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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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Retinoic acid-related orphan nuclear hormone receptor C (RORC) gene expression in intestinal lymphocyte subsets.(A) We measured gene expression levels of RORC by quantitative RT-PCR in 15 NEC tissue sections and 7 controls by quantitative RT-PCR array. RORC gene expression was significantly decreased in NEC samples versus controls (p<0.001). Relative level of mRNA expression of RORC in each sample was normalized to the expression level of reference gene GAPDH. (B) To determine whether loss of IEL contributed to reduction of RORC expression, we compared IEL with lamina propria lymphocytes (LPL) from the same tissue source using 10 non-NEC controls. RORC gene expression was significantly higher in IEL compared to LPL (p = 0.01). (C) RORC gene expression and proportions of total TCRγδ IEL correlated positively with each other (p = 0.02).
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pone-0099042-g006: Retinoic acid-related orphan nuclear hormone receptor C (RORC) gene expression in intestinal lymphocyte subsets.(A) We measured gene expression levels of RORC by quantitative RT-PCR in 15 NEC tissue sections and 7 controls by quantitative RT-PCR array. RORC gene expression was significantly decreased in NEC samples versus controls (p<0.001). Relative level of mRNA expression of RORC in each sample was normalized to the expression level of reference gene GAPDH. (B) To determine whether loss of IEL contributed to reduction of RORC expression, we compared IEL with lamina propria lymphocytes (LPL) from the same tissue source using 10 non-NEC controls. RORC gene expression was significantly higher in IEL compared to LPL (p = 0.01). (C) RORC gene expression and proportions of total TCRγδ IEL correlated positively with each other (p = 0.02).

Mentions: TCRγδ cells have been attributed an important role in innate mucosal immune responses, partially mediated through the production of IL17 [35], [36]. TCRγδ IEL have been specifically shown to produce IL17 under inflammatory conditions [37], [38]. To determine whether a similar mechanism may play a role in the human neonatal gut, we measured the gene expression of retinoic acid-related orphan nuclear hormone receptor C (RORC) in the small intestinal mucosa of 15 NEC patients compared to 7 surgical controls. Human RORC is an analogue to the murine retinoid orphan receptor (RORγt), which drives expression of IL17 in γδ IEL [36]. Since expression of IL17 is dependent on cell stimulation and IEL numbers were too low to isolate sufficient cells for stimulation assays, we used RORC gene expression as a correlate for IL17 production [39]. By quantitative RT-PCR, RORC gene expression in NEC samples was reduced by a median of 10 fold (p<0.001, Figure 6A). Next, we sought to determine if the reduction of RORC expression in NEC could be explained by loss of γδ IEL. We measured RORC gene expression in LPL and IEL isolated from identical tissue sections from non-NEC controls. RORC gene expression was significantly higher in IEL compared to LPL (p = 0.01, Figure 6B). In addition, we found a statistically significant positive correlation between total TCRγδ+ IEL proportions and RORC gene expression (Pearson R2 = 0.41, p = 0.02 (Figure 6C). Cumulatively, these data suggest that loss of γδ IEL in NEC may limit intestinal barrier defense through decreased production of IL17.


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)

Retinoic acid-related orphan nuclear hormone receptor C (RORC) gene expression in intestinal lymphocyte subsets.(A) We measured gene expression levels of RORC by quantitative RT-PCR in 15 NEC tissue sections and 7 controls by quantitative RT-PCR array. RORC gene expression was significantly decreased in NEC samples versus controls (p<0.001). Relative level of mRNA expression of RORC in each sample was normalized to the expression level of reference gene GAPDH. (B) To determine whether loss of IEL contributed to reduction of RORC expression, we compared IEL with lamina propria lymphocytes (LPL) from the same tissue source using 10 non-NEC controls. RORC gene expression was significantly higher in IEL compared to LPL (p = 0.01). (C) RORC gene expression and proportions of total TCRγδ IEL correlated positively with each other (p = 0.02).
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pone-0099042-g006: Retinoic acid-related orphan nuclear hormone receptor C (RORC) gene expression in intestinal lymphocyte subsets.(A) We measured gene expression levels of RORC by quantitative RT-PCR in 15 NEC tissue sections and 7 controls by quantitative RT-PCR array. RORC gene expression was significantly decreased in NEC samples versus controls (p<0.001). Relative level of mRNA expression of RORC in each sample was normalized to the expression level of reference gene GAPDH. (B) To determine whether loss of IEL contributed to reduction of RORC expression, we compared IEL with lamina propria lymphocytes (LPL) from the same tissue source using 10 non-NEC controls. RORC gene expression was significantly higher in IEL compared to LPL (p = 0.01). (C) RORC gene expression and proportions of total TCRγδ IEL correlated positively with each other (p = 0.02).
Mentions: TCRγδ cells have been attributed an important role in innate mucosal immune responses, partially mediated through the production of IL17 [35], [36]. TCRγδ IEL have been specifically shown to produce IL17 under inflammatory conditions [37], [38]. To determine whether a similar mechanism may play a role in the human neonatal gut, we measured the gene expression of retinoic acid-related orphan nuclear hormone receptor C (RORC) in the small intestinal mucosa of 15 NEC patients compared to 7 surgical controls. Human RORC is an analogue to the murine retinoid orphan receptor (RORγt), which drives expression of IL17 in γδ IEL [36]. Since expression of IL17 is dependent on cell stimulation and IEL numbers were too low to isolate sufficient cells for stimulation assays, we used RORC gene expression as a correlate for IL17 production [39]. By quantitative RT-PCR, RORC gene expression in NEC samples was reduced by a median of 10 fold (p<0.001, Figure 6A). Next, we sought to determine if the reduction of RORC expression in NEC could be explained by loss of γδ IEL. We measured RORC gene expression in LPL and IEL isolated from identical tissue sections from non-NEC controls. RORC gene expression was significantly higher in IEL compared to LPL (p = 0.01, Figure 6B). In addition, we found a statistically significant positive correlation between total TCRγδ+ IEL proportions and RORC gene expression (Pearson R2 = 0.41, p = 0.02 (Figure 6C). Cumulatively, these data suggest that loss of γδ IEL in NEC may limit intestinal barrier defense through decreased production of IL17.

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