Highlights d Lactobacillus species sense intestinal iron levels and attenuate host iron absorption d Microbial metabolites DAP and reuterin are novel HIF-2a inhibitors d Gut microbial metabolites regulate intestinal iron storage via ferritin regulation d Gut microbiota can be therapeutically targeted for ironrelated disorders

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Tendon is a dynamic tissue whose structure and function is influenced by mechanical loading, but little is known about the fundamental mechanisms that regulate tendon growth and remodeling in vivo. Data from cultured tendon fibroblasts indicated that the p38 MAPK pathway plays an important role in tendon fibroblast proliferation and collagen synthesis in vitro. To gain greater insight into the mechanisms of tendon growth, and explore the role of p38 MAPK signaling in this process, we tested the hypotheses that inducing plantaris tendon growth through the ablation of the synergist Achilles tendon would result in rapid expansion of a neotendon matrix surrounding the original tendon, and that treatment with the p38 MAPK inhibitor SB203580 would prevent this growth. Rats were treated with vehicle or SB203580, and subjected to synergist ablation by bilateral tenectomy of the Achilles tendon. Changes in histological and biochemical properties of plantaris tendons were analyzed 3, 7, or 28 days after overload, and comparisons were made to non-overloaded animals. By 28 days after overload, tendon mass had increased by 30% compared to non-overloaded samples, and cross-sectional area (CSA) increased by around 50%, with most of the change occurring in the neotendon. The expansion in CSA initially occurred through the synthesis of a hyaluronic acid rich matrix that was progressively replaced with mature collagen. Pericytes were present in areas of active tendon growth, but never in the original tendon ECM. Inhibition of p38 MAPK resulted in a profound decrease in IL6 expression, and had a modest effect on the expression of other ECM and cell proliferation genes, but had a negligible impact on overall tendon growth. The combined results from this study provided novel insights into tendon mechanobiology, and suggest that p38 MAPK signaling does not appear to be necessary for tendon growth in vivo.

Microfold cells (M-cells) are specialized cells of the intestine that sample luminal microbiota and dietary antigens to educate the immune cells of the intestinal lymphoid follicles. The function of M-cells in systemic inflammatory responses are still unclear. Here we show that epithelial non-canonical NFkB signaling mediated by NFkB-inducing kinase (NIK) is highly active in intestinal lymphoid follicles, and is required for M-cell maintenance. Intestinal NIK signaling modulates M-cell differentiation and elicits both local and systemic IL-17A and IgA production. Importantly, intestinal NIK signaling is active in mouse models of colitis and patients with inflammatory bowel diseases; meanwhile, constitutive NIK signaling increases the susceptibility to inflammatory injury by inducing ectopic M-cell differentiation and a chronic increase of IL-17A. Our work thus defines an important function of non-canonical NFkB and M-cells in immune homeostasis, inflammation and polymicrobial sepsis.

Inflammation is a significant risk factor for colon cancer. Recent work has demonstrated essential roles for several infiltrating immune populations in the metaplastic progression following inflammation. Hypoxia and stabilization of hypoxiainducible factors (HIFs) are hallmark features of inflammation and solid tumors. Previously, we demonstrated an important role for tumor epithelial HIF-2␣ in colon tumors; however, the function of epithelial HIF-2␣ as a critical link in the progression of inflammation to cancer has not been elucidated. In colitis-associated colon cancer models, epithelial HIF-2␣ was essential in tumor growth. Concurrently, epithelial disruption of HIF-2␣ significantly decreased neutrophils in the colon tumor microenvironment. Intestinal epithelial HIF-2␣-overexpressing mice demonstrated that neutrophil recruitment was a direct response to increased epithelial HIF-2␣ signaling. High-throughput RNA sequencing (RNA-seq) analysis of HIF-2␣-overexpressing mice in conjunction with data mining from the Cancer Genome Atlas showed that the neutrophil chemokine CXCL1 gene was highly upregulated in colon tumor epithelium in a HIF-2␣-dependent manner. Using selective peptide inhibitors of the CXCL1-CXCR2 signaling axis identified HIF-2␣-dependent neutrophil recruitment as an essential mechanism to increase colon carcinogenesis. These studies demonstrate that HIF-2␣ is a novel regulator of neutrophil recruitment to colon tumors and that it is essential in shaping the protumorigenic inflammatory microenvironment in colon cancer.KEYWORDS HIF-2␣, inflammation, colon cancer, cancer, colon, hypoxia, neutrophils C olon cancer remains a significant public health concern and is the second leading cause of cancer-associated deaths in the United States (1). Patients with chronic inflammation associated with inflammatory bowel disease (IBD), comprising ulcerative colitis and Crohn's disease, are at an increased lifetime risk of developing colon cancer; these tumors are termed colitis-associated cancers (CAC) (2). The genetic changes of sporadic colon cancer have been well defined, and a comprehensive genetic analysis of CAC was recently reported (3). In contrast to sporadic colon cancer, CAC are associated with early loss of the TP53 tumor suppressor and less frequent inactivation of adenomatous polyposis coli (APC) (4). Inflammation is an important component of the progression of sporadic cancer, and the inflammatory response is essential in the initiation and progression of CAC (5). The precise mechanisms that initiate the protumorigenic response following inflammation remain unknown.Hypoxia is a characteristic feature of IBD and nearly all solid tumors, including those of the colon (6). Hypoxia promotes activation of the hypoxia-inducible factors (HIFs). HIFs consist of a heterodimer of an O 2 -labile ␣-subunit (HIF-1␣, HIF-2␣, and HIF-3␣) and an O 2 -stable ␤-subunit (ARNT) (7). HIFs regulate transcription of target genes that mediate cellular responses to hypoxic microenvironments. HIFs are also essenti...

Colorectal cancer (CRC) requires massive iron stores, but the complete mechanisms by which CRC modulates local iron handling and metabolically leverages iron are poorly understood. We demonstrate that the liver-derived, endocrine regulator of systemic iron balance, hepcidin, is activated ectopically in CRC. Hepcidin binds to the only known mammalian iron exporter ferroportin, resulting in degradation of ferroportin and intracellular iron trapping. Mice deficient for the hepcidin gene specifically in colon tumor epithelium exhibited significant decreases in tumor number, burden, and size compared to wild-type littermates in a sporadic model of CRC, whereas ferroportin deletion exacerbated these tumor parameters. To further understand the biochemical and metabolic utilization of iron in CRC, we subjected a three-dimensional patient-derived CRC tumor enteroid model to metabolomics and found that iron is prioritized in CRC for the production of nucleotides. These metabolomics findings were recapitulated in our hepcidin/ferroportin mouse CRC models. Mechanistically, our data suggest that a decrease in mitochondrial function alters nucleotide synthesis following iron chelation. Restoration of nucleotide metabolism with exogenous supplementation of nucleosides led to a partial rescue of growth in patient-derived tumor enteroids and CRC cell lines in the presence of an iron chelator. Moreover, aspartate, a critical metabolite which links mitochondrial respiration and nucleotide synthesis, also partially rescued growth of iron deficient CRC cells. Collectively, these data suggest that ectopic hepcidin in the tumor epithelium establishes an axis to degrade ferroportin and sequester iron in colorectal tumors in order to maintain the nucleotide pool and sustain proliferation.

Skeletal muscle can adapt to increased mechanical loads by undergoing hypertrophy. Transient reductions in whole muscle force production have been reported during the onset of hypertrophy, but contractile changes in individual muscle fibers have not been previously studied. Additionally, the extracellular matrix (ECM) stores and transmits forces from muscle fibers to tendons and bones, and determining how the ECM changes during hypertrophy is important in understanding the adaptation of muscle tissue to mechanical loading. Using the synergist ablation model, we sought to measure changes in muscle fiber contractility, collagen content, and cross-linking, and in the expression of several genes and activation of signaling proteins that regulate critical components of myogenesis and ECM synthesis and remodeling during muscle hypertrophy. Tissues were harvested 3, 7, and 28 days after induction of hypertrophy, and nonoverloaded rats served as controls. Muscle fiber specific force (sF), which is the maximum isometric force normalized to cross-sectional area, was reduced 3 and 7 days after the onset of mechanical overload, but returned to control levels by 28 days. Collagen abundance displayed a similar pattern of change. Nearly a quarter of the transcriptome changed over the course of overload, as well as the activation of signaling pathways related to hypertrophy and atrophy. Overall, this study provides insight into fundamental mechanisms of muscle and ECM growth, and indicates that although muscle fibers appear to have completed remodeling and regeneration 1 mo after synergist ablation, the ECM continues to be actively remodeling at this time point. This study utilized a rat synergist ablation model to integrate changes in single muscle fiber contractility, extracellular matrix composition, activation of important signaling pathways in muscle adaption, and corresponding changes in the muscle transcriptome to provide novel insight into the basic biological mechanisms of muscle fiber hypertrophy.

Intestinal iron absorption is activated during increased systemic iron demand. The best-studied example is iron-deficiency anemia, which increases intestinal iron absorption. Interestingly, the intestinal response to anemia is very similar to that of iron overload disorders, as both the conditions activate a transcriptional program that leads to a hyperabsorption of iron via the transcription factor hypoxia-inducible factor (HIF)2a. However, pathways to selectively target intestinal-mediated iron overload remain unknown. Nuclear receptor co-activator 4 (NCOA4) is a critical cargo receptor for autophagic breakdown of ferritin (FTN) and subsequent release of iron, in a process termed ferritinophagy. Our work demonstrates that NCOA4-mediated intestinal ferritinophagy is integrated to systemic iron demand via HIF2a. To demonstrate the importance of intestinal HIF2a/ferritinophagy axis in systemic iron homeostasis, whole body and intestine-specific NCOA4-null mouse lines were generated and assessed. These analyses revealed that the intestinal and systemic response to iron deficiency was not altered following disruption of intestinal NCOA4. However, in a mouse model of hemochromatosis, ablation of intestinal NCOA4 was protective against iron overload. Therefore, NCOA4 can be selectively targeted for the management of iron overload disorders without disrupting the physiological processes involved in the response to systemic iron deficiency.