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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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Reduced proportions of γδ IEL subsets in patients with NEC compared to non-NEC surgical controls.(A) Example of the gating strategy used to calculate proportions of γδ IEL subsets. The control sample shown is from a 4 days old 26 weeks gestation infant with spontaneous (focal) intestinal perforation and the NEC sample is from a 15 days old 28 weeks gestation infant with surgical NEC. Gates were set on “live”, CD14−, CD19− (“Dump” negative) and CD3+ cells before applying to sub-populations. Next we identified CD3+ CD8+ T cells followed by differentiating conventional CD3+ CD103+ TCRαβ from TCRγδ IEL (γδ IEL). The patient with NEC showed significant reduction in γδ IEL with a corresponding greater proportion of αE integrin (CD103) negative, conventional T cells. Dot plot of total IEL (B) and γδ IEL (C) proportions were statistically significantly reduced in NEC tissue compared to non-NEC controls, p<0.001 and p = 0.02, respectively.
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pone-0099042-g001: Reduced proportions of γδ IEL subsets in patients with NEC compared to non-NEC surgical controls.(A) Example of the gating strategy used to calculate proportions of γδ IEL subsets. The control sample shown is from a 4 days old 26 weeks gestation infant with spontaneous (focal) intestinal perforation and the NEC sample is from a 15 days old 28 weeks gestation infant with surgical NEC. Gates were set on “live”, CD14−, CD19− (“Dump” negative) and CD3+ cells before applying to sub-populations. Next we identified CD3+ CD8+ T cells followed by differentiating conventional CD3+ CD103+ TCRαβ from TCRγδ IEL (γδ IEL). The patient with NEC showed significant reduction in γδ IEL with a corresponding greater proportion of αE integrin (CD103) negative, conventional T cells. Dot plot of total IEL (B) and γδ IEL (C) proportions were statistically significantly reduced in NEC tissue compared to non-NEC controls, p<0.001 and p = 0.02, respectively.

Mentions: We compared the proportions of total IEL and γδ IEL as demonstrated in Figure 1A. Using flow cytometry we defined IEL as life CD3+ CD8+ CD103+ lymphocytes and characterized as γδ IEL if cells were also TCRαβ- and TCRγδ+. Compared to non-NEC surgical controls, NEC samples exhibited significantly lower numbers of total IEL (mean 2,342 versus 124 cells per tissue section, p<0.01). Because NEC is associated with necrosis and intestinal epithelium loss likely explaining reduction in total IEL, we calculated percentages of IEL based on total CD3+ CD8+ cells isolated in tissue epithelium preparations. The mean fraction of IEL within epithelial CD3+ CD8+ cells in non-NEC surgical controls was 64% compared to 23% in NEC, Figure 1B, p<0.001). Within the IEL compartment of the control group, a sizable proportion of cells were γδ IEL (mean 27%), which was significantly decreased in NEC patients (mean 15%) (Figure 1C, p = 0.02). Therefore surgical NEC was characterized by a preferential reduction in γδ IEL over αβ IEL.


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)

Reduced proportions of γδ IEL subsets in patients with NEC compared to non-NEC surgical controls.(A) Example of the gating strategy used to calculate proportions of γδ IEL subsets. The control sample shown is from a 4 days old 26 weeks gestation infant with spontaneous (focal) intestinal perforation and the NEC sample is from a 15 days old 28 weeks gestation infant with surgical NEC. Gates were set on “live”, CD14−, CD19− (“Dump” negative) and CD3+ cells before applying to sub-populations. Next we identified CD3+ CD8+ T cells followed by differentiating conventional CD3+ CD103+ TCRαβ from TCRγδ IEL (γδ IEL). The patient with NEC showed significant reduction in γδ IEL with a corresponding greater proportion of αE integrin (CD103) negative, conventional T cells. Dot plot of total IEL (B) and γδ IEL (C) proportions were statistically significantly reduced in NEC tissue compared to non-NEC controls, p<0.001 and p = 0.02, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099042-g001: Reduced proportions of γδ IEL subsets in patients with NEC compared to non-NEC surgical controls.(A) Example of the gating strategy used to calculate proportions of γδ IEL subsets. The control sample shown is from a 4 days old 26 weeks gestation infant with spontaneous (focal) intestinal perforation and the NEC sample is from a 15 days old 28 weeks gestation infant with surgical NEC. Gates were set on “live”, CD14−, CD19− (“Dump” negative) and CD3+ cells before applying to sub-populations. Next we identified CD3+ CD8+ T cells followed by differentiating conventional CD3+ CD103+ TCRαβ from TCRγδ IEL (γδ IEL). The patient with NEC showed significant reduction in γδ IEL with a corresponding greater proportion of αE integrin (CD103) negative, conventional T cells. Dot plot of total IEL (B) and γδ IEL (C) proportions were statistically significantly reduced in NEC tissue compared to non-NEC controls, p<0.001 and p = 0.02, respectively.
Mentions: We compared the proportions of total IEL and γδ IEL as demonstrated in Figure 1A. Using flow cytometry we defined IEL as life CD3+ CD8+ CD103+ lymphocytes and characterized as γδ IEL if cells were also TCRαβ- and TCRγδ+. Compared to non-NEC surgical controls, NEC samples exhibited significantly lower numbers of total IEL (mean 2,342 versus 124 cells per tissue section, p<0.01). Because NEC is associated with necrosis and intestinal epithelium loss likely explaining reduction in total IEL, we calculated percentages of IEL based on total CD3+ CD8+ cells isolated in tissue epithelium preparations. The mean fraction of IEL within epithelial CD3+ CD8+ cells in non-NEC surgical controls was 64% compared to 23% in NEC, Figure 1B, p<0.001). Within the IEL compartment of the control group, a sizable proportion of cells were γδ IEL (mean 27%), which was significantly decreased in NEC patients (mean 15%) (Figure 1C, p = 0.02). Therefore surgical NEC was characterized by a preferential reduction in γδ IEL over αβ IEL.

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