Iron deficiency anemia (IDA) affects > 500 million people worldwide, and is linked to impaired cognitive development and function in children. Helicobacter pylori, a class 1 carcinogen, infects about half of the world's population, thus creating a high likelihood of overlapping risk. This study determined the effect of H. pylori infection on iron homeostasis in INS-GAS mice. Two replicates of INS-GAS/FVB male mice (n = 9-12/group) were dosed with H. pylori (Hp) strain SS1 or sham dosed at 6-9 weeks of age, and were necropsied at 27-29 weeks of age. Hematologic and serum iron parameters were evaluated, as was gene expression in gastric and brain tissues. Serum ferritin was lower in Hp SS1-infected mice than uninfected mice (p < 0.0001). Infected mice had a lower red blood cell count (p<0.0001), hematocrit (p < 0.001), and hemoglobin concentration (p <0.0001) than uninfected mice. Relative expression of gastric hepcidin antimicrobial peptide (Hamp) was downregulated in mice infected with Hp SS1 compared to sham-dosed controls (p<0.001). Expression of bone morphogenic protein 4 (Bmp4), a growth factor upstream of hepcidin, was downregulated in gastric tissue of Hp SS1-infected mice (p<0.001). Hp SS1-infected mice had downregulated brain expression of tyrosine hydroxylase (Th) (p = 0.02). Expression of iron-responsive genes involved in myelination (myelin basic protein (Mbp) and proteolipid protein 2 (Plp2)) was downregulated in infected mice (p = 0.001 and p = 0.02). Expression of synaptic plasticity markers (brain derived neurotrophic factor 3 (Bdnf3), Psd95 (a membrane associated guanylate kinase), and insulin-like growth factor 1 (Igf1)) was also downregulated in Hp SS1-infected mice (p = 0.09, p = 0.04, p = 0.02 respectively). Infection of male INS-GAS mice with Hp SS1, without concurrent dietary iron deficiency, depleted serum ferritin, deregulated gastric and hepatic expression of iron regulatory genes, and altered iron-dependent neural processes. The use of Hp SS1-infected INS-GAS mice will be an appropriate animal model for further study of the effects of concurrent H. pylori infection and anemia on iron homeostasis and adult iron-dependent brain gene expression.

Histamine is a biogenic amine that is widely distributed and has multiple functions, but the role it plays in acute myocardial infarction (AMI) remains unclear. In this study, we investigated the origin and contribution of endogenous histamine to AMI. Histidine decarboxylase (HDC) is the unique enzyme responsible for histamine generation. Using HDC-EGFP bacterial artificial chromosome (BAC) transgenic mice in which EGFP expression is controlled by the HDC promoter, we identified HDC expression primarily in CD11b(+)Gr-1(+) immature myeloid cells (IMCs) that markedly increase in the early stages of AMI. Deficiency of histamine in HDC knockout mice (HDC(-/-)) reduced cardiac function and exacerbated the injury of infarcted heart. Furthermore, administering either an H1 receptor antagonist (pyrilamine) or an H2 receptor antagonist (cimetidine) demonstrated a protective effect of histamine against myocardial injury. The results of in vivo and in vitro assays showed that histamine deficiency promotes the apoptosis of cardiomyocytes and inhibits macrophage infiltration. In conclusion, CD11b(+)Gr-1(+) IMCs are the predominant HDC-expressing sites in AMI, and histamine plays a protective role in the process of AMI through inhibition of cardiomyocyte apoptosis and facilitation of macrophage infiltration.

Food allergy can influence the development of colorectal cancer, although the underlying mechanisms are unclear. While mast cells (MC) store and secrete histamine, immature myeloid cells (IMC) are the major site of histidine decarboxylase (HDC) expression, the enzyme responsible for histamine production. From our earlier work, we hypothesized that histamine is central to the association between allergy and colorectal carcinogenesis through its influence on the MC-MDSC axis. Here, we show that in wild type (WT) mice, ovalbumin (OVA) immunization elicits a typical TH2 response. In contrast, in HDC-/- mice, the response to OVA allergy is skewed towards infiltration by IL-17 expressing MCs. This response is inhibited by histamine treatment. The HDC-/- allergic IL-17-expressing MCs promote MDSC proliferation and upregulation of Cox-2 and Arg-1. OVA allergy in HDC-/- mice increases the growth of colon tumor cells in both the MC38 tumor cell implantation model and the AOM/DSS carcinogenesis model. Taken together, our results show that histamine represses IL-17-expressing MCs and their subsequent activation of MDSCs, attenuating the risk of colorectal cancer in the setting of food allergy. Targeting the MC-MDSC axis may be useful for cancer prevention and treatment in patients, particularly in those with food allergy.

Commensal gastrointestinal bacteria resist the expansion of pathogens and are lost during critical illness, facilitating pathogen colonization and infection. We performed a prospective, ICU-based study to determine risk factors for loss of gut colonization resistance during the initial period of critical illness. Rectal swabs were taken from adult ICU patients within 4 hours of admission and 72 hours later, and analyzed using 16S rRNA gene sequencing and selective culture for vancomycin-resistant Enterococcus (VRE). Microbiome data was visualized using principal coordinate analyses (PCoA) and assessed using a linear discriminant analysis algorithm and logistic regression modeling. 93 ICU patients were analyzed. At 72 hours following ICU admission, there was a significant decrease in the proportion of Clostridial Clusters IV/XIVa, taxa that produce short chain fatty acids (SCFAs). At the same time, there was a significant expansion in Enterococcus. Decreases in Cluster IV/XIVa Clostridia were associated with loss of gut microbiome colonization resistance (reduced diversity and community stability over time). In multivariable analysis, both decreased Cluster IV/XIVa Clostridia and increased Enterococcus after 72 hours were associated with receipt of antibiotics. Cluster IV/XIVa Clostridia, although a small fraction of the overall gastrointestinal microbiome, drove distinct clustering on PCoA. During initial treatment for critical illness, there was a loss of Cluster IV/XIVa Clostridia within the distal gut microbiome which associated with an expansion of VRE and with a loss of gut microbiome colonization resistance. Receipt of broad-spectrum antibiotics was associated with these changes.

Stromal cells such as myofibroblasts influence tumor progression. The mechanisms are unclear but may involve effects on both tumor cells and recruitment of bone marrow-derived mesenchymal stromal cells (MSCs) which then colonize tumors. Using iTRAQ and LC-MS/MS we identified the adipokine, chemerin, as overexpressed in esophageal squamous cancer associated myofibroblasts (CAMs) compared with adjacent tissue myofibroblasts (ATMs). The chemerin receptor, ChemR23, is expressed by MSCs. Conditioned media (CM) from CAMs significantly increased MSC cell migration compared to ATM-CM; the action of CAM-CM was significantly reduced by chemerin-neutralising antibody, pretreatment of CAMs with chemerin siRNA, pretreatment of MSCs with ChemR23 siRNA, and by a ChemR23 receptor antagonist, CCX832. Stimulation of MSCs by chemerin increased phosphorylation of p42/44, p38 and JNK-II kinases and inhibitors of these kinases and PKC reversed chemerin-stimulated MSC migration. Chemerin stimulation of MSCs also induced expression and secretion of macrophage inhibitory factor (MIF) that tended to restrict migratory responses to low concentrations of chemerin but not higher concentrations. In a xenograft model consisting of OE21 esophageal cancer cells and CAMs, homing of MSCs administered i.v. was inhibited by CCX832. Thus, chemerin secreted from esophageal cancer myofibroblasts is a potential chemoattractant for MSCs and its inhibition may delay tumor progression.

The incidence of esophageal adenocarcinoma (EAC) has been increasing for decades without significant improvements in treatment. Barrett's esophagus (BE) is best established risk factor for EAC, but current surveillance with random biopsies cannot predict progression to cancer in most BE patients due to the low sensitivity and specificity of high-definition white light endoscopy.

Cancer is believed to arise from stem cells, but mechanisms that limit the acquisition of mutations and tumor development have not been well defined. We show that a +4 stem cell (SC) in the gastric antrum, marked by expression of Cck2r (a GPCR) and Delta-like ligand 1 (DLL1), is a label-retaining cell that undergoes predominant asymmetric cell division. This +4 antral SC is Notch1low/ Numb+ and repressed by signaling from gastrin-expressing endocrine (G) cells. Chemical carcinogenesis of the stomach is associated with loss of G cells, increased symmetric stem cell division, glandular fission, and more rapid stem cell lineage tracing, a process that can be suppressed by exogenous gastrin treatment. This hormonal suppression is associated with a marked reduction in gastric cancer mutational load, as revealed by exomic sequencing. Taken together, our results show that gastric tumorigenesis is associated with increased symmetric cell division that facilitates mutation and is suppressed by GPCR signaling.

Barrett's esophagus (BE) is a precursor to esophageal adenocarcinoma (EAC), but its cellular origin and mechanism of neoplastic progression remain unresolved. Notch signaling, which plays a key role in regulating intestinal stem cell maintenance, has been implicated in a number of cancers. The kinase Dclk1 labels epithelial post-mitotic tuft cells at the squamo-columnar junction (SCJ), and has also been proposed to contribute to epithelial tumor growth. Here, we find that genetic activation of intracellular Notch signaling in epithelial Dclk1-positive tuft cells resulted in the accelerated development of metaplasia and dysplasia in a mouse model of BE (pL2.Dclk1.N2IC mice). In contrast, genetic ablation of Notch receptor 2 in Dclk1-positive cells delayed BE progression (pL2.Dclk1.N2fl mice), and led to increased secretory cell differentiation. The accelerated BE progression in pL2.Dclk1.N2IC mice correlated with changes to the transcriptomic landscape, most notably for the activation of oncogenic, proliferative pathways in BE tissues, in contrast to upregulated Wnt signalling in pL2.Dclk1.N2fl mice. Collectively, our data show that Notch activation in Dclk1-positive tuft cells in the gastric cardia can contribute to BE development.

PDGFRA-expressing mesenchyme supports intestinal stem cells. Stomach epithelia have related niche dependencies, but their enabling mesenchymal cell populations are unknown, in part because previous studies pooled the gastric antrum and corpus. Our high-resolution imaging, transcriptional profiling, and organoid assays identify regional subpopulations and supportive capacities of purified mouse corpus and antral PDGFRA+ cells. Sub-epithelial PDGFRAHi myofibroblasts are principal sources of BMP ligands and two molecularly distinct pools distribute asymmetrically along antral glands but together fail to support epithelial growth in vitro. In contrast, PDGFRALo CD55+ cells strategically positioned beneath gastric glands promote epithelial expansion in the absence of other cells or factors. This population encompasses a small fraction expressing the BMP antagonist Grem1. Although Grem1+ cell ablation in vivo impairs intestinal stem cells, gastric stem cells are spared, implying that CD55+ cell activity in epithelial self-renewal derives from other subpopulations. Our findings shed light on spatial, molecular, and functional organization of gastric mesenchyme and the spectrum of signaling sources for epithelial support.

Osteoarthritis is a degenerative joint disease that causes pain, degradation, and dysfunction. Excessive canonical Wnt signaling in osteoarthritis contributes to chondrocyte phenotypic instability and loss of cartilage homeostasis; however, the regulatory niche is unknown. Using the temporomandibular joint as a model in multiple species, we identify Lgr5-expressing secretory cells as forming a Wnt inhibitory niche that instruct Wnt-inactive chondroprogenitors to form the nascent synovial joint and regulate chondrocyte lineage and identity. Lgr5 ablation or suppression during joint development, aging, or osteoarthritis results in depletion of Wnt-inactive chondroprogenitors and a surge of Wnt-activated, phenotypically unstable chondrocytes with osteoblast-like properties. We recapitulate the cartilage niche and create StemJEL, an injectable hydrogel therapy combining hyaluronic acid and sclerostin. Local delivery of StemJEL to post-traumatic osteoarthritic jaw and knee joints in rabbit, rat, and mini-pig models restores cartilage homeostasis, chondrocyte identity, and joint function. We provide proof of principal that StemJEL preserves the chondrocyte niche and alleviates osteoarthritis.

CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs) expand in the spleen during cancer and promote progression through suppression of cytotoxic T cells. An anti-inflammatory reflex arc involving the vagus nerve and memory T cells is necessary for resolution of acute inflammation. Failure of this neural circuit could promote procarcinogenic inflammation and altered tumour immunity. Here we show that splenic TFF2, a secreted anti-inflammatory peptide, is released by vagally modulated memory T cells to suppress the expansion of MDSCs through CXCR4. Splenic denervation interrupts the anti-inflammatory neural arc, resulting in the expansion of MDSCs and colorectal cancer. Deletion of Tff2 recapitulates splenic denervation to promote carcinogenesis. Colorectal carcinogenesis could be suppressed through transgenic overexpression of TFF2, adenoviral transfer of TFF2 or transplantation of TFF2-expressing bone marrow. TFF2 is important to the anti-inflammatory reflex arc and plays an essential role in arresting MDSC proliferation. TFF2 offers a potential approach to prevent and to treat cancer.

To date, no discrete genetic signature has been defined for isolated Dclk1+ tuft cells within the small intestine. Furthermore, recent reports on the functional significance of Dclk1+ cells in the small intestine have been inconsistent. These cells have been proposed to be fully differentiated cells, reserve stem cells, and tumor stem cells. In order to elucidate the potential function of Dclk1+ cells, we FACS-sorted Dclk1+ cells from mouse small intestinal epithelium using transgenic mice expressing YFP under the control of the Dclk1 promoter (Dclk1-CreER;Rosa26-YFP). Analysis of sorted YFP+ cells demonstrated marked enrichment (~6000 fold) for Dclk1 mRNA compared with YFP- cells. Dclk1+ population display ~6 fold enrichment for the putative quiescent stem cell marker Bmi1. We observed significantly greater expression of pluripotency genes, pro-survival genes, and quiescence markers in the Dclk1+ population. A significant increase in self-renewal capability (14-fold) was observed in in vitro isolated Dclk1+ cells. The unique genetic report presented in this manuscript suggests that Dclk1+ cells may maintain quiescence, pluripotency, and metabolic activity for survival/longevity. Functionally, these reserve characteristics manifest in vitro, with Dclk1+ cells exhibiting greater ability to self-renew. These findings indicate that quiescent stem-like functionality is a feature of Dclk1-expressing tuft cells.

The esophageal lumen is lined by a stratified squamous epithelium comprised of proliferative basal cells that differentiate while migrating toward the luminal surface and eventually desquamate. Rapid epithelial renewal occurs, but the specific cell of origin that supports this high proliferative demand remains unknown. Herein, we have described a long-lived progenitor cell population in the mouse esophageal epithelium that is characterized by expression of keratin 15 (Krt15). Genetic in vivo lineage tracing revealed that the Krt15 promoter marks a long-lived basal cell population able to self-renew, proliferate, and generate differentiated cells, consistent with a progenitor/stem cell population. Transcriptional profiling demonstrated that Krt15+ basal cells are molecularly distinct from Krt15- basal cells. Depletion of Krt15-derived cells resulted in decreased proliferation, thereby leading to atrophy of the esophageal epithelium. Further, Krt15+ cells were radioresistant and contributed to esophageal epithelial regeneration following radiation-induced injury. These results establish the presence of a long-lived and indispensable Krt15+ progenitor cell population that provides additional perspective on esophageal epithelial biology and the widely prevalent diseases that afflict this epithelium.

Histamine has pleiotropic pathophysiological effects, but its role in myocardial infarction (MI)-induced cardiac remodeling remains unclear. Histidine decarboxylase (HDC) is the main enzyme involved in histamine production. Here, we clarified the roles of HDC-expressing cells and histamine in heart failure post-MI using HDC-EGFP transgenic mice and HDC-knockout (HDC-/-) mice. HDC+CD11b+ myeloid cell numbers markedly increased in the injured hearts, and histamine levels were up-regulated in the circulation post-MI. HDC-/- mice exhibited more adverse cardiac remodeling, poorer left ventricular function and higher mortality by increasing cardiac fibrogenesis post-MI. In vitro assays further confirmed that histamine inhibited heart fibroblast proliferation. Furthermore, histamine enhanced the signal transducer and activator of transcription (STAT)-6 phosphorylation level in murine heart fibroblasts, and the inhibitive effects of histamine on fibroblast proliferation could be blocked by JAK3/STAT6 signaling selective antagonist. STAT6-knockout (STAT6-/-) mice had a phenotype similar to that of HDC-/- mice post-MI; however, in contrast to HDC-/- mice, the beneficial effects of exogenous histamine injections were abrogated in STAT6-/- mice. These data suggest that histamine exerts protective effects by modulating cardiac fibrosis and remodeling post-MI, in part through the STAT6-dependent signaling pathway.

In several organ systems, the transitional zone between different types of epithelium is a hotspot for pre-neoplastic metaplasia and malignancy, but the cells of origin for these metaplastic epithelia and subsequent malignancies remain unknown. In the case of Barrett's oesophagus, intestinal metaplasia occurs at the gastro-oesophageal junction, where stratified squamous epithelium transitions into simple columnar cells. On the basis of a number of experimental models, several alternative cell types have been proposed as the source of this metaplasia but in all cases the evidence is inconclusive: no model completely mimics Barrett's oesophagus in terms of the presence of intestinal goblet cells. Here we describe a transitional columnar epithelium with distinct basal progenitor cells (p63+KRT5+KRT7+) at the squamous-columnar junction of the upper gastrointestinal tract in a mouse model. We use multiple models and lineage tracing strategies to show that this squamous-columnar junction basal cell population serves as a source of progenitors for the transitional epithelium. On ectopic expression of CDX2, these transitional basal progenitors differentiate into intestinal-like epithelium (including goblet cells) and thereby reproduce Barrett's metaplasia. A similar transitional columnar epithelium is present at the transitional zones of other mouse tissues (including the anorectal junction) as well as in the gastro-oesophageal junction in the human gut. Acid reflux-induced oesophagitis and the multilayered epithelium (believed to be a precursor of Barrett's oesophagus) are both characterized by the expansion of the transitional basal progenitor cells. Our findings reveal a previously unidentified transitional zone in the epithelium of the upper gastrointestinal tract and provide evidence that the p63+KRT5+KRT7+ basal cells in this zone are the cells of origin for multi-layered epithelium and Barrett's oesophagus.

Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor that plays a vital role in regulating cell lineage differentiation during development and maintaining epithelial homeostasis in the intestine. In normal intestine, KLF4 is predominantly expressed in the differentiated epithelial cells. It has been identified as a tumor suppressor in colorectal cancer. KLF4 knockout mice demonstrated a decrease in number of goblet cells in the colon, and conditional ablation of KLF4 from the intestinal epithelium led to altered epithelial homeostasis. However, the role of KLF4 in differentiated intestinal cells and colon cancer cells, as well as the mechanism by which it regulates homeostasis and represses tumorigenesis in the intestine is not well understood. In our study, KLF4 was partially depleted in the differentiated intestinal epithelial cells by a tamoxifen-inducible Cre recombinase. We found a significant increase in the number of goblet cells in the KLF4-deleted small intestine, suggesting that KLF4 is not only required for goblet cell differentiation, but also required for maintaining goblet cell numbers through its function in inhibiting cell proliferation. The number and position of Paneth cells also changed. This is consistent with the KLF4 knockout study using villin-Cre [1]. Through immunohistochemistry (IHC) staining and statistical analysis, we found that a stem cell and/or tuft cell marker, DCAMKL1, and a proliferation marker, Ki67, are affected by KLF4 depletion, while an enteroendocrine cell marker, neurotensin (NT), was not affected. In addition, we found KLF4 depletion altered the morphology and polarity of the intestinal epithelial cells. Using a three-dimensional (3D) intestinal epithelial cyst formation assay, we found that KLF4 is essential for cell polarity and crypt-cyst formation in human colon cancer cells. These findings suggest that, as a tumor suppressor in colorectal cancer, KLF4 affects intestinal epithelial cell morphology by regulating proliferation, differentiation and polarity of the cells.

Murine bone marrow-derived myofibroblasts (BMFs) have previously been shown to promote gastric cancer growth. However, whether BMFs promote gastric cancer cell metastasis remains largely unknown.

Histidine decarboxylase (HDC), the histamine-synthesizing enzyme, is expressed in a subset of myeloid cells but also marks quiescent myeloid-biased hematopoietic stem cells (MB-HSCs) that are activated upon myeloid demand injury. However, the role of MB-HSCs in dextran sulfate sodium (DSS)-induced acute colitis has not been addressed.

The enteric neurotransmitter acetylcholine governs important intestinal epithelial secretory and immune functions through its actions on epithelial muscarinic Gq-coupled receptors such as M3R. Its role in the regulation of intestinal stem cell function and differentiation, however, has not been clarified. Here, we find that nonselective muscarinic receptor antagonism in mice as well as epithelial-specific ablation of M3R induces a selective expansion of DCLK1-positive tuft cells, suggesting a model of feedback inhibition. Cholinergic blockade reduces Lgr5-positive intestinal stem cell tracing and cell number. In contrast, Prox1-positive endocrine cells appear as primary sensors of cholinergic blockade inducing the expansion of tuft cells, which adopt an enteroendocrine phenotype and contribute to increased mucosal levels of acetylcholine. This compensatory mechanism is lost with acute irradiation injury, resulting in a paucity of tuft cells and acetylcholine production. Thus, enteroendocrine tuft cells appear essential to maintain epithelial homeostasis following modifications of the cholinergic intestinal niche.

Enterochromaffin-like (ECL) cells in the stomach express gastrin/cholecystokinin 2 receptor CCK2R and are known to expand under hypergastrinemia, but whether this results from expansion of existing ECL cells or increased production from progenitors has not been clarified.