Slc11a1 gene polymorphism influences dextran sulfate sodium (DSS)-induced colitis in a murine model of acute inflammation.

Ulcerative Colitis (UC) is an inflammatory disease characterized by colonic mucosal lesions associated with an increased risk of carcinogenesis. UC pathogenesis involves environmental and genetic factors. Genetic studies have indicated the association of gene variants coding for the divalent metal ion transporter SLC11A1 protein (formerly NRAMP1) with UC susceptibility in several animal species. Two mouse lines were genetically selected for high (AIRmax) or low (AIRmin) acute inflammatory responses (AIR). AIRmax is susceptible, and AIRmin is resistant to DSS-induced colitis and colon carcinogenesis. Furthermore, AIRmin mice present polymorphism of the Slc11a1 gene. Here we investigated the possible modulating effect of the Slc11a1 R and S variants in DSS-induced colitis by using AIRmin mice homozygous for Slc11a1 R (AIRminRR) or S (AIRminSS) alleles. We evaluated UC by the disease activity index (DAI), considering weight loss, diarrhea, blood in the anus or feces, cytokines, histopathology, and cell populations in the distal colon epithelium. AIRminSS mice have become susceptible to DSS effects, with higher DAI, IL6, G-CSF, and MCP-1 production and morphological and colon histopathological alterations than AIRminRR mice. The results point to a role of the Slc11a1 S allele in DSS colitis induction in the genetic background of AIRmin mice.

Mapping of novel loci involved in lung and colon tumor susceptibility by the use of genetically selected mouse strains.

Two non-inbred mouse lines, phenotypically selected for maximal (AIRmin) and minimal (AIRmax) acute inflammatory response, show differential susceptibility/resistance to the development of several chemically-induced tumor types. An intercross pedigree of these mice was generated and treated with the chemical carcinogen dimethylhydrazine, which induces lung and intestinal tumors. Genome wide high-density genotyping with the Restriction Site-Associated DNA genotyping (2B-RAD) technique was used to map genetic loci modulating individual genetic susceptibility to both lung and intestinal cancer. Our results evidence new common quantitative trait loci (QTL) for those phenotypes and provide an improved understanding of the relationship between genomic variation and individual genetic predisposition to tumorigenesis in different organs.

A liver-on-a-chip platform with bioprinted hepatic spheroids.

The inadequacy of animal models in correctly predicting drug and biothreat agent toxicity in humans has resulted in a pressing need for in vitro models that can recreate the in vivo scenario. One of the most important organs in the assessment of drug toxicity is liver. Here, we report the development of a liver-on-a-chip platform for long-term culture of three-dimensional (3D) human HepG2/C3A spheroids for drug toxicity assessment. The bioreactor design allowed for in situ monitoring of the culture environment by enabling direct access to the hepatic construct during the experiment without compromising the platform operation. The engineered bioreactor could be interfaced with a bioprinter to fabricate 3D hepatic constructs of spheroids encapsulated within photocrosslinkable gelatin methacryloyl (GelMA) hydrogel. The engineered hepatic construct remained functional during the 30 days culture period as assessed by monitoring the secretion rates of albumin, alpha-1 antitrypsin, transferrin, and ceruloplasmin, as well as immunostaining for the hepatocyte markers, cytokeratin 18, MRP2 bile canalicular protein and tight junction protein ZO-1. Treatment with 15 mM acetaminophen induced a toxic response in the hepatic construct that was similar to published studies on animal and other in vitro models, thus providing a proof-of-concept demonstration of the utility of this liver-on-a-chip platform for toxicity assessment.

7,12-Dimethylbenz(a)anthracene-induced genotoxicity on bone marrow cells from mice phenotypically selected for low acute inflammatory response.

Exposure to polycyclic aromatic hydrocarbon (PAH) environmental contaminants has been associated with the development of mutations and cancer. 7,12-Dimethylbenz(a)anthracene ( DMBA), a genotoxic agent, reacts with DNA directly, inducing p53-dependent cytotoxicity resulting in cell death by apoptosis or giving rise to cancer. DMBA metabolism largely depends on activation of the aryl hydrocarbon receptor (AhR). Mice phenotypically selected for high (AIRmax) or low (AIRmin) acute inflammatory response present a complete segregation of Ahr alleles endowed with low (Ahr(d)) or high (Ahr(b1)) affinity to PAHs, respectively. To evaluate the role of AhR genetic polymorphism on the bone marrow susceptibility to DMBA, AIRmax and AIRmin mice were treated with a single intraperitoneal injection of DMBA (50mg/kg b.w.) in olive oil. Bone marrow cells (BMCs) were phenotyped by both flow cytometry and cytoslide preparations. Despite a significant decrease in total cell count in BM from AIRmin mice, there was an increase of blast cells and immature neutrophils at 1 and 50 days after DMBA treatment, probably due to a cell-cycle blockade at the G1/S transition leading to immature stage cell production. A panel of proteins related to cell cycle regulation was evaluated in immature BM cells (Lin(-)) by Western Blot, and DNA damage and repair were measured using an alkaline version of the Comet assay. In Lin(-) cells isolated from AIRmin mice, high levels were found in both p53 and p21 protein contents in contrast with the low levels of CDK4 and Ciclin D1. Evaluation of DNA repair in DMBA-treated BMCs, indicated long-lasting genotoxicity and cytotoxicity in BMC from AIRmin mice and a blockade of cell cycle progression. On the other hand, AIRmax mice have a high capacity of DNA damage repair and protection. These mechanisms can be associated with the differential susceptibility to the toxic and carcinogenic effects of DMBA observed in these mice.

Aryl hydrocarbon receptor polymorphism modulates DMBA-induced inflammation and carcinogenesis in phenotypically selected mice.

We tested the role of aryl hydrocarbon receptor (Ahr) gene polymorphism in the inflammatory response and in skin and lung tumorigenesis in 2 lines of mice phenotypically selected for maximum or minimum acute inflammatory reaction (AIRmax and AIRmin, respectively). Following 7,12-dimethylbenz[a]anthracene (DMBA) treatment, AIRmin but not AIRmax mice showed early skin reactions and eventually developed malignant skin tumors and lung adenocarcinomas. In skin tissue, transcript levels of IL1beta, Tnf, Il6, Tgfbeta1 and Cyp1b1 genes were upregulated in AIRmin but not AIRmax mice, consistent with the inflammatory responses to the carcinogen. These findings appeared to be related to the homozygosity status of the Ahr functional A375V polymorphism, which influences the binding capability of the receptor for DMBA: the 375A allele, encoding the high-affinity ligand-binding receptor (Ahr(b1)), segregated in AIRmin mice, whereas AIRmax mice carried the 375V, corresponding to the low-affinity binding receptor (Ahr(d)), to DMBA. The differential segregation of Ahr functional Ahr(d)versus Ahr(b1) alleles in AIRmax and AIRmin suggests a role for the Ahr gene in the control of inflammatory responsiveness and tumor development of these mouse lines.

Inverse genetic predisposition to colon versus lung carcinogenesis in mouse lines selected based on acute inflammatory responsiveness.

Mouse lines produced by bidirectional selection on the basis of maximum (AIRmax) or minimum (AIRmin) acute inflammatory reactions were examined for the development of chemically induced acute colitis and colon tumors and the development of lung tumors. AIRmax mice were more susceptible than AIRmin to acute colitis induced by ingestion of dextran sodium sulfate showing a 3-fold higher disease activity index and presenting an intense inflammatory infiltrate in the base of colon crypts as well as elevated expression of IL-1beta, TNFalpha, IFNgamma and IL-6 mRNA in colon tissue. AIRmax were also more susceptible than AIRmin to colon cancer induced by 2 or 7 weekly doses of 1,2-dimethylhydrazine (DMH), showing significantly higher numbers of colonic aberrant crypt foci (ACF) at 150 days after DMH treatment (P = 0.01) and significantly higher numbers of tumors affecting larger intestinal areas at 300-475 days. At the latter time point, however, multiple lung adenomas and large adenocarcinomas were found in AIRmin but not in AIRmax mice. Treatment of mice with nimesulide for 60 days beginning 24 h before the first of two DMH doses almost completely inhibited the appearance of ACF in both lines. Furthermore, ACF numbers and the degree of acute inflammation directly co-segregated in an F2 (AIRmax x AIRmin) intercross population. The results demonstrate that genetic determinants of the inflammatory response differentially influence susceptibility to colon and lung carcinogenesis in the AIRmax and AIRmin mouse model.

Genetic selection for high acute inflammatory response confers resistance to lung carcinogenesis in the mouse.

Mice selected for a high acute inflammatory response (AIRmax) are resistant to chemically induced lung tumorigenesis, whereas the low responders (AIRmin) are susceptible. In urethane-treated mice, anti-inflammatory drugs increased the tumor incidence in AIRmax but not AIRmin mice, and an inverse correlation (P<.001) between the degree of acute inflammatory response (AIR) and lung tumorigenesis was found in an F2 (AIRmax x AIRmin) intercross population. The results provide evidence for the involvement of lung tumor modifier loci in AIR regulation and implicate AIR quantitative trait loci in the inherited predisposition to lung cancer.

Genetic control of innate and acquired immunity

Host defense against infection depends on both specific and nonspecific mechanisms.The lines of mice genetically selected for high (H) or low (L) antibody responsiveness and for the maximal (AIR max.) or minimal (AIR min.) acute inflammatory response, in which the opposite extreme potentialities have been clearly defined, ofter an appropriate model for investigation of the major genetic and environmental factors of resistance to infections. The alternative advantagens of the extreme phenotypes such as efficacy of specific and nonspecific immunity in natural populations are discussed.