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1.  Gliadin Induces Neutrophil Migration via Engagement of the Formyl Peptide Receptor, FPR1 
PLoS ONE  2015;10(9):e0138338.
Gliadin, the immunogenic component within gluten and trigger of celiac disease, is known to induce the production of Interleukin-8, a potent neutrophil-activating and chemoattractant chemokine. We sought to study the involvement of neutrophils in the early immunological changes following gliadin exposure.
Utilizing immunofluorescence microscopy and flow cytometry, the redistribution of major tight junction protein, Zonula occludens (ZO)-1, and neutrophil recruitment were assessed in duodenal tissues of gliadin-gavaged C57BL/6 wild-type and Lys-GFP reporter mice, respectively. Intravital microscopy with Lys-GFP mice allowed monitoring of neutrophil recruitment in response to luminal gliadin exposure in real time. In vitro chemotaxis assays were used to study murine and human neutrophil chemotaxis to gliadin, synthetic alpha-gliadin peptides and the neutrophil chemoattractant, fMet-Leu-Phe, in the presence or absence of a specific inhibitor of the fMet-Leu-Phe receptor-1 (FPR1), cyclosporine H. An irrelevant protein, zein, served as a control.
Redistribution of ZO-1 and an influx of CD11b+Lys6G+ cells in the lamina propria of the small intestine were observed upon oral gavage of gliadin. In vivo intravital microscopy revealed a slowing down of GFP+ cells within the vessels and influx in the mucosal tissue within 2 hours after challenge. In vitro chemotaxis assays showed that gliadin strongly induced neutrophil migration, similar to fMet-Leu-Phe. We identified thirteen synthetic gliadin peptide motifs that induced cell migration. Blocking of FPR1 completely abrogated the fMet-Leu-Phe-, gliadin- and synthetic peptide-induced migration.
Gliadin possesses neutrophil chemoattractant properties similar to the classical neutrophil chemoattractant, fMet-Leu-Phe, and likewise uses FPR1 in the process.
PMCID: PMC4574934  PMID: 26378785
2.  Nutritional Keys for Intestinal Barrier Modulation 
The intestinal tract represents the largest interface between the external environment and the human body. Nutrient uptake mostly happens in the intestinal tract, where the epithelial surface is constantly exposed to dietary antigens. Since inflammatory response toward these antigens may be deleterious for the host, a plethora of protective mechanisms take place to avoid or attenuate local damage. For instance, the intestinal barrier is able to elicit a dynamic response that either promotes or impairs luminal antigens adhesion and crossing. Regulation of intestinal barrier is crucial to control intestinal permeability whose increase is associated with chronic inflammatory conditions. The cross talk among bacteria, immune, and dietary factors is able to modulate the mucosal barrier function, as well as the intestinal permeability. Several nutritional products have recently been proposed as regulators of the epithelial barrier, even if their effects are in part contradictory. At the same time, the metabolic function of the microbiota generates new products with different effects based on the dietary content. Besides conventional treatments, novel therapies based on complementary nutrients are now growing. Fecal therapy has been recently used for the clinical treatment of refractory Clostridium difficile infection instead of the classical antibiotic therapy. In the present review, we will outline the epithelial response to nutritional components derived from dietary intake and microbial fermentation focusing on the consequent effects on the integrity of the epithelial barrier.
PMCID: PMC4670985  PMID: 26697008
inflammation; intestinal permeability; nutrition; mucosal immunity; microbiota
3.  Administration of Reconstituted Polyphenol Oil Bodies Efficiently Suppresses Dendritic Cell Inflammatory Pathways and Acute Intestinal Inflammation 
PLoS ONE  2014;9(2):e88898.
Polyphenols are natural compounds capable of interfering with the inflammatory pathways of several in vitro model systems. In this study, we developed a stable and effective strategy to administer polyphenols to treat in vivo models of acute intestinal inflammation. The in vitro suppressive properties of several polyphenols were first tested and compared for dendritic cells (DCs) production of inflammatory cytokines. A combination of the polyphenols, quercetin and piperine, were then encapsulated into reconstituted oil bodies (OBs) in order to increase their stability. Our results showed that administration of low dose reconstituted polyphenol OBs inhibited LPS-mediated inflammatory cytokine secretion, including IL-6, IL-23, and IL-12, while increasing IL-10 and IL-1Rα production. Mice treated with the polyphenol-containing reconstituted OBs (ROBs) were partially protected from dextran sodium sulfate (DSS)-induced colitis and associated weight loss, while mortality and inflammatory scores revealed an overall anti-inflammatory effect that was likely mediated by impaired DC immune responses. Our study indicates that the administration of reconstituted quercetin and piperine-containing OBs may represent an effective and potent anti-inflammatory strategy to treat acute intestinal inflammation.
PMCID: PMC3928302  PMID: 24558444
4.  Messages from the Inside. The Dynamic Environment that Favors Intestinal Homeostasis 
An organism is defined as “an individual living thing capable of responding to stimuli, growing, reproducing, and maintaining homeostasis.” Early during evolution multicellular organisms explored the advantages of a symbiotic life. Mammals harbor a complex aggregate of microorganisms (called microbiota) that includes bacteria, fungi, and archaea. Some of these bacteria have already defined beneficial roles for the human host that include the ability to break down nutrients that could not otherwise be digested, preventing the growth of harmful species, as well as the ability to produce vitamins or hormones. It is intuitive that along the evolutionary path several mechanisms favored bacteria that provided advantages to the host which, in return, avoided launching an aggressive immunological response against them. The intestinal immunological response does not ignore the lumenal content, on the contrary, immune surveillance is favored by continuous antigen sampling. Some intestinal epithelial cells (ECs) are crucial during the sampling process, others actively participate in the defense mechanism. In essence the epithelium acts as a traffic light, communicating to the inside world whether conditions are safe or dangerous, and thus influencing immunological response. In this review we will discuss the dynamic factors that act on the intestinal ECs and how they directly or indirectly influence immune cells during states of health and disease.
PMCID: PMC3793270  PMID: 24130559
mucosal immunology; intestinal evolution; DCs; intestinal epithelial cells; intestinal inflammation
5.  Intestinal Epithelium and Autophagy: Partners in Gut Homeostasis 
One of the most significant challenges of cell biology is to understand how each type of cell copes with its specific workload without suffering damage. Among the most intriguing questions concerns intestinal epithelial cells in mammals; these cells act as a barrier between the internally protected region and the external environment that is exposed constantly to food and microbes. A major process involved in the processing of microbes is autophagy. In the intestine, through multiple, complex signaling pathways, autophagy including macroautophagy and xenophagy is pivotal in mounting appropriate intestinal immune responses and anti-microbial protection. Dysfunctional autophagy mechanism leads to chronic intestinal inflammation, such as inflammatory bowel disease (IBD). Studies involving a number of in vitro and in vivo mouse models in addition to human clinical studies have revealed a detailed role for autophagy in the generation of chronic intestinal inflammation. A number of genome-wide association studies identified roles for numerous autophagy genes in IBD, especially in Crohn’s disease. In this review, we will explore in detail the latest research linking autophagy to intestinal homeostasis and how alterations in autophagy pathways lead to intestinal inflammation.
PMCID: PMC3786390  PMID: 24137160
IBD; autophagy; intestinal epithelium; ATG16L1; IRGM
6.  The SAMP1/YitFc Mouse Strain: A Spontaneous Model of Crohn’s Disease-Like Ileitis 
Inflammatory bowel diseases  2011;17(12):2566-2584.
The SAMP1/YitFc mouse strain represents a model of Crohn’s disease (CD)-like ileitis that is ideal for investigating the pathogenesis of chronic intestinal inflammation. Differently from the vast majority of animal models of colitis, the ileal-specific phenotype characteristic of SAMP1/YitFc mice occurs spontaneously, without genetic, chemical or immunological manipulation. In addition, SAMP1/YitFc mice possess remarkable similarities to the human condition in regard to disease location, histologic features, incidence of extra-intestinal manifestations, and response to conventional therapies. SAMP1/YitFc mice also display a well-defined time course of a pre-disease state, and phases of acute and chronic ileitis. As such, the SAMP1/YitFc model is particularly suitable for elucidating pathways that precede the clinical phenotype that may lead to preventive, and therefore more efficacious, intervention with the natural course of disease, or alternatively, for the development of therapeutic strategies directed against chronic, established ileitis. In the following review, we summarize important contributions made by our group and others that uncover potential mechanisms in the pathogenesis of CD using this unique murine model of chronic intestinal inflammation.
PMCID: PMC3154989  PMID: 21557393
spontaneous animal model of IBD; Crohn’s disease; genetics; epithelial barrier function; cytokines; intestinal microflora; adhesion molecules; leukocyte trafficking
7.  Probiotic Bacteria Regulate Intestinal Epithelial Permeability in Experimental Ileitis by a TNF-Dependent Mechanism 
PLoS ONE  2012;7(7):e42067.
We previously showed that the probiotic mixture, VSL#3, prevents the onset of ileitis in SAMP/YitFc (SAMP) mice, and this effect was associated with stimulation of epithelial-derived TNF. The aim of this study was to determine the mechanism(s) of VSL#3-mediated protection on epithelial barrier function and to further investigate the “paradoxical” effects of TNF in preventing SAMP ileitis.
Permeability was evaluated in SAMP mice prior to the onset of inflammation and during established disease by measuring transepithelial electrical resistance (TEER) on ex vivo-cultured ilea following exposure to VSL#3 conditioned media (CM), TNF or VSL#3-CM + anti-TNF. Tight junction (TJ) proteins were assessed by qRT-PCR, Western blot, and confocal microscopy, and TNFRI/TNFRII expression measured in freshly isolated intestinal epithelial cells (IEC) from SAMP and control AKR mice.
Culture with either VSL#3-CM or TNF resulted in decreased ileal paracellular permeability in pre-inflamed SAMP, but not SAMP with established disease, while addition of anti-TNF abrogated these effects. Modulation of the TJ proteins, claudin-2 and occludin, occurred with a significant decrease in claudin-2 and increase in occludin following stimulation with VSL#3-CM or TNF. TNF protein levels increased in supernatants of SAMP ilea incubated with VSL#3-CM compared to vehicle, while IEC-derived TNFR mRNA expression decreased in young, and was elevated in inflamed, SAMP versus AKR mice.
Our data demonstrate that the previously established efficacy of VSL#3 in preventing SAMP ileitis is due to direct innate and homeostatic effects of TNF on the gut epithelium, modulation of the TJ proteins, claudin-2 and occludin, and overall improvement of intestinal permeability.
PMCID: PMC3405026  PMID: 22848704
8.  House dust mite allergen induces asthma via TLR4 triggering of airway structural cells 
Nature medicine  2009;15(4):410-416.
Barrier epithelial cells and airway dendritic cells (DC) make up the first line of defence against inhaled substances like house dust mite (HDM) allergen and endotoxin. We hypothesized that these cells need to communicate to cause allergic disease. Using irradiated chimeric mice, we demonstrate that TLR4 expression on radioresistant lung structural cells is required and sufficient for DC activation in the lung and for priming of effector T helper responses to HDM. TLR4 triggering on structural cells caused production of the innate proallergic cytokines thymic stromal lymphopoietin, granulocyte-macrophage colony stimulating factor, interleukin-25 and IL-33. The absence of TLR4 on structural cells, but not on hematopoietic cells, abolished HDM driven allergic airway inflammation. Finally, inhalation of a TLR4 antagonist to target exposed epithelial cells suppressed the salient features of asthma including bronchial hyperreactivity. Our data identify an innate immune function of airway epithelial cells that drives allergic inflammation via activation of mucosal DCs.
PMCID: PMC2789255  PMID: 19330007
9.  An extended vision for dynamic high-resolution intravital immune imaging 
Seminars in immunology  2005;17(6):431-441.
The past few years have seen the application of confocal and especially two-photon microscopy to the dynamic high-resolution imaging of lymphocytes and antigen presenting cells within organs such as lymph nodes and thymus. After summarizing some of the published results obtained to date using these methods, we describe our view of how this technology will develop and be applied in the near future. This includes its extension to a wide variety of non-lymphoid tissues, to the tracking of functional responses in addition to migratory behavior, to the analysis of molecular events previously studied only in vitro, to dissection of the interplay between hematopoietic and stromal elements, to visualization of a wider array of cell types including neutrophils, macrophages, NK cells, NKT cells and others, and to the interaction of the host with infectious agents. Reaching these goals will depend on a combination of new tools for genetic manipulations, novel fluorescent reporters, enhanced instrumentation, and better surgical techniques for the extended imaging of live animals. The end result will be a new level of understanding of how orchestrated cell movement and interaction contribute to the physiological and pathological activities of the immune system.
PMCID: PMC1462950  PMID: 16216522
Antigen presenting cell; Dendritic cell; T cell; Chemokine; Lymph node; Microscopy
11.  Microbial translocation augments the function of adoptively transferred self/tumor-specific CD8+ T cells via TLR4 signaling  
Journal of Clinical Investigation  2007;117(8):2197-2204.
Lymphodepletion with total body irradiation (TBI) increases the efficacy of adoptively transferred tumor-specific CD8+ T cells by depleting inhibitory lymphocytes and increasing homeostatic cytokine levels. We found that TBI augmented the function of adoptively transferred CD8+ T cells in mice genetically deficient in all lymphocytes, indicating the existence of another TBI mechanism of action. Additional investigation revealed commensal gut microflora in the mesenteric lymph nodes and elevated LPS levels in the sera of irradiated mice. These findings correlated with increased dendritic cell activation and heightened levels of systemic inflammatory cytokines. Reduction of host microflora using antibiotics, neutralization of serum LPS using polymyxin B, or removal of LPS signaling components using mice genetically deficient in CD14 and TLR4 reduced the beneficial effects of TBI on tumor regression. Conversely, administration of microbial ligand–containing serum or ultrapure LPS from irradiated animals to nonirradiated antibody-lymphodepleted mice enhanced CD8+ T cell activation and improved tumor regression. Administration of ultrapure LPS to irradiated animals further enhanced the number and function of the adoptively transferred cells, leading to long-term cure of mice with large B16F10 tumors and enhanced autoimmune vitiligo. Thus, disruption of the homeostatic balance between the host and microbes can enhance cell-based tumor immunotherapy.
PMCID: PMC1924500  PMID: 17657310
12.  Dynamic imaging of dendritic cell extension into the small bowel lumen in response to epithelial cell TLR engagement 
The Journal of Experimental Medicine  2006;203(13):2841-2852.
Cells lining the gastrointestinal tract serve as both a barrier to and a pathway for infectious agent entry. Dendritic cells (DCs) present in the lamina propria under the columnar villus epithelium of the small bowel extend processes across this epithelium and capture bacteria, but previous studies provided limited information on the nature of the stimuli, receptors, and signaling events involved in promoting this phenomenon. Here, we use immunohistochemical as well as dynamic explant and intravital two-photon imaging to investigate this issue. Analysis of CD11c–enhanced green fluorescent protein (EGFP) or major histocompatibility complex CII-EGFP mice revealed that the number of trans-epithelial DC extensions, many with an unusual “balloon” shape, varies along the length of the small bowel. High numbers of such extensions were found in the proximal jejunum, but only a few were present in the terminal ileum. The extensions in the terminal ileum markedly increased upon the introduction of invasive or noninvasive Salmonella organisms, and chimeric mouse studies revealed the key role of MyD88-dependent Toll-like receptor (TLR) signaling by nonhematopoietic (epithelial) elements in the DC extension response. Collectively, these findings support a model in which epithelial cell TLR signaling upon exposure to microbial stimuli induces active DC sampling of the gut lumen at sites distant from organized lymphoid tissues.
PMCID: PMC2118178  PMID: 17145958
13.  Fractalkine (CX3CL1) as an amplification circuit of polarized Th1 responses 
Journal of Clinical Investigation  2001;107(9):1173-1181.
Fractalkine (FKN, CX3CL1) is a membrane-bound CX3C chemokine induced by primary proinflammatory signals in vascular endothelial cells (ECs). Here we examined the role of FKN in polarized Th1 or Th2 responses. Proinflammatory signals, including LPS, IL-1, TNF, and CD40 ligand, induced FKN, as did IFN-γ, which had synergistic activity with TNF. IL-4 and IL-13 did not stimulate the expression of FKN and markedly reduced induction by TNF and IFN-γ. TNF alone or combined with IFN-γ also induced release of soluble FKN, which was inhibited by IL-4 and IL-13. In light of this differential regulation of FKN by the master cytokines that control polarized responses, we analyzed the interaction of FKN with natural killer (NK) cells and polarized T-cell populations. NK cells expressed high levels of the FKN receptor CX3CR1 and responded to FKN. CX3CR1 was preferentially expressed in Th1 compared with Th2 cells. Th1 but not Th2 cells responded to FKN. By immunohistochemistry, FKN was expressed on ECs in psoriasis, a Th1-dominated skin disorder, but not in Th2-driven atopic dermatitis. Similarly, ECs in Mycobacterium tuberculosis granulomatous lymphadenitis, but not those in reactive lymph node hyperplasia or in Castelman’s disease, showed immunoreactive FKN. These results indicate that regulated expression of FKN in ECs participates in an amplification circuit of polarized type I responses.
PMCID: PMC209276  PMID: 11342581

Results 1-13 (13)