Intestinal Epithelial TBK1 Prevents Differentiation of T-helper 17 Cells and Tumorigenesis in Mice.

BACKGROUND & AIMS: Intestinal epithelial cells (IECs) regulate intestinal immune cells, particularly development of T-helper 17 (Th17) cells. Deregulation of this process leads to intestinal inflammation and tumorigenesis, via unknown mechanisms. TANK-binding kinase 1 (TBK1) is expressed by IECs and cells in the innate immune system. We studied the functions of TBK1 in the intestinal immune response and tumorigenesis in mice. METHODS: We performed studies of wild-type mice, mice with conditional disruption of Tbk1 (Tbk1IEC-KO), Tbk1IEC-KO mice crossed with ApcMin/+ mice, and Mt-/- mice crossed with ApcMin/+ mice. Some mice were given intraperitoneal injections of a neutralizing antibody against interleukin 17 (IL17) or IL1ß. Intestine tissues were collected from mice and analyzed by histology, for numbers of adenomas and Th17 cells, and expression of inflammatory cytokines by real-time PCR. IECs were isolated from wild-type and Tbk1IEC-KO mice, stimulated with lipopolysaccharide, co-cultured for with bone marrow-derived macrophages, and analyzed by RNA sequencing and biochemical analyses. RESULTS: Compared to ApcMin/+Tbk1WT mice, ApcMin/+Tbk1IEC-KO mice had significant increases in number and size of intestinal polyps, and significantly more Th17 cells in lamina propria. Administration of an antibody against IL17 reduced the number of intestinal polyps in ApcMin/+Tbk1IEC-KO mice to that observed in ApcMin/+Tbk1WT mice. In culture, TBK1-deficient IECs promoted expression of IL1ß by macrophages, which induced differentiation of naïve CD4+ T cells into Th17 cells. RNA sequencing analysis revealed that the TBK1-deficient IECs had increased expression of metallothionein 1 (MT1), an immune regulator that promotes intestinal inflammation. Intestine tissues from ApcMin/+Mt-/- mice had significant fewer Th17 cells than ApcMin/+Mt+/+ mice, and a significantly lower number of polyps. Analyses of colorectal tumors in the Cancer Genome Atlas found colorectal tumors with high levels of MT1 and IL17 mRNAs to be associated with reduced survival times of patients. CONCLUSIONS: Expression of TBK1 by IECs suppresses expression of MT1 and prevents expression of IL1ß by macrophages and differentiation of Th17 cells, to prevent inflammation and tumorigenesis. Strategies to block this pathway might be developed for colorectal tumorigenesis.

Dihydrofolate Reductase and Thymidylate Synthase Transgenes Resistant to Methotrexate Interact to Permit Novel Transgene Regulation.

Methotrexate (MTX) is an anti-folate that inhibits de novo purine and thymidine nucleotide synthesis. MTX induces death in rapidly replicating cells and is used in the treatment of multiple cancers. MTX inhibits thymidine synthesis by targeting dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS). The use of MTX to treat cancer also causes bone marrow suppression and inhibits the immune system. This has led to the development of an MTX-resistant DHFR, DHFR L22F, F31S (DHFR(FS)), to rescue healthy cells. 5-Fluorouracil-resistant TYMS T51S, G52S (TYMS(SS)) is resistant to MTX and improves MTX resistance of DHFR(FS) in primary T cells. Here we find that a known mechanism of MTX-induced increase in DHFR expression persists with DHFR(FS) and cis-expressed transgenes. We also find that TYMS(SS) expression of cis-expressed transgenes is similarly decreased in an MTX-inducible manner. MTX-inducible changes in DHFR(FS) and TYMS(SS) expression changes are lost when both genes are expressed together. In fact, expression of the DHFR(FS) and TYMS(SS) cis-expressed transgenes becomes correlated. These findings provide the basis for an unrecognized post-transcriptional mechanism that functionally links expression of DHFR and TYMS. These findings were made in genetically modified primary human T cells and have a clear potential for use in clinical applications where gene expression needs to be regulated by drug or maintained at a specific expression level. We demonstrate a potential application of this system in the controlled expression of systemically toxic cytokine IL-12.

Progesterone receptor and PTEN expression predict survival in patients with low- and intermediate-grade pancreatic neuroendocrine tumors.

CONTEXT: The PI3K-AKT-mTOR (phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin) pathway plays a crucial role in a subset of advanced pancreatic neuroendocrine tumors (PanNETs). In breast and endometrial carcinoma, activation of this pathway inhibits progesterone receptor (PR) expression. OBJECTIVE: To determine whether combined low expression of PR and phosphatase and tensin homologue (PTEN), a negative regulator of the PI3K-AKT-mTOR pathway, is a prognostic factor. DESIGN: A total of 160 resected PanNETs (89 low grade and 71 intermediate grade) were analyzed for PR and PTEN immunohistochemical positivity and staining was correlated with metastasis-free survival (MFS) and overall survival (OS). Progesterone receptor staining was scored as positive by using 1% or greater as cutoff. Weak/faint staining in greater than 90% of tumor cells was considered low PTEN positivity. RESULTS: Most PanNETs (110 cases, 69%) were both PR and PTEN positive, 45 (28%) were either PR or PTEN positive, and only 5 (3%) had a PR-negative and PTEN-low profile. Combined PR-PTEN positivity was significantly associated with MFS in patients with stage I and II disease (P <.001) and OS in all patients (P < .001) and remained a significant predictor of survival after adjusting for other factors. Patients with PR-negative-PTEN-low PanNETs had the shortest median MFS and OS, compared to those with tumors that were either PR or PTEN positive and with tumors positive for both PR and PTEN (P ≤ .001). CONCLUSION: Combined immunohistochemical assessment of PR and PTEN may help identify a small subset of PanNETs with more aggressive behavior and may aid in risk stratification.

Innate immune responses to Encephalitozoon species infections.

Microsporidia are obligate intracellular, eukaryotic fungi, which have gained recognition as opportunistic parasites in immunocompromised patients. Resistance to lethal microsporidia infections requires a Th1 immune response; how this protection is initiated against Encephalitozoon species is the focus of this review article.