An immunological signature to predict outcome in patients with triple-negative breast cancer with residual disease after neoadjuvant chemotherapy.

BACKGROUND: When triple-negative breast cancer (TNBC) patients have residual disease after neoadjuvant chemotherapy (NACT), they have a high risk of metastatic relapse. With immune infiltrate in TNBC being prognostic and predictive of response to treatment, our aim was to develop an immunologic transcriptomic signature using post-NACT samples to predict relapse. MATERIALS AND METHODS: We identified 115 samples of residual tumors from post-NACT TNBC patients. We profiled the expression of 770 genes related to cancer microenvironment using the NanoString PanCancer IO360 panel to develop a prognostic transcriptomic signature, and we describe the immune microenvironments of the residual tumors. RESULTS: Thirty-eight (33%) patients experienced metastatic relapse. Hierarchical clustering separated patients into five clusters with distinct prognosis based on pathways linked to immune activation, epithelial-to-mesenchymal transition and cell cycle. The immune microenvironment of the residual disease was significantly different between patients who experienced relapse compared to those who did not, the latter having significantly more effector antitumoral immune cells, with significant differences in lymphoid subpopulations. We selected eight genes linked to immunity (BLK, GZMM, CXCR6, LILRA1, SPIB, CCL4, CXCR4, SLAMF7) to develop a transcriptomic signature which could predict relapse in our cohort. This signature was validated in two external cohorts (KMplot and METABRIC). CONCLUSIONS: Lack of immune activation after NACT is associated with a high risk of distant relapse. We propose a prognostic signature based on immune infiltrate that could lead to targeted therapeutic strategies to improve patient prognosis.

High vitamin D3 diet administered during active colitis negatively affects bone metabolism in an adoptive T cell transfer model.

Decreased bone mineral density (BMD) represents an extraintestinal complication of inflammatory bowel disease (IBD). Vitamin D3 has been considered a viable adjunctive therapy in IBD. However, vitamin D3 plays a pleiotropic role in bone modeling and regulates the bone formation-resorption balance, depending on the physiological environment, and supplementation during active IBD may have unintended consequences. We evaluated the effects of vitamin D3 supplementation during the active phase of disease on colonic inflammation, BMD, and bone metabolism in an adoptive IL-10-/- CD4⁺ T cell transfer model of chronic colitis. High-dose vitamin D3 supplementation for 12 days during established disease had negligible effects on mucosal inflammation. Plasma vitamin D3 metabolites correlated with diet, but not disease, status. Colitis significantly reduced BMD. High-dose vitamin D3 supplementation did not affect cortical bone but led to a further deterioration of trabecular bone morphology. In mice fed a high vitamin D3 diet, colitis more severely impacted bone formation markers (osteocalcin and bone alkaline phosphatase) and increased bone resorption markers, ratio of receptor activator of NF-κB ligand to osteoprotegrin transcript, plasma osteoprotegrin level, and the osteoclast activation marker tartrate-resistant acid phosphatase (ACp5). Bone vitamin D receptor expression was increased in mice with chronic colitis, especially in the high vitamin D3 group. Our data suggest that vitamin D3, at a dose that does not improve inflammation, has no beneficial effects on bone metabolism and density during active colitis or may adversely affect BMD and bone turnover. These observations should be taken into consideration in the planning of further clinical studies with high-dose vitamin D3 supplementation in patients with active IBD.

NHE3 modulates the severity of colitis in IL-10-deficient mice.

NHE3, the major intestinal Na(+)/H(+) exchanger, was shown to be downregulated and/or inhibited in patients with inflammatory bowel disease (IBD), a phenomenon believed to contribute to inflammation-associated diarrhea. NHE3(-/-) mice spontaneously develop colitis and demonstrate high susceptibility to dextran sulfate-induced mucosal injury. We investigated the effects of NHE3 deficiency on the development of chronic colitis in an IL-10 knockout (KO) mouse model of Crohn's disease. NHE3(-/-) mice were first backcrossed to 129/SvEv mice for >10 generations, with no apparent changes in their survival or phenotype. These mice were crossed with IL-10(-/-) mice on the same genetic background, and the phenotypes of 10-wk-old wild-type (WT), IL-10(-/-), NHE3(-/-), and IL-10(-/-)/NHE3(-/-) (double-KO) mice were studied. Histological and immunohistochemical examination of the colon established important architectural alterations, including increased neutrophilic and mononuclear cell infiltration in double- compared with single-KO mice. Double-KO mice demonstrated increased colonic expression of neutrophil collagenase matrix metalloproteinase-8 and the chemokines macrophage inflammatory protein-2, CXCL1, CXCL10, and CXCL11. Colonic IFNγ, IL-17, and IL-12/23 p40 protein secretion was significantly increased in double- compared with single-KO mice. IL-10(-/-)/NHE3(-/-) mouse colonic epithelium exhibited increased hallmarks of apoptosis, including a significantly increased number of cleaved caspase-3-positive surface epithelial cells. These results highlight the importance of NHE3 in the maintenance of intestinal barrier integrity and in modulating the inflammatory process in IL-10-deficient mice. Chronic NHE3 inhibition or underexpression observed in IBD may therefore contribute to the pathogenesis of IBD by influencing the extent of the epithelial barrier defect and affect the ultimate degree of inflammation.

Modulation of neutrophil motility by curcumin: implications for inflammatory bowel disease.

BACKGROUND: Neutrophils (PMN) are the first cells recruited at the site of inflammation. They play a key role in the innate immune response by recognizing, ingesting, and eliminating pathogens and participate in the orientation of the adaptive immune responses. However, in inflammatory bowel disease (IBD) transepithelial neutrophil migration leads to an impaired epithelial barrier function, perpetuation of inflammation, and tissue destruction via oxidative and proteolytic damage. Curcumin (diferulolylmethane) displays a protective role in mouse models of IBD and in human ulcerative colitis, a phenomenon consistently accompanied by a reduced mucosal neutrophil infiltration. METHODS: We investigated the effect of curcumin on mouse and human neutrophil polarization and motility in vitro and in vivo. RESULTS: Curcumin attenuated lipopolysaccharide (LPS)-stimulated expression and secretion of macrophage inflammatory protein (MIP)-2, interleukin (IL)-1ß, keratinocyte chemoattractant (KC), and MIP-1α in colonic epithelial cells (CECs) and in macrophages. Curcumin significantly inhibited PMN chemotaxis against MIP-2, KC, or against conditioned media from LPS-treated macrophages or CEC, a well as the IL-8-mediated chemotaxis of human neutrophils. At nontoxic concentrations, curcumin inhibited random neutrophil migration, suggesting a direct effect on neutrophil chemokinesis. Curcumin-mediated inhibition of PMN motility could be attributed to a downregulation of PI3K activity, AKT phosphorylation, and F-actin polymerization at the leading edge. The inhibitory effect of curcumin on neutrophil motility was further demonstrated in vivo in a model of aseptic peritonitis. CONCLUSIONS: Our results indicate that curcumin interferes with colonic inflammation partly through inhibition of the chemokine expression and through direct inhibition of neutrophil chemotaxis and chemokinesis.

Limited effects of dietary curcumin on Th-1 driven colitis in IL-10 deficient mice suggest an IL-10-dependent mechanism of protection.

Curcumin (diferulolylmethane) demonstrates profound anti-inflammatory effects in intestinal epithelial cells (IEC) and in immune cells in vitro and exhibits a protective role in rodent models of chemically induced colitis, with its presumed primary mechanism of action via inhibition of NF-kappaB. Although it has been demonstrated effective in reducing relapse rate in ulcerative colitis patients, curcumin's effectiveness in Crohn's disease (CD) or in Th-1/Th-17 mediated immune models of CD has not been evaluated. Therefore, we investigated the effects of dietary curcumin (0.1-1%) on the development of colitis, immune activation, and in vivo NF-kappaB activity in germ-free IL-10(-/-) or IL-10(-/-);NF-kappaB(EGFP) mice colonized with specific pathogen-free microflora. Proximal and distal colon morphology showed a mild protective effect of curcumin only at 0.1%. Colonic IFN-gamma and IL-12/23p40 mRNA expression followed similar pattern ( approximately 50% inhibition at 0.1%). Secretion of IL-12/23p40 and IFN-gamma by colonic explants and mesenteric lymph node cells was elevated in IL-10(-/-) mice and was not decreased by dietary curcumin. Surprisingly, activation of NF-kappaB in IL-10(-/-) mice (phospho-NF-kappaBp65) or in IL-10(-/-);NF-kappaB(EGFP) mice (whole organ or confocal imaging) was not noticeably inhibited by curcumin. Furthermore, we demonstrate that IL-10 and curcumin act synergistically to downregulate NF-kappaB activity in IEC and IL-12/23p40 production by splenocytes and dendritic cells. In conclusion, curcumin demonstrates limited effectiveness on Th-1 mediated colitis in IL-10(-/-) mice, with moderately improved colonic morphology, but with no significant effect on pathogenic T cell responses and in situ NF-kappaB activity. In vitro studies suggest that the protective effects of curcumin are IL-10 dependent.