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Gliadin Induces Neutrophil Migration via Engagement of the Formyl Peptide Receptor, FPR1.

Lammers KM, Chieppa M, Liu L, Liu S, Omatsu T, Janka-Junttila M, Casolaro V, Reinecker HC, Parent CA, Fasano A - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Mucosal Immunology and Biology Research Center, Massachusetts General Hospital East, Charlestown, Massachusetts, United States of America.

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusions: Gliadin possesses neutrophil chemoattractant properties similar to the classical neutrophil chemoattractant, fMet-Leu-Phe, and likewise uses FPR1 in the process.

No MeSH data available.


Related in: MedlinePlus

In-vivo intestinal luminal injection of PT-gliadin induces an immediate and considerable neutrophil migration.(A) Image stills at different time points from S1 movie. Intravital microscopy of the duodenum of Lys-GFP mice showed a rapid extravasation and recruitment of neutrophils (bright green) within 1 hour after PT-gliadin luminal administration, which was absent in mice treated with the same volume of PBS. Data are representative of results obtained from eight independent experiments (n = 8 for PT-gliadin, n = 8 for PBS). (B) Quantitative analysis of neutrophil recruitment in response to intestinal luminal PT-gliadin challenge. The green fluorescent channel in the original images was extracted, converted and processed into 16 bit binary images using the Image J software. The number of bright spots, which represents the number of neutrophils in the movies, was calculated with Image J using the Analyze Particles function. Neutrophil recruitment started immediately upon intestinal gliadin exposure, became significant after 15 minutes (P = .006) and increased further over time (P = .019 at t = 30 minutes).
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pone.0138338.g001: In-vivo intestinal luminal injection of PT-gliadin induces an immediate and considerable neutrophil migration.(A) Image stills at different time points from S1 movie. Intravital microscopy of the duodenum of Lys-GFP mice showed a rapid extravasation and recruitment of neutrophils (bright green) within 1 hour after PT-gliadin luminal administration, which was absent in mice treated with the same volume of PBS. Data are representative of results obtained from eight independent experiments (n = 8 for PT-gliadin, n = 8 for PBS). (B) Quantitative analysis of neutrophil recruitment in response to intestinal luminal PT-gliadin challenge. The green fluorescent channel in the original images was extracted, converted and processed into 16 bit binary images using the Image J software. The number of bright spots, which represents the number of neutrophils in the movies, was calculated with Image J using the Analyze Particles function. Neutrophil recruitment started immediately upon intestinal gliadin exposure, became significant after 15 minutes (P = .006) and increased further over time (P = .019 at t = 30 minutes).

Mentions: Increased intestinal permeability favors the access of gliadin and other macromolecular antigens from the intestinal lumen into the lamina propria where the host, by interpreting them as danger signals, will respond with first-line defense mechanisms such as neutrophil recruitment to the site of exposure. To understand the dynamics of neutrophil recruitment upon pepsin/trypsin digested (PT)-gliadin exposure, we performed in vivo intravital microscopy using Lys-GFP transgenic mice [20]. In these mice GFP is expressed selectively in neutrophils and macrophages. We first imaged neutrophil recruitment from the luminal side of the intestine. Even if limited by the acquisition field, we observed that numerous green fluorescent cells were recruited following PT-gliadin administration (data not shown). We then visualized a larger field of the duodenum by imaging from the serosal side of the intestine. Shortly after PT-gliadin administration we observed that green fluorescent cells were slowing down, rolling along the endothelium and accumulating into the tissue (Fig 1, S1 movie-gliadin). Importantly, recruitment started within 30 minutes after injection of PT-gliadin, consistent with the timing of PT-gliadin-induced increase in intestinal permeability [16]. Since the surgical procedure could have been responsible for cell recruitment, parallel experiments were performed in mice treated with the same volume of PBS as negative control. No significant recruitment of green fluorescent cells was observed in these mice (Fig 1, S1 movie-control).


Gliadin Induces Neutrophil Migration via Engagement of the Formyl Peptide Receptor, FPR1.

Lammers KM, Chieppa M, Liu L, Liu S, Omatsu T, Janka-Junttila M, Casolaro V, Reinecker HC, Parent CA, Fasano A - PLoS ONE (2015)

In-vivo intestinal luminal injection of PT-gliadin induces an immediate and considerable neutrophil migration.(A) Image stills at different time points from S1 movie. Intravital microscopy of the duodenum of Lys-GFP mice showed a rapid extravasation and recruitment of neutrophils (bright green) within 1 hour after PT-gliadin luminal administration, which was absent in mice treated with the same volume of PBS. Data are representative of results obtained from eight independent experiments (n = 8 for PT-gliadin, n = 8 for PBS). (B) Quantitative analysis of neutrophil recruitment in response to intestinal luminal PT-gliadin challenge. The green fluorescent channel in the original images was extracted, converted and processed into 16 bit binary images using the Image J software. The number of bright spots, which represents the number of neutrophils in the movies, was calculated with Image J using the Analyze Particles function. Neutrophil recruitment started immediately upon intestinal gliadin exposure, became significant after 15 minutes (P = .006) and increased further over time (P = .019 at t = 30 minutes).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4574934&req=5

pone.0138338.g001: In-vivo intestinal luminal injection of PT-gliadin induces an immediate and considerable neutrophil migration.(A) Image stills at different time points from S1 movie. Intravital microscopy of the duodenum of Lys-GFP mice showed a rapid extravasation and recruitment of neutrophils (bright green) within 1 hour after PT-gliadin luminal administration, which was absent in mice treated with the same volume of PBS. Data are representative of results obtained from eight independent experiments (n = 8 for PT-gliadin, n = 8 for PBS). (B) Quantitative analysis of neutrophil recruitment in response to intestinal luminal PT-gliadin challenge. The green fluorescent channel in the original images was extracted, converted and processed into 16 bit binary images using the Image J software. The number of bright spots, which represents the number of neutrophils in the movies, was calculated with Image J using the Analyze Particles function. Neutrophil recruitment started immediately upon intestinal gliadin exposure, became significant after 15 minutes (P = .006) and increased further over time (P = .019 at t = 30 minutes).
Mentions: Increased intestinal permeability favors the access of gliadin and other macromolecular antigens from the intestinal lumen into the lamina propria where the host, by interpreting them as danger signals, will respond with first-line defense mechanisms such as neutrophil recruitment to the site of exposure. To understand the dynamics of neutrophil recruitment upon pepsin/trypsin digested (PT)-gliadin exposure, we performed in vivo intravital microscopy using Lys-GFP transgenic mice [20]. In these mice GFP is expressed selectively in neutrophils and macrophages. We first imaged neutrophil recruitment from the luminal side of the intestine. Even if limited by the acquisition field, we observed that numerous green fluorescent cells were recruited following PT-gliadin administration (data not shown). We then visualized a larger field of the duodenum by imaging from the serosal side of the intestine. Shortly after PT-gliadin administration we observed that green fluorescent cells were slowing down, rolling along the endothelium and accumulating into the tissue (Fig 1, S1 movie-gliadin). Importantly, recruitment started within 30 minutes after injection of PT-gliadin, consistent with the timing of PT-gliadin-induced increase in intestinal permeability [16]. Since the surgical procedure could have been responsible for cell recruitment, parallel experiments were performed in mice treated with the same volume of PBS as negative control. No significant recruitment of green fluorescent cells was observed in these mice (Fig 1, S1 movie-control).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Mucosal Immunology and Biology Research Center, Massachusetts General Hospital East, Charlestown, Massachusetts, United States of America.

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusions: Gliadin possesses neutrophil chemoattractant properties similar to the classical neutrophil chemoattractant, fMet-Leu-Phe, and likewise uses FPR1 in the process.

No MeSH data available.


Related in: MedlinePlus