Background: Studies evaluating sex differences in colorectal cancer (CRC) tumor microenvironment are limited, and no previous study has focused on rectal cancer patients’ constitutive immune surveillance mechanisms. The authors aimed to assess gender-related differences in the immune microenvironment of rectal cancer patients. Methods: A systematic review and meta-analysis were conducted up to 31 May 2021, including studies focusing on gender-related differences in the CRC tumor microenvironment. Data on the mutational profile of rectal cancer were extracted from the Cancer Genome Atlas (TCGA). A subanalysis of the two IMMUNOREACT trials (NCT04915326 and NCT04917263) was performed, aiming to detect gender-related differences in the immune microenvironment of the healthy mucosa in patients with early (IMMUNOREACT 1 cohort) and locally advanced rectal cancer following neoadjuvant therapy (IMMUNOREACT 2 cohort). In the retrospective IMMUNOREACT 1 cohort (therapy naive), the authors enrolled 442 patients (177 female and 265 male), while in the retrospective IMMUNOREACT 2 cohort (patients who had neoadjuvant therapy), we enrolled 264 patients (80 female and 184 male). In the prospective IMMUNOREACT 1 cohort (therapy naive), the authors enrolled 72 patients (26 female and 46 male), while in the prospective IMMUNOREACT 2 cohort (patients who had neoadjuvant therapy), the authors enrolled 105 patients (42 female and 63 male). Results: Seven studies reported PD-L1 expression in the CRC microenvironment, but no significant difference could be identified between the sexes. In the TGCA series, mutations of SYNE1 and RYR2 were significantly more frequent in male patients with rectal cancer. In the IMMUNOREACT 1 cohort, male patients had a higher expression of epithelial cells expressing HLA class I, while female patients had a higher number of activated CD4+Th1 cells. Female patients in the IMMUNOREACT 2 cohort showed a higher infiltration of epithelial cells expressing CD86 and activated cytotoxic T cells (P=0.01). Conclusions: Male patients have more frequent oncogene mutations associated with a lower expression of T-cell activation genes. In the healthy mucosa of female patients, more Th1 cells and cytotoxic T cells suggest a potentially better immune response to the tumor. Sex should be considered when defining the treatment strategy for rectal cancer patients or designing prognostic scores.

Appendectomy has been associated with reduced risk of developing ulcerative colitis (UC) or experiencing flares after diagnosis, suggesting the appendix may play a role in UC pathogenesis. Given its function in microbial homeostasis and gut immunity, we investigated the relationship between mucosal microbiota and immune environment of the appendix in UC. Appendix tissue was collected from 85 patients undergoing surgery for UC, acute appendicitis (APA) or colon cancer (CC). Immunophenotyping of dendritic cells (DC), macrophages and T lymphocytes was performed using flow cytometry. Microbiota composition was analyzed via 16S rRNA gene amplicon sequencing. Although alpha diversity did not differ between UC and non-UC appendices, beta diversity indicated significant compositional differences. Five bacterial species (Actinomyces hyovaginalis, Mogibacterium sp. Fusobacterium sp. Pseudoramibacter eubacterium, and Streptococcus anginosus) were significantly reduced in the UC appendix compared to APA. However, no species were associated with UC disease activity. In contrast, UC patients showed a significantly higher frequency of activated DCs (CD1a⁺ HLAdr⁺ CD86⁺). DC activation levels correlated with daily stool frequency and T-cell activation. These findings suggest that the appendix may contribute to UC pathogenesis through immune, rather than microbial, mechanisms - supporting a role for dendritic cell-mediated T-cell priming in colonic inflammation.

Pouchitis is an idiopathic inflammatory disease that may occur in ileal pouches, and it can lead to ileal pouch failure. This was a single-center, randomized, double-blinded, placebo-controlled trial that assessed the effect of Lactobacillus casei (L. casei) DG®, a probiotic strain, on the ileal pouch mucosa to determine the crosstalk between microbiota and mucosal immune system. Fifty-two patients undergoing restorative proctocolectomy were recruited and randomly assigned to receive a daily oral supplementation of L. casei DG® (n = 26) or placebo (n = 26) for 8 weeks from the ileostomy closure (T0) to a pouch endoscopy after 8 weeks (T1) and 1 year (T2). Ileal pouch mucosa samples were collected at T0, T1, and T2. At T1, the L. casei DG®-supplemented group showed a significant reduction of inflammatory cytokines levels compared to T0 baseline levels in the pouch mucosa, whereas in the placebo group cytokines levels resulted stable. In conclusion, probiotic manipulation of mucosal microbiota by L. casei DG®-supplementation after stoma closure in patients who underwent restorative proctocolectomy has a beneficial impact on the ileal pouch microenvironment. Registration number: NCT03136419 (http://www.clinicaltrials.gov).

Background: Anastomotic leaks (ALs) remain a critical complication after rectal cancer surgery. Emerging evidence suggests that local immune dysregulation may play a key role in anastomotic healing. We investigated the immune microenvironment of histologically normal, tumor-adjacent rectal mucosa—a tumor-conditioned field—as a potential substrate for AL predisposition. Methods: IMMUNOREACT 4 is a sub-analysis of the IMMUNOREACT project (clinicaltrials.gov NCT04915326 and NCT04915326), a multicenter translational study evaluating immune features of histologically normal, tumor-adjacent rectal mucosa of patients undergoing colorectal anastomosis. A prospective cohort (n = 121) was analyzed using flow cytometry, in addition to a retrospective cohort (n = 262) using immunohistochemistry. Immune markers of epithelial activation and lymphocyte subsets were compared between patients with and without postoperative ALs. Exploratory predictive models combining immune and clinical variables were developed and evaluated using discrimination, calibration and decision curve analyses. Results: At flow cytometry, the CK⁺HLAabc⁺ MFI (AUC 0.66, 95% CI 0.52–0.80), CD8⁺CD38⁺ cell rate (AUC 0.65, 95% CI 0.52–0.78) and CD3⁺CTLA4⁺ cell rate (AUC 0.65, 95% CI 0.51–0.80) showed moderate predictive potential for ALs. In immunohistochemistry, CD3⁺ (AUC 0.57, 95% CI 0.54–0.60), CD8⁺ (AUC 0.57, 95% CI 0.52–0.62), CD8β⁺ (AUC 0.59, 95% CI 0.53–0.65) and Tbet⁺ (AUC 0.60, 95% CI 0.56–0.64) showed some predictive ability for ALs. The model including CD8β⁺, the BMI, neutrophile/lymphocyte ratio and tumor location had an AUC of 0.67 (95% CI 0.62–0.72). Conclusions: Immune activation within histologically normal, tumor-adjacent rectal mucosa—characterized by epithelial HLA upregulation and cytotoxic or Th1 T cell infiltration—is associated with postoperative ALs. Although predictive accuracy is limited, these findings support the concept that a tumor-conditioned immune microenvironment may predispose patients to impaired anastomotic healing. Integration of mucosal immune profiling with clinical variables represents a promising exploratory approach that warrants further prospective validation.

Viral infections with gastrointestinal involvement remain a significant global health burden with limited therapeutic options. While probiotics show antiviral potential, their impact on primary human intestinal epithelial defenses is poorly defined. This study utilized human intestinal organoid-derived monolayers (ODMs), generated from the non-inflamed mucosa of patients with inflammatory bowel disease, to examine how Bifidobacterium animalis ssp. lactis BB-12 (BB-12) and Lacticaseibacillus rhamnosus GG (LGG) modulate mucosal antiviral pathways. Unlike conventional Caco-2 cells, ODMs preserved physiological cellular diversity and intact innate signaling. Expression of viral receptors and interferon (IFN)-stimulated genes (ISGs) was quantified by RT-qPCR, while the effector 2′-5′-oligoadenylate synthetase 1 (OAS1) was also assessed by immunofluorescence and flow cytometry. Both probiotic strains modulated IFN-associated pathways; however, BB-12 induced a markedly stronger antiviral transcriptional response than LGG. Notably, OAS1 exhibited cell type-specific regulation; while goblet cells showed high basal levels, both probiotics enhanced OAS1 expression selectively in ileal enterocytes. Despite this shared effect, only BB-12 pretreatment significantly restricted Influenza A (H1N1) replication in ileal ODMs, whereas LGG did not significantly affect viral replication. These findings establish human ODMs as a superior platform for probiotic immunology, suggesting that BB-12 more effectively shapes epithelial antiviral “set-points” and highlighting OAS1 as a sensitive component of a broader antiviral program.