BEL1-LIKE HOMEODOMAIN 11 regulates chloroplast development and chlorophyll synthesis in tomato fruit.

Chloroplast development and chlorophyll(Chl)metabolism in unripe tomato contribute to the growth and quality of the fruit, however these mechanisms are poorly understood. In this study, we initially investigated seven homeobox-containing transcription factors (TFs) with specific ripening-associated expression patterns using virus-induced gene silencing (VIGS) technology and found that inhibiting the expression of one of these TFs, BEL1-LIKE HOMEODOMAIN11 (SlBEL11), significantly increased Chl levels in unripe tomato fruit. This enhanced Chl accumulation was further validated by generating stable RNA interference (RNAi) transgenic lines. RNA sequencing (RNA-seq) of RNAi-SlBEL11 fruit at the mature green (MG) stage showed that 48 genes involved in Chl biosynthesis, photosynthesis and chloroplast development were significantly upregulated compared with the wild type (WT) fruit. Genomic global scanning for Homeobox TF binding sites combined with RNA-seq differential gene expression analysis showed that 22 of these 48 genes were potential target genes of SlBEL11 protein. These genes included Chl biosynthesis-related genes encoding for protochlorophyllide reductase (POR), magnesium chelatase H subunit (CHLH) and chlorophyllide a oxygenase (CAO), and chloroplast development-related genes encoding for chlorophyll a/b binding protein (CAB), homeobox protein knotted 2 (TKN2) and ARABIDOPSIS PSEUDO RESPONSE REGULATOR 2-LIKE (APRR2-like). Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation quantitative polymerase chain reaction (PCR) (ChIP-qPCR) assays were employed to verify that SlBEL11 protein could bind to the promoters for TKN2, CAB and POR. Taken together, our findings demonstrated that SlBEL11 plays an important role in chloroplast development and Chl synthesis in tomato fruit.

The intestinal microbiota affect central levels of brain-derived neurotropic factor and behavior in mice.

BACKGROUND & AIMS: Alterations in the microbial composition of the gastrointestinal tract (dysbiosis) are believed to contribute to inflammatory and functional bowel disorders and psychiatric comorbidities. We examined whether the intestinal microbiota affects behavior and brain biochemistry in mice. METHODS: Specific pathogen-free (SPF) BALB/c mice, with or without subdiaphragmatic vagotomy or chemical sympathectomy, or germ-free BALB/c mice received a mixture of nonabsorbable antimicrobials (neomycin, bacitracin, and pimaricin) in their drinking water for 7 days. Germ-free BALB/c and NIH Swiss mice were colonized with microbiota from SPF NIH Swiss or BALB/c mice. Behavior was evaluated using step-down and light preference tests. Gastrointestinal microbiota were assessed using denaturing gradient gel electrophoresis and sequencing. Gut samples were analyzed by histologic, myeloperoxidase, and cytokine analyses; levels of serotonin, noradrenaline, dopamine, and brain-derived neurotropic factor (BDNF) were assessed by enzyme-linked immunosorbent assay. RESULTS: Administration of oral antimicrobials to SPF mice transiently altered the composition of the microbiota and increased exploratory behavior and hippocampal expression of BDNF. These changes were independent of inflammatory activity, changes in levels of gastrointestinal neurotransmitters, and vagal or sympathetic integrity. Intraperitoneal administration of antimicrobials to SPF mice or oral administration to germ-free mice did not affect behavior. Colonization of germ-free BALB/c mice with microbiota from NIH Swiss mice increased exploratory behavior and hippocampal levels of BDNF, whereas colonization of germ-free NIH Swiss mice with BALB/c microbiota reduced exploratory behavior. CONCLUSIONS: The intestinal microbiota influences brain chemistry and behavior independently of the autonomic nervous system, gastrointestinal-specific neurotransmitters, or inflammation. Intestinal dysbiosis might contribute to psychiatric disorders in patients with bowel disorders.

Mucin gene deficiency in mice impairs host resistance to an enteric parasitic infection.

BACKGROUND & AIMS: Hyperplasia of mucin-secreting intestinal goblet cells accompanies a number of enteric infections, including infections by nematode parasites. Nevertheless, the precise role of mucins in host defense in nematode infection is not known. We investigated the role of the mucin (Muc2) in worm expulsion and host immunity in a model of nematode infection. METHODS: Resistant (BALB/c, C57BL/6), susceptible (AKR), and Muc2-deficient mouse strains were infected with the nematode, Trichuris muris, and worm expulsion, energy status of the whipworms, changes in mucus/mucins, and inflammatory and immune responses were investigated after infection. RESULTS: The increase in Muc2 production, observed exclusively in resistant mice, correlated with worm expulsion. Moreover, expulsion of the worms from the intestine was significantly delayed in the Muc2-deficient mice. Although a marked impairment in the development of periodic acid Schiff (PAS)-stained intestinal goblet cells was observed in Muc2-deficient mice, as infection progressed a significant increase in the number of PAS-positive goblet cells was observed in these mice. Surprisingly, an increase in Muc5ac, a mucin normally expressed in the airways and stomach, was observed after infection of only the resistant animals. Overall, the mucus barrier in the resistant mice was less permeable than that of susceptible mice. Furthermore, the worms isolated from the resistant mice had a lower energy status. CONCLUSIONS: Mucins are an important component of innate defense in enteric infection; this is the first demonstration of the important functional contribution of mucins to host protection from nematode infection.

Serotonin has a key role in pathogenesis of experimental colitis.

BACKGROUND & AIMS: Mucosal changes in inflammatory bowel disease are characterized by ulcerative lesions accompanied by a prominent infiltrate of immune cells as well as alteration in serotonin (5-hydroxytryptamine [5-HT])-producing enterochromaffin cells. We investigated the role of 5-HT in colonic inflammation in mice. METHODS: Colitis was induced with dextran sulfate sodium or dinitrobenzene sulfonic acid in tryptophan hydroxylase 1-deficient (TPH1(-/-)) mice, which have markedly reduced 5-HT in the gastrointestinal tract, and in mice given the 5-HT synthesis inhibitor parachlorophenylalanine. RESULTS: Delayed onset, decreased severity of clinical disease, and significantly lower macroscopic and histologic damage scores were observed in TPH1(-/-) mice, compared with wild-type mice, and in mice given parachlorophenylalanine after induction of colitis by dextran sulfate sodium. This was associated with down-regulation of macrophage infiltration and production of proinflammatory cytokines. 5-HT stimulated production of proinflammatory cytokines from macrophages collected from the peritoneal cavity of wild-type mice; this process was inhibited by a nuclear factor kappaB inhibitor, indicating a critical role for nuclear factor kappaB signaling in 5-HT-mediated activation of immune cells. Restoration of 5-HT levels in TPH1(-/-) mice by the 5-HT precursor 5-hydroxytryptophan increased the severity of DSS-induced colitis. We also observed significant reduction in severity of colitis in TPH1(-/-) mice after induction of dinitrobenzene sulfonic acid-induced colitis. CONCLUSIONS: 5-HT is involved in the pathogenesis of inflammation in experimental colitis. These findings provide insight into the mechanisms of gastrointestinal inflammation and could lead to new therapeutic strategies for inflammatory disorders.

Transplantation of fecal microbiota from patients with irritable bowel syndrome alters gut function and behavior in recipient mice.

Irritable bowel syndrome (IBS) is a common disorder characterized by altered gut function and often is accompanied by comorbid anxiety. Although changes in the gut microbiota have been documented, their relevance to the clinical expression of IBS is unknown. To evaluate a functional role for commensal gut bacteria in IBS, we colonized germ-free mice with the fecal microbiota from healthy control individuals or IBS patients with diarrhea (IBS-D), with or without anxiety, and monitored gut function and behavior in the transplanted mice. Microbiota profiles in recipient mice clustered according to the microbiota profiles of the human donors. Mice receiving the IBS-D fecal microbiota showed a taxonomically similar microbial composition to that of mice receiving the healthy control fecal microbiota. However, IBS-D mice showed different serum metabolomic profiles. Mice receiving the IBS-D fecal microbiota, but not the healthy control fecal microbiota, exhibited faster gastrointestinal transit, intestinal barrier dysfunction, innate immune activation, and anxiety-like behavior. These results indicate the potential of the gut microbiota to contribute to both intestinal and behavioral manifestations of IBS-D and suggest the potential value of microbiota-directed therapies in IBS patients.

IL-4 gene transfer to the small bowel serosa leads to intestinal inflammation and smooth muscle hyperresponsiveness.

Intestinal mucosal inflammation can lead to altered function of the underlying smooth muscle, which becomes hyperreactive to most contractile stimuli. Through nematode parasite infection models, T helper type 2 (Th2) cytokines have been implicated in intestinal muscle dysfunction; however, the mechanisms involved and the relevance of these findings to other forms of intestinal inflammation are unclear. Through gene transfer, we explored whether the Th2 cytokine IL-4 can mediate changes in longitudinal muscle function in the context of an adenoviral infection. Following abdominal surgery on mice, control beta-galactosidase-encoding recombinant adenoviruses and IL-4-encoding adenoviruses were applied to the serosal surface of the jejunum, leading to infection of cells in the serosa and in the mesentery. Marker transgene expression lasted for 3 wk and was accompanied by the recruitment of macrophages, lymphocytes, and neutrophils into the peritoneal cavity and mild inflammation at the site of infection. IL-4 transgene expression led to a stronger inflammatory response characterized by tissue eosinophilia and increased numbers of peritoneal mast cells and plasma cells. Whereas control virus infection had no effect on intestinal muscle function, infection with the IL-4 virus led to significant jejunal muscle hypercontractility, evident by day 7 postinfection. This modulation of smooth muscle function was shown to be IL-4 specific, since the application of an IL-5-encoding adenovirus induced tissue eosinophilia but did not alter muscle function. These results highlight an important causal role for IL-4 in the pathological regulation of enteric smooth muscle function and identify a novel strategy for gene transfer to the intestine.

Cytokine modulation of muscarinic receptors in the murine intestine.

The extent to which gut motility and smooth muscle contractility are altered by intestinal inflammation depends on the nature of the underlying immune activation. The muscarinic receptor on smooth muscle plays a critical role in mediating acetylcholine-driven motor function. We examined the ability of cytokines to influence muscarinic receptor characteristics on intestinal longitudinal muscle and related the findings to studies on carbachol-induced contraction. Cells were isolated from longitudinal muscle myenteric plexus (LMMP). Cytokine receptor expression, muscle contractility, and muscarinic agonist receptor characteristics were examined by agonist displacement of [N-methyl-(3)H]scopolamine ([(3)H]NMS) binding. The TGF-beta1 receptor (543 bp) and the IFN-gamma receptor 1 (660 bp) were identified on smooth muscle cells. Scatchard analysis revealed dissociation constant and maximum binding values for [(3)H]NMS of 2.6 nM and 2.4 x 10(4) sites/cell, respectively, in control cells. Nematode infection was accompanied by a reduction in inhibitory constant of the high-affinity sites (K(H)), and this was independent of signal transduction and activator of transcription 6. Preincubation with TGF-beta1 enhanced longitudinal muscle contractility and decreased the K(H) to 2.2 pM (increased muscarinic receptor affinity), whereas preincubation with IFN-gamma increased the K(H) to 0.4 muM (decreased muscarinic receptor affinity) and decreased longitudinal muscle contractility. Preincubation of LMMP with IL-13 decreased the K(H) to 0.2 nM. Cytokines exert differential effects on the muscarinic receptor on intestinal longitudinal smooth muscle. These findings explain the basis for altered muscle contractility observed in Th1 and Th2 models of inflammation, as well as in the post-nematode-infected state.

CD4+ T cell-mediated immunological control of enterochromaffin cell hyperplasia and 5-hydroxytryptamine production in enteric infection.

BACKGROUND: Enterochromaffin (EC) cells are dispersed throughout the gastrointestinal (GI) mucosa and are the main source of 5-hydroxytryptamine (5-HT) in the gut. 5-HT has been implicated in the pathophysiology of several GI disorders, but the mechanisms regulating 5-HT production in the gut are unknown. AIM: To investigate the role of CD4(+) T cells in the production of 5-HT using a model of enteric parasitic infection. METHODS AND RESULTS: Severe combined immunodeficient (SCID) mice and their wild-type controls were infected with the nematode Trichuris muris and killed on various days after infection to study colonic EC cells and 5-HT production. The number of EC cells and the amount of 5-HT produced were significantly higher in infected wild-type mice than in non-infected mice. The number of EC cells and the amount of 5-HT after infection were significantly lower in SCID mice after infection than in wild-type mice. The number of EC cells and the amount of 5-HT was significantly increased after reconstitution of SCID mice with CD4(+) T cells from infected mice and this was accompanied by an upregulation of colonic CD3 T cells and T helper 2 (Th2) cytokines. Laser capture microdissection-based molecular and immunofluorescence techniques revealed the presence of interleukin 13 receptor alpha1-chain on EC cells. CONCLUSION: These results show an important immunoendocrine axis in the gut, where secretory products from CD4(+) T cells interact with EC cells to enhance the production of 5-HT in the gut via Th2-based mechanisms. These results show new insights into the mechanisms of gut function, which may ultimately lead to improved therapeutic strategies in functional and inflammatory disorders of the GI tract.

Mechanisms underlying the maintenance of muscle hypercontractility in a model of postinfective gut dysfunction.

BACKGROUND & AIMS: Acute gastroenteritis is a strong risk factor for the development of irritable bowel syndrome (IBS). We have developed an animal model in which transient acute infection leads to persistent muscle hypercontractility. Here, we investigate the mechanisms underlying the maintenance of this hypercontractility in the postinfective (PI) state. METHODS: Muscle contraction and messenger RNA (mRNA) or protein expression of cytokines were examined from jejunal longitudinal muscle cells of NIH Swiss mice infected with Trichinella spiralis or incubated with or without cytokines. RESULTS: During acute infection, interleukin (IL)-4 or IL-13, transforming growth factor (TGF)-beta1, and cyclooxygenase (COX)-2 were increased in the muscle layer ( P < .05). In the PI phase of the model, T helper (Th)2 cytokines returned to normal, but TGF-beta1 remained in the muscle ( P < .05). Exposure of muscle cells to IL-4 or IL-13 increased TGF-beta1 ( P < .01), COX-2 protein, and prostaglandin (PG)E 2 . Exposure of muscle cells to TGF-beta1 increased PGE 2 ( P < .05) and COX-2 protein. Incubation of tissue with IL-4, IL-13, TGF-beta1, or PGE 2 enhanced carbachol-induced muscle cell contractility ( P < .05). COX-2 inhibitor attenuated TGF-beta1-induced muscle hypercontractility ( P < .05). CONCLUSIONS: These results support the hypothesis that Th2 cytokines induce muscle hypercontractility during infection by a direct action on smooth muscle. The maintenance of hypercontractility results from Th2 cytokine-induced expression of TGF-beta1 and the subsequent up-regulation of COX-2 and PGE 2 at the level of the smooth muscle cell. We propose that PI gut dysfunction reflects mediator production in the neuromuscular tissues and that this may occur in PI-IBS.

Interleukin-4- and -13-induced hypercontractility of human intestinal muscle cells-implication for motility changes in Crohn's disease.

Crohn's disease is an idiopathic inflammatory condition. However, little is known about the changes that occur in the muscularis externa, despite the fact that this tissue contributes to motility changes and stricture formation. We characterized immune activity in the muscularis externa from intestinal segments of Crohn's disease patients and evaluated the role of IL-4 and -13 as well as signal transducer and activator of transcription (STAT)6 in the contractility of the cultured human intestinal smooth muscle cells. CD3+ve cells (P < 0.01) and IL-4 protein (P < 0.01) were significantly increased in the muscularis externa of Crohn's disease patients compared with noninflamed controls. Preincubation of human cultured smooth muscle cells with IL-4 (P < 0.001) or IL-13 (P < 0.05) significantly enhanced carbachol-induced contraction, and this was significantly inhibited by the STAT6 inhibitor leflunomide (P < 0.0001). A similar profile was observed in muscle cells isolated from Crohn's disease patients. Both IL-4 and IL-13 increased specific STAT6-DNA binding in control cells, and this was inhibited by anti-STAT6 Ab (P < 0.05) or leflunomide (P < 0.05). IL-4 and IL-13 mediate the hypercontractility of intestinal muscle via a STAT6 pathway at the level of the smooth muscle cell. The STAT6 pathway may contribute to the hypercontractility of intestinal muscle in Crohn's disease.

Role of IL-4, IL-13, and STAT6 in inflammation-induced hypercontractility of murine smooth muscle cells.

T helper 2 (Th2) cytokines interleukin (IL)-4 and IL-13, which activate signal transducer and activator of transcription 6 (STAT6) are expressed in the muscularis externa during nematode infection and are candidate mediators of the associated hypercontractility. To determine the locus of action of these cytokines, we examined the IL-4- and IL-13-induced hypercontractility of the isolated muscle cells from STAT6 +/+ and STAT6 -/- mice. We compared the results with cells isolated from Trichinella spiralis-infected STAT6 +/+ and STAT6 -/- mice. Carbamylcholine chloride (Carbachol) induced the contraction of jejunal muscle cells in a concentration-dependent manner maximal contraction (R(max) 26.7 +/- 1.9%). Cells from T. spiralis-infected STAT6 -/- mice showed the hypertrophy (cell lengths 41.4 +/- 0.8 to 89.0 +/- 8.7 microm) and hypercontractility (R(max) 37.5 +/- 1.3%) induced by infection. IL-4Ralpha mRNA was detected in dispersed smooth muscle cells. Incubation of longitudinal muscle-myenteric plexus (LMMP) with IL-4 and IL-13 enhanced Carbachol-induced muscle contraction (R(max) 35.5 +/- 1.9 and 32.4 +/- 2.9%, respectively). Incubation of LMMP from STAT6 -/- mice with IL-4 did not enhance the contraction. The hypercontractility in T. spiralis-infected mice was attenuated in STAT6 -/- mice (P < 0.02). These results indicate both IL-4 and IL-13 induce hypercontractility of muscle cells via the STAT6 pathway, and this is the basis for hypercontractility observed in T. spiralis-infected mice.

Modulatory effects of estrogen in two murine models of experimental colitis.

The association between oral contraceptives or pregnancy and inflammatory bowel disease is unclear. We investigated whether 17beta-estradiol modulates intestinal inflammation in two models of colitis. Female mice were treated with 17beta-estradiol alone or with tamoxifen, tamoxifen alone, 17 alpha-estradiol, or placebo. Dinitrobenzene sulfonic acid (DNB)- or dextran sodium sulfate (DSS)-induced colitis were assessed macroscopically, histologically, and by myeloperoxidase (MPO) activity. Malondialdehyde and mRNA levels of intercellular adhesion molecule-1 (ICAM-1), interferon-gamma (IFN-gamma), and interleukin-13 (IL-13) were determined. In DNB colitis, 17beta-estradiol alone, but not 17beta-estradiol plus tamoxifen, or 17 alpha-estradiol reduced macroscopic and histological scores, MPO activity and malondialdehyde levels. 17beta-Estradiol also decreased the expression of ICAM-1, IFN-gamma, and IL-13 mRNA levels compared with placebo. In contrast, 17beta-Estradiol increased the macroscopic and histological scores compared with placebo in mice with DSS colitis. These results demonstrate anti-inflammatory and proinflammatory effects of 17beta-estradiol in two different models of experimental colitis. The net modulatory effect most likely reflects a combination of estrogen receptor-mediated effects and antioxidant activity and may explain, in part, conflicting results from clinical trials.