exert anti-inflammatory effects, some of which may be mediated via modulation of WNT signaling (34, 35). In the Dietary Intervention, Stem cells and Colorectal cancer (DISC) Study, we supplemented healthy individuals with two types of dietary fiber, resistant starch (RS), and polydextrose (PD), and observed downregulation of β-catenin, c-MYC, SFRP1, and SFRP2 in the rectal mucosa (36).

Taken together, the evidence suggests that the WNT pathway mediates the effects of diet, including perhaps its inflammatory potential, on colorectal cancer risk. Therefore, this study had two aims: (i) to test the hypothesis that diet-associated inflammation is related to WNT pathway activity by investigating relationships between DII score and expression of WNT pathway components in the rectal mucosa of healthy individuals; and (ii) to investigate whether the inflammatory potential of habitual diet modulated the response to supplementation with RS and/or PD in the DISC Study. We also investigated relationships between DII score and crypt cell proliferative state (CCPS) as a functional outcome of WNT signaling, and biomarker of colorectal cancer risk (35, 36).

The DISC study participants

This study used data and samples from the DISC Study (ClinicalTrials.gov Identifier: NCT01214681), a randomized, placebo-controlled dietary intervention that investigated the effects of two types of dietary fiber (RS and PD) on markers of colorectal cancer risk (36, 37). The study was conducted according to the guidelines laid down in the Declaration of Helsinki, and all procedures involving human subjects were approved by the Newcastle and North Tyneside Research Ethics Committee (REC No. 09/H0907/77). Healthy participants were recruited from gastroenterology out-patients departments at North Tyneside General Hospital, North Shields, UK, and Wansbeck General Hospital, Ashington, UK, between May 2010 and July 2011. Written-informed consent was obtained from all participants.

Dietary intervention

Participants were supplemented with RS and/or PD or placebo for 50 days in a 2 × 2 factorial design. At least 1 week after their first endoscopy appointment, participants were randomized to one of four intervention groups: RS (23 g Hi-maize 260, Ingredion, Food Innovation), PD (12 g of Litesse Ultra, DuPont Danisco), RS and PD, or double placebo [12 g of Maltodextrin (RS placebo) and 23 g of Amioca starch (PD placebo)]. Randomization was stratified by endoscopy procedure (flexible sigmoidoscopy or colonoscopy).

Sample collection

Phenotypic data (e.g., height and body weight) and biological samples were collected before and after intervention. Rectal mucosal biopsies were collected at endoscopy (colonoscopy or flexible sigmoidoscopy for baseline samples and rigid sigmoidoscopy for postintervention samples) using Biobite Biopsy forceps (Medical Innovations) from the midrectum (10 cm from the ano-rectal verge). For the collection of stool samples, participants were given a sealable bucket pot, a disposable bedpan, two ice packs (to be frozen prior to sample collection), and a cool bag. Participants stored samples in cool bags containing the frozen ice packs. Preintervention stool samples were collected at least 7 days after the endoscopy appointment and picked up by the research team from the participants' homes, and postintervention samples were brought by the participant to the second endoscopy appointment. Samples were divided into aliquots and stored at −80°C until analysis.

Measurement of inflammatory markers

High-sensitivity CRP (hsCRP) in serum was quantified at Newcastle Laboratories, Freeman Hospital (Newcastle upon Tyne, UK) from blood samples collected in one 5 mL BD Vacutainer SST II Advance tube with gold hemogard closure (Becton Dickinson). Fecal calprotectin was quantified in extracts from 100 mg of stool using the Fecal Sample Preparation Kit (Calpro AS). Prior to preparation of fecal extracts, samples were defrosted overnight and mixed using Stomacher80 Biomaster (Seward Ltd.). Extracts were diluted 1:20 in sample dilution buffer and used to quantify fecal calprotectin using the Calprolab Calprotectin ELISA (ALP) kit (Calpro AS). Optical density was read after 40-minute incubation with enzyme substrate solution on a FLUOstar Omega microplate reader (BMG Labtech Ltd.) operated by BMG Omega software version 1.20.

Expression of WNT pathway components

RNA was extracted from rectal mucosal biopsies using the RNeasy Mini Kit (Qiagen) using five 3 mm glass beads (VWR) and QiaShredders (Qiagen) for tissue disruption and homogenization, respectively. cDNA was synthesized from 1 μg RNA using the QuantiTect Reverse Transcription Kit (Qiagen). The expression of 12 WNT pathway genes and two reference genes (18S and β2M) was quantified by qPCR using the StepOnePlus Real Time PCR system (Applied Biosystems). These target genes were selected by reviewing the literature to identify WNT genes that were (i) implicated in colorectal carcinogenesis (selection criterion 1) and (ii) whose expression is modified by butyrate (a product of dietary fiber fermentation; selection criterion 2; Supplementary Table S1). In addition, APC was chosen due to its key role in the WNT pathway and in colorectal cancer. We have found that the expression 18S and β2M reference genes is stable in rectal mucosal samples (36).

Quantification of CCND1, c-MYC, and SFRP1 was performed using primers designed and optimized by Dr. Nigel J. Belshaw and Dr. Wing Leung (Quadram Institute, Norwich, UK; Supplementary Table S2). For these three genes, together with two reference genes (18S and β2M), qPCR reactions contained 5 μL ImmoMix (2x; Bioline), 0.1 μL MgCl2 (50 mmol/L; Bioline), 1 μL BSA (10 mg/mL; Ambion), 0.2 μL ROX Reference Dye (50x; Invitrogen), 0.06 μL SYBR Green (100x; Invitrogen), 0.6 μL RNase-free water, 0.02 μL each of forward and reverse primers (100 μmol/L), and 3 μL of cDNA. The program was run for a 10-minute activation step at 95°C followed by 40 cycles of 30 seconds each, denaturation at 95°C, annealing at 60°C, and extension at 72°C. For the remaining nine genes, qPCR was performed using the QuantiTect SYBR Green PCR Kit (Qiagen) and QuantiTect primer assays (Qiagen, Supplementary Table S3), with reactions containing 15 μL of master mix and 5 μL of the sample cDNA. The program was run for a 15-minute activation step at 95°C followed by 40 cycles of 15-second denaturation at 94°C, 30-seconds annealin at 55°C, and 30-second extension at 72°C. All samples were run in duplicate. Each plate contained pre- and postintervention samples for each participant and representatives from each intervention group. Data collection was during the extension stage, and melting curve analysis was performed. Gene expression data are expressed as adjusted values (2−ΔCt × 10,000) relative to the geometric mean of 18S and β2M reference genes (38).

Assessment of rectal CCPS

Rectal CCPS was assessed in whole, microdissected, Schiff reagent–stained crypts (37). Briefly, Carnoy's fixed rectal mucosal biopsies were hydrated in 50% ethanol, followed by 25% ethanol, for 10 minutes each at room temperature. Biopsies were then hydrolyzed in 1 mol/L HCl for 10 minutes at 60°C and stained with Schiff reagent (Surgipath) for 1 hour at room temperature. The Schiff reagent was replaced with 1 mL of 45% acetic acid, and whole crypts were microdissected using an Olympus SZ40 dissecting microscope and Leica CLS 150X light source. On a microscope slide with a drop of 45% acetic acid, rows of individual crypts (bases of the crypts facing upward) were teased apart using fine gauge hypodermic needles (25G × 5/8″ Terumo, Belgium) and covered and sealed with a cover slip (Surgipath, Leica). Ten intact crypts were selected at random, and each divided into ten equal compartments longitudinally, starting from the base of the crypt. The number of mitotic cells in each compartment was counted, and from this, the proportion of mitotic cells in the upper half of the crypt was calculated, as well as crypt width and length measurements, from which crypt volumes were calculated.

Quantification of fecal SCFA concentrations

SCFA concentrations were quantified by gas chromatography using pivalic acid as an internal standard as described previously (39). Briefly, 1 mL 20 mmol/L pivalic acid and 5 mL water were added to 1 g of fecal sample, mixed thoroughly and centrifuged at 5,000 × g for 5 minutes. Note that 0.250 mL saturated oxalic acid solution was added to 0.5 mL of the supernatant and incubated at 48°C for 1 hour. This was centrifuged at 16,000 × g for 5 minutes, and the supernatant fraction was used for analysis as described previously (40).

Calculation of energy-adjusted DII

Habitual diet was assessed at baseline using a food frequency questionnaire (FFQ) adapted from that used in the EPIC–Norfolk Study (version 6, CAMB/PQ/6/1205; ref. 41), asking participants for their average consumption of foods over the last year. The inflammatory potential of diet was assessed by calculating the DII scores and energy-adjusted DII (E-DII) scores (10). Dietary intakes of 29 food components (alcohol, beta-carotene, carbohydrates, cholesterol, fiber, total fat, iron, trans fatty acids, folate, energy, magnesium, monosaturated fatty acids, niacin, polyunsaturated fatty acids, protein, retinol, riboflavin, saturated fatty acids, selenium, thiamine, vitamin B6, vitamin B12, vitamin C, vitamin D, vitamin E, zinc, onions, garlic, and tea) were included in the calculation (10). Intake from foods only, not supplements, was included in DII calculations. A total E-DII score was calculated by adding the scores for each of the 29 food parameters, and expressed per 1,000 kilocalories (4.187 MJ) consumed. Higher E-DII scores indicate more proinflammatory diets, whereas lower E-DII scores represent less inflammatory, or more anti-inflammatory, diets.

Statistical analyses

For descriptive statistical analyses, independent sample t tests and Fisher exact tests were used for comparisons between the lower and higher E-DII groups. In cross-sectional analyses, multivariable regression models were used to investigate relationships between E-DII and the measured outcomes, with model 2 adjusting for age, gender, endoscopy procedure, body mass index (BMI), and smoking status as covariates. For categorical analyses, participants were divided into a low E-DII (more anti-inflammatory) and a high E-DII (more pro-inflammatory) group by dichotomizing at the median E-DII (0.700). Differences in the measured outcomes between the low and high E-DII groups at baseline were investigated using the ANOVA General Linear Model (GLM) and adjusting for age, gender, endoscopy procedure, BMI, and smoking status as covariates. Models were not adjusted for total energy intake because it is one of the components of the DII and is explicitly accounted for in the calculation of E-DII scores.

For the randomized controlled trial (RCT), interactions between E-DII status at baseline and the effects of the dietary intervention (RS and/or PD) on the measured outcomes after intervention were investigated using the ANOVA GLM, adjusting for preintervention measurement, age, gender, endoscopy procedure, BMI, and smoking status as covariates. All statistical analyses were performed using IBM SPSS Statistics version 25. P < 0.05 was considered statistically significant.

Participant demographics

Seventy-five healthy participants were recruited to the DISC Study (Table 1). The mean age of participants was 52 years (range, 30–80 years), and 53% were female. Most of the participants (97%) were White. For more details, see the study by Malcomson and colleagues (36).

Inflammatory potential of diets of DISC study participants

The mean E-DII score was slightly proinflammatory (0.736 ± 0.253), and E-DII scores ranged from −4.480 to 5.030. Table 1 shows the participants' characteristics according to E-DII group. Participants with more proinflammatory diets, i.e., those in the higher-E-DII group, were more likely to be former or current smokers (P = 0.03).

Relationships between E-DII and inflammatory markers

hsCRP concentrations in the higher, more proinflammatory, E-DII group were approximately 2-fold greater compared with the lower E-DII group (P = 0.03; Table 2). Although fecal calprotectin concentrations were, on average, 32% higher in individuals in the higher E-DII group, the considerable interindividual variation within groups meant that this difference was not statistically significant (P = 0.46). There were no significant relationships between E-DII and fecal calprotectin or hsCRP concentrations when investigated using the regression models (Supplementary Table S4).

Relationships between E-DII and WNT pathway markers

In the unadjusted multilevel linear regression model, E-DII score was significantly associated with baseline (preintervention) rectal expression of Fos-related antigen 1 (FOSL1; β = 0.414, P = 0.01) and WNT11 (β = 0.365, P = 0.009; Table 3). These findings were strengthened in the fully adjusted model [FOSL1 (β = 0.503, P = 0.003) and WNT11 (β = 0.472, P = 0.006)]. Furthermore, participants in the higher E-DII group had more than 2-fold higher expression of WNT11 compared with those in the lower E-DII group (least squares means 0.131 vs. 0.059, P = 0.002, Fig. 1).

There were no significant associations observed between E-DII and the remaining 10 WNT pathway components (Table 3), nor differences in their expression between the lower and higher E-DII groups (Supplementary Table S5).

Interestingly, there was a weak but significant correlation between rectal mucosal WNT11 expression and fecal calprotectin concentrations (Spearman's correlation coefficient = 0.362, P = 0.01). No such relationship was observed, however, for hsCRP (Spearman's correlation coefficient = 0.142, P = 0.33), and there were no significant correlations between rectal FOSL1 expression and the inflammatory markers measured in this study [hsCRP (Spearman's correlation coefficient = 0.234, P = 0.16) and fecal calprotectin (Spearman's correlation coefficient = -0.248, P = 0.15)].

Relationships between E-DII and rectal CCPS at baseline

There were no significant associations between E-DII score and total mitoses in the rectal epithelium, proportion of mitoses in the top half of the crypts (CCPS outcomes measured in this study), or crypt dimensions (length, width, and volume; Table 4). In addition, crypt dimensions and rectal CCPS outcomes did not differ between participants with lower and higher E-DII scores (Supplementary Table S6). Furthermore, there were no significant correlations between expression of FOSL1 and WNT11 (that were associated with E-DII; Table 3), and CCPS outcomes or crypt dimensions (Supplementary Table S7).

Interaction between baseline E-DII and the effects of supplementation with RS and PD on the measured outcomes

The effects of RS and PD on WNT pathway–related outcomes have been published previously (36, 37). In the present study, we investigated whether E-DII scores, derived from habitual diet data assessed at baseline, modulated the response to RS and PD. There were no significant differences in the inflammatory potential of habitual diet (i.e., E-DII score) according to dietary intervention group at baseline (P = 0.64; Supplementary Table S8). We observed a significant interaction effect of E-DII and PD supplementation on postintervention rectal FOSL1 expression (P = 0.04, Fig. 2). Individuals in the higher E-DII group at baseline, with a more proinflammatory diet, had a lower postintervention FOSL1 expression when given PD compared with those with less inflammatory E-DII scores. In individuals given the placebo, individuals with higher E-DII scores had higher postintervention FOSL1 expression compared with those with less inflammatory diets in the lower E-DII group. There were no interaction effects between EDII and RS and/or PD on the other quantified genes or inflammatory and CCPS markers measured (Supplementary Table S9).

Chronic inflammation is a key risk factor for colorectal cancer by causing mutations, chromosomal alterations, and aberrant patterns of DNA methylation which lead to oncogene activation, tumor suppressor inactivation, dysregulated DNA repair, and chromosomal instability (42). In addition, both inflammatory state and colorectal cancer risk are influenced by environmental and lifestyle factors, especially diet (3, 43). Aberrant WNT signaling occurs early in the tumorigenic process (44) and provides both a selective advantage for the initial clonal expansion and genetic instability for subsequent tumor progression and malignant transformation (45). WNT signaling is modulated by dietary factors including dietary fiber (34), and there may be cross-talk between WNT signaling and inflammatory pathways (24). The inflammatory potential of individual diets can be assessed using the DII (10); higher DII values indicate a more proinflammatory diet and have been associated with increased expression of inflammatory markers (11, 12, 14, 15) as well as greater colorectal cancer risk (18). However, little is known about the relationships between DII scores and molecular markers of colorectal cancer risk. This study is the first to report associations between DII and WNT pathway activity, CCPS, and crypt dimensions in the healthy rectal mucosa, and to explore the potential modulation by habitual DII of the response to supplementation with dietary fiber.

E-DII is associated with expression of WNT11 and FOSL1 in the rectal mucosa of healthy adults

We observed significant positive correlations between the E-DII scores and expression of WNT11 and FOSL1 in the rectal mucosa of DISC Study participants. WNT11 is a ligand that regulates the activation of both canonical and noncanonical WNT signaling pathways (46), and its expression is induced by WNT pathway activation and by factors such as TGFβ (47). In the intestinal epithelium, WNT11 regulates cell proliferation, intercellular adhesion, and migration and, consequently, is implicated in tumorigenesis (48). WNT11 is upregulated in colorectal cancer (49) and is involved in cancer progression (50). Upregulation of WNT11 in colorectal adenocarcinomas may play a role in colorectal tumorigenesis through stimulation of WNT signaling (49), and greater expression of WNT11 has been reported in patients with UC (22). In the present study, a “less inflammatory diet,” as assessed by a lower E-DII score, was associated with reduced WNT11 expression. When stratified by age, the difference between E-DII groups remains statistically significant for the younger (<50 years) group only (P = 0.03; Supplementary Table S10). It is probable that the reduction in group sizes coupled with the greater interindividual variability in participants aged ≥50 years limited our ability to detect the between E-DII groups among older participants. In addition, rectal WNT11 expression correlated positively with fecal calprotectin, a marker of gastrointestinal inflammation. Interestingly, we have previously reported lower rectal mucosal expression of WNT11 in participants with greater adherence to the WCRF Cancer Prevention Recommendations (33). Furthermore, adherence to the recommendation to consume ≥25 g dietary fiber/day, an anti-inflammatory component of the DII (10), and to the recommendation to be physically active was associated with lower WNT11 expression (33). These findings suggest that WNT11 may be particularly sensitive to modulation by environmental and lifestyle factors, including diet. Although they will require confirmation in independent studies, our findings suggest that such relationships between lifestyle factors and rectal mucosal markers may be affected by age, which is particularly important as this is the strongest risk factor for colorectal cancer. Furthermore, because the molecular characteristics of sporadic colorectal cancer cases in early-onset (age < 50 years) differ from those developing colorectal cancer at an older age (51), and age-dependent effects on other markers of colorectal cancer risk have been reported (32, 37, 52), further studies investigating these age-dependent processes are warranted.

Greater expression of FOSL1 (also known as FRA-1) was also associated with higher E-DII scores, i.e., more inflammatory diets, in the rectal mucosa of healthy individuals. FOSL1 is a member of the FOS oncogene family and a target gene of the WNT pathway. Increased FOSL1 protein and greater β-catenin accumulation occur in human colorectal adenocarcinomas (53). Interestingly, increased IL6 secretion as a consequence of activation of STAT3 signaling promotes FOSL1 deacetylation in colorectal cancer cell lines, resulting in increased FOSL1 expression. Furthermore, increased FOSL1 protein was observed in cancer tissue from patients with colorectal cancer, and this correlated with abundance of the proinflammatory cytokine IL6 (54). Aberrant FOSL1 expression has also been reported in patients with mild UC, and expression levels were positively correlated with concentrations of IL11 in biopsies from patients with UC (55). To our knowledge, this is the first study to report relationships between diet quality and FOSL1 expression in the rectal mucosa.

E-DII and expression of other WNT signaling genes in the rectal mucosa

In the present study, we did not detect relationships between E-DII and the expression of the other 10 quantified WNT pathway–related genes. As this is the first study to explore such relationships, we could not be specific about which WNT genes would be modulated by E-DII. Because these target genes were selected because of their potential modulation by dietary fiber (36), it is possible that not all are responsive to differences in the inflammatory potential of diet. However, a previous mouse study suggested that high fat diet–induced inflammation was associated with downregulation of Apc and increased expression of Ctnnb1 and target genes, e.g., c-Jun and Ccnd1 in the colon (56). In the present study performed in healthy human adults, we observed no relationships between the inflammatory potential of habitual diet and expression of these four genes in the rectal mucosa.

Dietary fiber supplementation may modulate the relationships between E-DII and FOSL1 in the rectal mucosa

We investigated whether baseline E-DII modulated the effects of supplementing healthy individuals with dietary fiber (provided as RS and/or PD) on WNT pathway–related markers of colorectal cancer risk. We observed a significant interaction between E-DII and supplementation with PD on rectal expression of FOSL1, in which those with poorer, more inflammatory diets (i.e., higher E-DII scores) had lower postintervention FOSL1 expression. Because lower FOSL1 expression may be associated with lower colorectal cancer risk, this finding suggests that those with poorer diets may benefit more from supplementation with PD. The opposite was observed for those given placebo; those with higher E-DII scores had higher postintervention FOSL1 expression. To our knowledge, this is the first study to explore whether baseline E-DII modulates the response to a dietary intervention. However, there is evidence of a poorer response to bariatric surgery (smaller weight and fat mass changes) in individuals with more inflammatory baseline DII scores (57). We explored whether the observed differences in FOSL1 expression in response to PD supplementation between lower and higher E-DII groups could have resulted from differences in fecal SCFA concentrations. We hypothesized that individuals with poorer diets, indicated by higher E-DII scores, may have lower SCFA concentrations at baseline, which may lead to greater relative change in SCFA with PD supplementation, and therefore respond better to the dietary intervention. However, there were no significant differences between individuals with lower and higher E-DII scores in the fecal concentrations or proportions of SCFAs at baseline (Supplementary Table S11) nor in the change in SCFAs after intervention. The potential mechanisms underpinning the observed effects, and why these were observed for PD supplementation but not for RS, are unclear. Therefore, further research is warranted to substantiate this novel finding.

Greater CCPS, and especially a higher proportion of mitotic cells in the top half of the crypt, is a biomarker of colorectal cancer risk (58, 59). In the present study, for the first time, we investigated relationships between E-DII score and rectal CCPS in healthy participants, but we did not observe any significant relationship. Previous studies suggest that dietary components, such as dietary fiber, that modulate inflammation may mediate colorectal cancer risk via effects on cell proliferation (37, 52, 60, 61). Butyrate, produced from bacterial fermentation of dietary fiber, activates T-regulatory cells that block proinflammatory T cells, leading to reduced production of cytokines associated with the stimulation of cell proliferation (62). Chronic inflammation is associated with activation of WNT signaling, induced by the STAT3 pathway, which stimulates cell proliferation in the colorectal epithelium (63). In a mouse model of chronic colitis, supplementation with red raspberries, which contain anti-inflammatory compounds, was associated with reduced expression of WNT pathway components that regulate the cell cycle (CCND1 or c-MYC) as well as cell proliferation in colonic tissue (64). Furthermore, WNT pathway activity, assessed by the quantification of β-catenin expression, and STAT3 signaling were also reduced by red raspberry supplementation (64).

Strengths and limitations of study

This was a tightly controlled study with careful measurement of exposures, covariates, and outcomes. The DISC Study is one of the largest studies assessing a variety of molecular and functional markers of large bowel health and of colorectal cancer risk in the macroscopically normal rectal mucosa, and the largest RCT investigating these effects of dietary fiber in healthy people. All participants were recruited from the same region (two hospitals in the North East of England) using stringent inclusion and exclusion criteria, such as excluding any participants on anti-inflammatory medication, thus minimizing the effects of potential confounders. However, this study is limited by its relatively small sample size and lack of ethnic diversity. Although the relatively homogenous population within this study reduces the effects of potential confounders, this may limit the generalizability of findings to other populations, with different dietary patterns, socioeconomic status, education, ethnicity, and geographical location.

Estimation of habitual dietary intake using self-reported data from FFQs, which are prone to recall bias and misreporting, was used to calculate E-DII scores. At the individual level, BMI has well-recognized limitations as an index of adiposity. Future studies should investigate potential modifying effects of adiposity on E-DII links with colorectal cancer risk. Further, baseline biopsies were collected by two different endoscopy procedures, with different bowel preparation requirements. However, for the RCT, randomization was stratified according to baseline endoscopy procedure, and this was included as a covariate during statistical analyses. In addition, all of the biopsies were collected from the same anatomical site, so reducing potential confounding. Our use of data from a cross-sectional study means that we cannot attribute causality to the observed relationships between E-DII and expression of WNT pathways genes in the rectal mucosa. Such relationships will need to be confirmed in future intervention studies.

Our findings suggest that the WNT signaling pathway may mediate some effects of inflammatory dietary components on markers of large bowel health in the healthy rectal mucosa. More specifically, more proinflammatory diets are associated with greater expression of FOSL1 and WNT11, both of which are more highly expressed in colorectal cancer tissue and in tissue from patients with IBD. Furthermore, individuals with greater E-DII scores had reduced rectal FOSL1 expression after PD supplementation. Expression of both FOSL1 and WNT11 has been associated with levels of inflammatory cytokines such as IL6 (47, 54). Interestingly, we observed a weak but significant correlation between rectal WNT11 expression and the concentration of fecal calprotectin, a local marker of intestinal inflammation. Therefore, FOSL1 and WNT11, putative markers of colorectal cancer risk, may be responsive to dietary factors and may have potential as surrogate endpoints in dietary intervention studies. Because WNT signaling is also modulated by adipose tissue, and obesity-induced inflammation is a risk factor for colorectal cancer, further investigations exploring molecular changes in adipose tissue may be of interest (65). Furthermore, investigations into the potential modulation of diet-related inflammation and WNT signaling by obesity and/or body mass change are warranted (29).

To our knowledge, this is the first study to investigate relationships between the inflammatory potential of diet, assessed using the E-DII, and molecular markers in the target tissue (i.e., rectal tissue) of healthy individuals and the first to explore whether E-DII modulates the response to supplementation with dietary fiber. Further investigations, using transcriptome-wide and multiomic approaches studies, of how the inflammatory potential of habitual diet, assessed using the DII, modulates the response to dietary and other lifestyle interventions are warranted.

F.C. Malcomson reports grants from Biotechnology and Biological Sciences Research Council (BBSRC) during the conduct of the study. N.D. Willis reports grants from BBSRC during the conduct of the study. I. McCallum reports grants from BBSRC during the conduct of the study. N. Shivappa reports non-financial support and other from Connecting Health Innovations outside the submitted work. M.D. Wirth reports personal fees from Connecting Health Innovations outside the submitted work. J.R. Hébert reports grants and other from Connecting Health Innovations LLC (CHI) outside the submitted work; in addition, J.R. Hébert has a patent for Federally registered trademark for the DII; issued, licensed, and with royalties paid from Connecting Health Innovations LLC (CHI). I.T. Johnson reports grants from BBSRC during the conduct of the study. J.C. Mathers reports grants from BBSRC during the conduct of the study. No disclosures were reported by the other authors.

F.C. Malcomson: Conceptualization, resources, data curation, formal analysis, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing. N.D. Willis: Resources, investigation, project administration. I. McCallum: Resources, investigation. L. Xie: Resources. N. Shivappa: Resources, investigation, methodology, writing–review and editing. M.D. Wirth: Resources, investigation, methodology, writing–review and editing. J.R. Hébert: Conceptualization, resources, supervision, methodology, writing–review and editing. B. Kocaadam-Bozkurt: Resources. A. Özturan-Sirin: Resources. S.B. Kelly: Conceptualization, supervision. D.M. Bradburn: Conceptualization, supervision. N.J. Belshaw: Conceptualization. I.T. Johnson: Conceptualization, supervision, funding acquisition, project administration, writing–review and editing. J.C. Mathers: Conceptualization, resources, supervision, funding acquisition, writing–original draft, project administration, writing–review and editing.

This work was supported by an award from the BBSRC Diet and Health Research Industry Club (DRINC; grant number BB/H005013/1) to J.C. Mathers, I.T. Johnson, N.J. Belshaw, and S.B. Kelly. I. McCallum was funded by a fellowship from Northumbria NHS Foundation Trust. The authors acknowledge further support from the Newcastle University Centre for Ageing and Vitality, which is funded by the Medical Research Council and BBSRC as part of the cross-council Lifelong Health and Wellbeing Initiative (grant no. MR/L016354/1) and from the Wellcome Trust. Further funding was awarded by the Wellcome Trust Broadening Our Horizons scheme to support the collaboration between Newcastle University and the University of South Carolina.

We are very grateful to DISC Study participants without whom this study would have been impossible. We acknowledge the staff at the gastroenterology units at Wansbeck and North Tyneside General Hospitals for their help and support with participant recruitment and study visits. We thank Ingredion, formerly National Starch, Food Innovation, USA, and DuPont Danisco, Finland, for supplying the RS and PD, respectively. We are very grateful to Julie Coaker for her very kind assistance with training in the assessment of CCPS.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data