Nichole R. Blatner

T-Regulatory Cells Shift from a Protective Anti-Inflammatory to a Cancer-Promoting Proinflammatory Phenotype in Polyposis

T-regulatory (Treg) cells play a major role in cancer by suppressing protective antitumor immune responses. A series of observations (from a single laboratory) suggest that Treg cells are protective in cancer by virtue of their ability to control cancer-associated inflammation in an interleukin (IL)-10-dependent manner. Here, we report that the ability of Treg cells to produce IL-10 and control inflammation is lost in the course of progressive disease in a mouse model of hereditary colon cancer. Treg cells that expand in adenomatous polyps no longer produce IL-10 and instead switch to production of IL-17. Aberrant Treg cells from polyp-ridden mice promote rather than suppress focal mastocytosis, a critical tumor-promoting inflammatory response. The cells, however, maintain other Treg characteristics, including their inability to produce IL-2 and ability to suppress proliferation of stimulated CD4 T cells. By promoting inflammation and suppressing T-helper functions, these cells act as a double-edged knife propagating tumor growth.

The significant role of mast cells in cancer

Mast cells (MC) are a bone marrow-derived, long-lived, heterogeneous cellular population that function both as positive and negative regulators of immune responses. They are arguably the most productive chemical factory in the body and influence other cells through both soluble mediators and cell-to-cell interaction. MC are commonly seen in various tumors and have been attributed alternatively with tumor rejection or tumor promotion. Tumor-infiltrating MC are derived both from sentinel and recruited progenitor cells. MC can directly influence tumor cell proliferation and invasion but also help tumors indirectly by organizing its microenvironment and modulating immune responses to tumor cells. Best known for orchestrating inflammation and angiogenesis, the role of MC in shaping adaptive immune responses has become a focus of recent investigations. MC mobilize T cells and antigen-presenting dendritic cells. They function as intermediaries in regulatory T cells (Treg)-induced tolerance but can also modify or reverse Treg-suppressive properties. The central role of MC in the control of innate and adaptive immunity endows them with the ability to tune the nature of host responses to cancer and ultimately influence the outcome of disease and fate of the cancer patient.

Abating colon cancer polyposis by Lactobacillus acidophilus deficient in lipoteichoic acid

An imbalance of commensal bacteria and their gene products underlies mucosal and, in particular, gastrointestinal inflammation and a predisposition to cancer. Lactobacillus species have received considerable attention as examples of beneficial microbiota. We have reported previously that deletion of the phosphoglycerol transferase gene that is responsible for lipoteichoic acid (LTA) biosynthesis in Lactobacillus acidophilus (NCK2025) rendered this bacterium able to significantly protect mice against induced colitis when delivered orally. Here we report that oral treatment with LTA-deficient NCK2025 normalizes innate and adaptive pathogenic immune responses and causes regression of established colonic polyps. This study reveals the proinflammatory role of LTA and the ability of LTA-deficient L. acidophilus to regulate inflammation and protect against colonic polyposis in a unique mouse model.

In colorectal cancer mast cells contribute to systemic regulatory T-cell dysfunction.

T-regulatory cells (Treg) and mast cells (MC) are abundant in colorectal cancer (CRC) tumors. Interaction between the two is known to promote immune suppression or loss of Treg functions and autoimmunity. Here, we demonstrate that in both human CRC and murine polyposis the outcome of this interaction is the generation of potently immune suppressive but proinflammatory Treg (ΔTreg). These Treg shut down IL10, gain potential to express IL17, and switch from suppressing to promoting MC expansion and degranulation. This change is also brought about by direct coculture of MC and Treg, or culture of Treg in medium containing IL6 and IL2. IL6 deficiency in the bone marrow of mice susceptible to polyposis eliminated IL17 production by the polyp infiltrating Treg, but did not significantly affect the growth of polyps or the generation of proinflammatory Treg. IL6-deficient MC could generate proinflammatory Treg. Thus, MC induce Treg to switch function and escalate inflammation in CRC without losing T-cell–suppressive properties. IL6 and IL17 are not needed in this process.

Expression of RORγt Marks a Pathogenic Regulatory T Cell Subset in Human Colon Cancer

Tregs that expand in human colon cancer have proinflammatory properties and contribute to tumor progression. A Treg Melting Pot Some things are not what they seem. Like the allegorical wolf in sheep’s clothing, cell populations that may seem homogeneous may actually contain subsets with different functions. Indeed, such hidden subpopulations may result in contradictory findings in different systems. Blatner et al. now find a subset of regulatory T cells (Tregs) in human colon cancer that may explain disparate clinical outcomes between studies. The authors found preferential expansion in human colon cancer of Tregs that can suppress T cells but are not anti-inflammatory like more classic Tregs. They then looked in a mouse model of hereditary polyposis and found that these cells, which express Foxp3 and RORγt, express the proinflammatory cytokine IL-17 and are directly associated with inflammation and disease progression. The balance between anti-inflammatory Tregs and these “pathogenic” proinflammatory Tregs may play a role in regulating cancer inflammation. Targeting these RORγt+ Tregs may influence disease outcome in colon cancer. The role of regulatory T cells (Tregs) in human colon cancer (CC) remains controversial: high densities of tumor-infiltrating Tregs can correlate with better or worse clinical outcomes depending on the study. In mouse models of cancer, Tregs have been reported to suppress inflammation and protect the host, suppress T cells and protect the tumor, or even have direct cancer-promoting attributes. These different effects may result from the presence of different Treg subsets. We report the preferential expansion of a Treg subset in human CC with potent T cell–suppressive, but compromised anti-inflammatory, properties; these cells are distinguished from Tregs present in healthy donors by their coexpression of Foxp3 and RORγt. Tregs with similar attributes were found to be expanded in mouse models of hereditary polyposis. Indeed, ablation of the RORγt gene in Foxp3+ cells in polyp-prone mice stabilized Treg anti-inflammatory functions, suppressed inflammation, improved polyp-specific immune surveillance, and severely attenuated polyposis. Ablation of interleukin-6 (IL-6), IL-23, IL-17, or tumor necrosis factor–α in polyp-prone mice reduced polyp number but not to the same extent as loss of RORγt. Surprisingly, loss of IL-17A had a dual effect: IL-17A–deficient mice had fewer polyps but continued to have RORγt+ Tregs and developed invasive cancer. Thus, we conclude that RORγt has a central role in determining the balance between protective and pathogenic Tregs in CC and that Treg subtype regulates inflammation, potency of immune surveillance, and severity of disease outcome.

Adenomatous polyps are driven by microbe-instigated focal inflammation and are controlled by IL-10-producing T cells.

Interleukin (IL)-10 is elevated in cancer and is thought to contribute to immune tolerance and tumor growth. Defying these expectations, the adoptive transfer of IL-10-expressing T cells to mice with polyposis attenuates microbial-induced inflammation and suppresses polyposis. To gain better insights into how IL-10 impacts polyposis, we genetically ablated IL-10 in T cells in APC(Δ468) mice and compared the effects of treatment with broad-spectrum antibiotics. We found that T cells and regulatory T cells (Treg) were a major cellular source of IL-10 in both the healthy and polyp-bearing colon. Notably, T cell-specific ablation of IL-10 produced pathologies that were identical to mice with a systemic deficiency in IL-10, in both cases increasing the numbers and growth of colon polyps. Eosinophils were found to densely infiltrate colon polyps, which were enriched similarly for microbiota associated previously with colon cancer. In mice receiving broad-spectrum antibiotics, we observed reductions in microbiota, inflammation, and polyposis. Together, our findings establish that colon polyposis is driven by high densities of microbes that accumulate within polyps and trigger local inflammatory responses. Inflammation, local microbe densities, and polyp growth are suppressed by IL-10 derived specifically from T cells and Tregs.

Current status of interleukin-10 and regulatory T-cells in cancer.

Purpose of review Tumor growth elicits antigen-specific cytotoxic as well as immune suppressive responses. Interleukin-10 (IL-10) is a key immune-suppressive cytokine produced by regulatory T-cells and by helper T-cells. Here, we review pleiotropic functions of IL-10 that impact the immune pathology of cancer. Recent findings The role of IL-10 in cancer has become less certain with the knowledge of its immune stimulatory functions. IL-10 is needed for T-helper cell functions, T-cell immune surveillance, and suppression of cancer-associated inflammation. By promoting tumor-specific immune surveillance and hindering pathogenic inflammation, IL-10 is emerging as a key cytokine in the battle of the host against cancer. Summary IL-10 functions at the cross-roads of immune stimulation and immune suppression in cancer. Immunological mechanisms of action of IL-10 can be ultimately exploited to develop novel and effective cancer therapies.

Mast cell 5-lipoxygenase activity promotes intestinal polyposis in APC Δ468 mice

Arachidonic acid metabolism has been implicated in colon carcinogenesis, but the role of hematopoietic 5-lipoxygenase (5LO) that may impact tumor immunity in development of colon cancer has not been explored. Here we show that tissue-specific deletion of the 5LO gene in hematopoietic cells profoundly attenuates polyp development in the APC(Δ468) murine model of colon polyposis. In vitro analyses indicated that mast cells in particular utilized 5LO to limit proliferation of intestinal epithelial cells and to mobilize myeloid-derived suppressor cells (MDSCs). Mice lacking hemapoietic expression of 5LO exhibited reduced recruitment of MDSCs to the spleen, mesenteric lymph nodes, and primary tumor site. 5LO deficiency also reduced the activity in MDSCs of arginase-1, which is thought to be critical for MDSC function. Together, our results establish a pro-tumorigenic role of hematopoietic 5LO in the immune microenvironment and suggest 5LO inhibition as an avenue for future investigation in treatment of colorectal polyposis and cancer.

β-Catenin Promotes Colitis and Colon Cancer Through Imprinting of Proinflammatory Properties in T Cells

Wnt/β-catenin signaling in T cells is activated during polyposis and colon cancer and drives inflammation and tumor growth by promoting expression of TH17-associated genes including RORγt. β-Catenin, Corrupter of the Tregs It is well known that the numbers and types of lymphocytes that infiltrate colon tumors are relevant to the clinical outcome. However, the reasons for this association are complex and not yet fully understood. Here, Keerthivasan and colleagues identify β-catenin as a culprit and show that it directly contributes to inflammation and colon carcinogenesis in patients and mice with underlying colitis. Keerthivasan and coauthors also provide a mechanistic explanation for the observed effects, showing that the expression of β-catenin in T cells induces the expression of T helper 17 (TH17) genes. This activation of TH17 genes converts T cells to a proinflammatory phenotype and impairs Treg development. Mice overexpressing β-catenin all develop colitis and then colitis-induced cancer, even when the β-catenin is overexpressed only in Tregs. Similarly, human patients with colitis-induced or sporadic colon cancer have abnormally high amounts of β-catenin–expressing T cells in their tumors, confirming the relevance of the mouse findings to human disease. The density and type of lymphocytes that infiltrate colon tumors are predictive of the clinical outcome of colon cancer. High densities of T helper 17 (TH17) cells and inflammation predict poor outcome, whereas infiltration by T regulatory cells (Tregs) that naturally suppress inflammation is associated with longer patient survival. However, the role of Tregs in cancer remains controversial. We recently reported that Tregs in colon cancer patients can become proinflammatory and tumor-promoting. These properties were directly linked with their expression of RORγt (retinoic acid–related orphan receptor-γt), the signature transcription factor of TH17 cells. We report that Wnt/β-catenin signaling in T cells promotes expression of RORγt. Expression of β-catenin was elevated in T cells, including Tregs, of patients with colon cancer. Genetically engineered activation of β-catenin in mouse T cells resulted in enhanced chromatin accessibility in the proximity of T cell factor-1 (Tcf-1) binding sites genome-wide, induced expression of TH17 signature genes including RORγt, and promoted TH17-mediated inflammation. Strikingly, the mice had inflammation of small intestine and colon and developed lesions indistinguishable from colitis-induced cancer. Activation of β-catenin only in Tregs was sufficient to produce inflammation and initiate cancer. On the basis of these findings, we conclude that activation of Wnt/β-catenin signaling in effector T cells and/or Tregs is causatively linked with the imprinting of proinflammatory properties and the promotion of colon cancer.

PI3K/AKT Signaling Is Essential for Communication between Tissue-Infiltrating Mast Cells, Macrophages, and Epithelial Cells in Colitis-Induced Cancer

Purpose: To understand signaling pathways that shape inflamed tissue and predispose to cancer is critical for effective prevention and therapy for chronic inflammatory diseases. We have explored phosphoinositide 3-kinase (PI3K) activity in human inflammatory bowel diseases and mouse colitis models. Experimental Design: We conducted immunostaining of phosphorylated AKT (pAKT) and unbiased high-throughput image acquisition and quantitative analysis of samples of noninflamed normal colon, colitis, dysplasia, and colorectal cancer. Mechanistic insights were gained from ex vivo studies of cell interactions, the piroxicam/IL-10−/− mouse model of progressive colitis, and use of the PI3K inhibitor LY294002. Results: Progressive increase in densities of pAKT-positive tumor-associated macrophages (TAM) and increase in densities of mast cells in the colonic submucosa were noted with colitis and progression to dysplasia and cancer. Mast cells recruited macrophages in ex vivo migration assays, and both mast cells and TAMs promoted invasion of cancer cells. Pretreatment of mast cells with LY294002 blocked recruitment of TAMs. LY294002 inhibited mast cell and TAM-mediated tumor invasion, and in mice, blocked stromal PI3K, colitis, and cancer. Conclusion: The PI3K/AKT pathway is active in cells infiltrating inflamed human colon tissue. This pathway sustains the recruitment of inflammatory cells through a positive feedback loop. The PI3K/AKT pathway is essential for tumor invasion and the malignant features of the piroxicam/IL-10−/− mouse model. LY294002 targets the PI3K pathway and hinders progressive colitis. These findings indicate that colitis and progression to cancer are dependent on stromal PI3K and sensitive to treatment with LY294002. Clin Cancer Res; 19(9); 2342–54. ©2013 AACR.