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Small intestinal intraepithelial TCRγδ+ T lymphocytes are present in the premature intestine but selectively reduced in surgical necrotizing enterocolitis.

Weitkamp JH, Rosen MJ, Zhao Z, Koyama T, Geem D, Denning TL, Rock MT, Moore DJ, Halpern MD, Matta P, Denning PW - PLoS ONE (2014)

Bottom Line: In human preterm infants, the proportion of IEL was reduced by 66% in 11 NEC ileum resections compared to 30 non-NEC controls (p<0.001).While γδ IEL dominated over conventional αβ IEL early in gestation in mice and in humans, γδ IEL were preferential decreased in the ileum of surgical NEC patients compared to non-NEC controls (50% reduction, p<0.05).Loss of IEL in human NEC was associated with downregulation of the Th17 transcription factor retinoic acid-related orphan nuclear hormone receptor C (RORC, p<0.001).

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Vanderbilt University School of Medicine and Monroe Carell Jr. Children's Hospital at Vanderbilt, Nashville, Tennessee, United States of America.

ABSTRACT

Background: Gastrointestinal barrier immaturity predisposes preterm infants to necrotizing enterocolitis (NEC). Intraepithelial lymphocytes (IEL) bearing the unconventional T cell receptor (TCR) γδ (γδ IEL) maintain intestinal integrity and prevent bacterial translocation in part through production of interleukin (IL) 17.

Objective: We sought to study the development of γδ IEL in the ileum of human infants and examine their role in NEC pathogenesis. We defined the ontogeny of γδ IEL proportions in murine and human intestine and subjected tcrδ-/- mice to experimental gut injury. In addition, we used polychromatic flow cytometry to calculate percentages of viable IEL (defined as CD3+ CD8+ CD103+ lymphocytes) and the fraction of γδ IEL in surgically resected tissue from infants with NEC and gestational age matched non-NEC surgical controls.

Results: In human preterm infants, the proportion of IEL was reduced by 66% in 11 NEC ileum resections compared to 30 non-NEC controls (p<0.001). While γδ IEL dominated over conventional αβ IEL early in gestation in mice and in humans, γδ IEL were preferential decreased in the ileum of surgical NEC patients compared to non-NEC controls (50% reduction, p<0.05). Loss of IEL in human NEC was associated with downregulation of the Th17 transcription factor retinoic acid-related orphan nuclear hormone receptor C (RORC, p<0.001). TCRδ-deficient mice showed increased severity of experimental gut injury (p<0.05) with higher TNFα expression but downregulation of IL17A.

Conclusion: Complimentary mouse and human data suggest a role of γδ IEL in IL17 production and intestinal barrier production early in life. Specific loss of the γδ IEL fraction may contribute to NEC pathogenesis. Nutritional or pharmacological interventions to support γδ IEL maintenance in the developing small intestine could serve as novel strategies for NEC prevention.

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Intestinal injury in wild-type mice is not associated with a selective reduction in γδ IEL.A) Flow cytometry of small intestinal intraepithelial cells isolated from 2 weeks old dam fed wild-type (WT control, n = 2) or wild-type mice subjected to experimental gut injury as described (WT+PAF/LPS, n = 2). C57BL/6J mice stained for CD103, CD3, CD8α, TCRγδ and TCRβ as described above. Intraepithelial cells were pregated on CD103+, CD3+ to depict IEL and then further gated on TCRγδ and TCRβ as shown. B) Percent (mean ±SE) γδ IEL (defined as percent of total IEL that were TCRγδ+, TCRβ- IEL). Data are representative of 3 independent experiments (NS indicates no statistical difference between groups).
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pone-0099042-g005: Intestinal injury in wild-type mice is not associated with a selective reduction in γδ IEL.A) Flow cytometry of small intestinal intraepithelial cells isolated from 2 weeks old dam fed wild-type (WT control, n = 2) or wild-type mice subjected to experimental gut injury as described (WT+PAF/LPS, n = 2). C57BL/6J mice stained for CD103, CD3, CD8α, TCRγδ and TCRβ as described above. Intraepithelial cells were pregated on CD103+, CD3+ to depict IEL and then further gated on TCRγδ and TCRβ as shown. B) Percent (mean ±SE) γδ IEL (defined as percent of total IEL that were TCRγδ+, TCRβ- IEL). Data are representative of 3 independent experiments (NS indicates no statistical difference between groups).

Mentions: For ethical reasons, it is not possible to determine definitively whether the selective reduction of γδ IEL in human NEC occurred prior to or as a result of intestinal injury. Therefore we sought to determine whether experimental intestinal injury in a murine model causes selective reduction in γδ IEL. To induce intestinal injury, we injected 2 weeks old C57BL/6J or TCRδ−/− mice intraperitoneally with 100 µg/kg PAF and 1 mg/kg E. coli 0128:B12 LPS or PBS vehicle control as described above. Pups were sacrificed two hours later and small intestinal epithelial-associated immune cells were isolated as stated above. We detected no differences in percentages of γδ IEL between control mice and those subjected to experimental intestinal injury (Figure 5). These data suggest that the selective reduction in γδ IEL associated with human NEC is not a secondary finding following injury but may indicate a specific risk factor.


Small intestinal intraepithelial TCRγδ+ T lymphocytes are present in the premature intestine but selectively reduced in surgical necrotizing enterocolitis.

Weitkamp JH, Rosen MJ, Zhao Z, Koyama T, Geem D, Denning TL, Rock MT, Moore DJ, Halpern MD, Matta P, Denning PW - PLoS ONE (2014)

Intestinal injury in wild-type mice is not associated with a selective reduction in γδ IEL.A) Flow cytometry of small intestinal intraepithelial cells isolated from 2 weeks old dam fed wild-type (WT control, n = 2) or wild-type mice subjected to experimental gut injury as described (WT+PAF/LPS, n = 2). C57BL/6J mice stained for CD103, CD3, CD8α, TCRγδ and TCRβ as described above. Intraepithelial cells were pregated on CD103+, CD3+ to depict IEL and then further gated on TCRγδ and TCRβ as shown. B) Percent (mean ±SE) γδ IEL (defined as percent of total IEL that were TCRγδ+, TCRβ- IEL). Data are representative of 3 independent experiments (NS indicates no statistical difference between groups).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048281&req=5

pone-0099042-g005: Intestinal injury in wild-type mice is not associated with a selective reduction in γδ IEL.A) Flow cytometry of small intestinal intraepithelial cells isolated from 2 weeks old dam fed wild-type (WT control, n = 2) or wild-type mice subjected to experimental gut injury as described (WT+PAF/LPS, n = 2). C57BL/6J mice stained for CD103, CD3, CD8α, TCRγδ and TCRβ as described above. Intraepithelial cells were pregated on CD103+, CD3+ to depict IEL and then further gated on TCRγδ and TCRβ as shown. B) Percent (mean ±SE) γδ IEL (defined as percent of total IEL that were TCRγδ+, TCRβ- IEL). Data are representative of 3 independent experiments (NS indicates no statistical difference between groups).
Mentions: For ethical reasons, it is not possible to determine definitively whether the selective reduction of γδ IEL in human NEC occurred prior to or as a result of intestinal injury. Therefore we sought to determine whether experimental intestinal injury in a murine model causes selective reduction in γδ IEL. To induce intestinal injury, we injected 2 weeks old C57BL/6J or TCRδ−/− mice intraperitoneally with 100 µg/kg PAF and 1 mg/kg E. coli 0128:B12 LPS or PBS vehicle control as described above. Pups were sacrificed two hours later and small intestinal epithelial-associated immune cells were isolated as stated above. We detected no differences in percentages of γδ IEL between control mice and those subjected to experimental intestinal injury (Figure 5). These data suggest that the selective reduction in γδ IEL associated with human NEC is not a secondary finding following injury but may indicate a specific risk factor.

Bottom Line: In human preterm infants, the proportion of IEL was reduced by 66% in 11 NEC ileum resections compared to 30 non-NEC controls (p<0.001).While γδ IEL dominated over conventional αβ IEL early in gestation in mice and in humans, γδ IEL were preferential decreased in the ileum of surgical NEC patients compared to non-NEC controls (50% reduction, p<0.05).Loss of IEL in human NEC was associated with downregulation of the Th17 transcription factor retinoic acid-related orphan nuclear hormone receptor C (RORC, p<0.001).

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Vanderbilt University School of Medicine and Monroe Carell Jr. Children's Hospital at Vanderbilt, Nashville, Tennessee, United States of America.

ABSTRACT

Background: Gastrointestinal barrier immaturity predisposes preterm infants to necrotizing enterocolitis (NEC). Intraepithelial lymphocytes (IEL) bearing the unconventional T cell receptor (TCR) γδ (γδ IEL) maintain intestinal integrity and prevent bacterial translocation in part through production of interleukin (IL) 17.

Objective: We sought to study the development of γδ IEL in the ileum of human infants and examine their role in NEC pathogenesis. We defined the ontogeny of γδ IEL proportions in murine and human intestine and subjected tcrδ-/- mice to experimental gut injury. In addition, we used polychromatic flow cytometry to calculate percentages of viable IEL (defined as CD3+ CD8+ CD103+ lymphocytes) and the fraction of γδ IEL in surgically resected tissue from infants with NEC and gestational age matched non-NEC surgical controls.

Results: In human preterm infants, the proportion of IEL was reduced by 66% in 11 NEC ileum resections compared to 30 non-NEC controls (p<0.001). While γδ IEL dominated over conventional αβ IEL early in gestation in mice and in humans, γδ IEL were preferential decreased in the ileum of surgical NEC patients compared to non-NEC controls (50% reduction, p<0.05). Loss of IEL in human NEC was associated with downregulation of the Th17 transcription factor retinoic acid-related orphan nuclear hormone receptor C (RORC, p<0.001). TCRδ-deficient mice showed increased severity of experimental gut injury (p<0.05) with higher TNFα expression but downregulation of IL17A.

Conclusion: Complimentary mouse and human data suggest a role of γδ IEL in IL17 production and intestinal barrier production early in life. Specific loss of the γδ IEL fraction may contribute to NEC pathogenesis. Nutritional or pharmacological interventions to support γδ IEL maintenance in the developing small intestine could serve as novel strategies for NEC prevention.

Show MeSH
Related in: MedlinePlus