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Invariant natural killer T-cell neutralization is a possible novel therapy for human eosinophilic esophagitis.

Rayapudi M, Rajavelu P, Zhu X, Kaul A, Niranjan R, Dynda S, Mishra A, Mattner J, Zaidi A, Dutt P, Mishra A - Clin Transl Immunology (2014)

Bottom Line: Herein, we show that iNKT cell number, their receptor subcomponents Vα24 and Vβ11 expression, and associated chemokine CXCL16 levels (or expression) are induced significantly in EoE patients compared with normal individuals.Furthermore, we show that anti-mCD1d- and anti-hVα24Jα18-neutralizing antibody treatment protects allergen-induced experimental EoE.Taken together, we have shown first time that iNKT cells have a critical pathogenic role in human and experimental EoE. iNKT cell neutralization by humanized anti-CD1d and anti-Vα24Jα18 antibodies might be a novel and potential therapy for human EoE.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cincinnati , Cincinnati, OH, USA.

ABSTRACT
Eosinophilic esophagitis (EoE) is a recently recognized inflammatory disorder that needs a potential therapeutic strategy. We earlier showed that iNKT cell-deficient mice are protected from allergen-induced EoE. Therefore, we now tested the hypothesis that iNKT cells are induced in the human EoE and is a novel possible target for the treatment of human EoE. Accordingly, we examine number of iNKT cells and eosinophils and expression of iNKT-associated cell surface receptors and chemokines by performing immunofluorescence, qPCR and ELISA in the esophageal biopsies and blood samples of normal subjects (comparison control) and EoE patients. Herein, we show that iNKT cell number, their receptor subcomponents Vα24 and Vβ11 expression, and associated chemokine CXCL16 levels (or expression) are induced significantly in EoE patients compared with normal individuals. In addition, we show that CXCL16 levels (or expression) correlate with the mRNA levels of Vα24 receptor but not well with esophageal eosinophilia in human EoE. Of note, we show that in vivo activation of iNKT cells is sufficient to induce EoE in mice. Furthermore, we show that anti-mCD1d- and anti-hVα24Jα18-neutralizing antibody treatment protects allergen-induced experimental EoE. Taken together, we have shown first time that iNKT cells have a critical pathogenic role in human and experimental EoE. iNKT cell neutralization by humanized anti-CD1d and anti-Vα24Jα18 antibodies might be a novel and potential therapy for human EoE.

No MeSH data available.


Related in: MedlinePlus

Analysis of iNKT cell-specific genes in human esophageal biopsies. The mRNA levels of iNKT cell surface molecule, TCR and T-cell components like CD1d (a), Vα24 (b), Vβ11 (c), Jα18, (d), CXCR6 (e) and chemokine CXCL16 (f) were examined by performing quantitative real-time PCR analysis. Each data point represents an individual patient (n=12–15 normal and 24–28 in EoE). The mRNA expression was normalized to GAPDH and expressed as relative expression. Statistical significance was calculated using the Mann–Whitney test. P-values for each experiment are provided in the figure (a–f).
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fig3: Analysis of iNKT cell-specific genes in human esophageal biopsies. The mRNA levels of iNKT cell surface molecule, TCR and T-cell components like CD1d (a), Vα24 (b), Vβ11 (c), Jα18, (d), CXCR6 (e) and chemokine CXCL16 (f) were examined by performing quantitative real-time PCR analysis. Each data point represents an individual patient (n=12–15 normal and 24–28 in EoE). The mRNA expression was normalized to GAPDH and expressed as relative expression. Statistical significance was calculated using the Mann–Whitney test. P-values for each experiment are provided in the figure (a–f).

Mentions: IL-15 is known as a growth and survival factor for iNKT cells,37, 38, 39 and we recently reported that IL-15 mRNA and protein levels are increased and correlate with esophageal eosinophilia in patients with EoE.27 In order to further explore the relationship of IL-15 and iNKT cells in the pathogenesis of human EoE, we performed immunofluorescence staining on frozen proximal and distal esophageal biopsy sections of normal subjects and EoE patients, using an iNKT cell-specific receptor anti-hVα24Jα18 antibody (eBioscience, San Diego, CA, USA). Our analysis indicated that induced numbers of iNKT cell receptor, anti-hVα24Jα18, positive cells are accumulated in the esophageal mucosa of human EoE (Figure 1A). In contrast, none to few anti-hVα24Jα18-iNKT-positive cells were detected in esophageal biopsies of normal subjects (Figure 1B). The specificity of the staining was demonstrated by the lack of positive staining of isotype-matched control antibodies on the EoE patient biopsy sections (Figure 1C). Further, anti-hVα24Jα18-positive iNKT cells were quantified in the esophageal biopsies of normal subjects and EoE patients. The analysis indicated that ∼20±5 iNKT cells (Vα24Jα18+) accumulated per high power field (hpf) in the esophageal biopsies of EoE patients compared with average <1 iNKT cells/hpf (Figure 1D). In addition, we examined the number of circulating iNKT cells in normal subjects (comparison control individual), EoE and non-EoE/CE patients by staining total blood cells with anti-CD3 and anti-hVα24Jα18 antibodies followed by flow cytometry analysis, and gated populations are shown in Figures 2a–d. The absolute number as well as percent of iNKT cells was decreased in the blood of EoE patients compared with normal subjects or non-EoE/CE patients (Figures 2e and f). The decrease of iNKT cells was confirmed by examining the mRNA level of iNKT cell specific T-cell components in the blood of normal and EoE patients. The levels of Vβ11 and Vα24 were found significantly reduced in EoE patients compared with normal (comparison control) subjects (Figures 2g and h). All EoE patients used in the analyses have active EoE (>15 eosinophil/hpf) without any steroids or other treatments. The decreased number of iNKT cells in the blood of human EoE patients may be due to the induced chemotaxis of iNKT cells in the esophagus. Notably, our flow cytometric analysis of comparison control normal subjects exhibit a large inter-donor variation of iNKT cell percent as well as in absolute cell number counts that suggests iNKT cell response varies from individual to individual even at healthy state. However, in the earlier studies investigators indicated that iNKT cells are rarely present in normal individuals.14 To further confirm the anatomical relationship of iNKT cells with esophageal epithelial cells, we measured mRNA levels of iNKT cell-specific genes in the esophageal biopsies from normal subjects and EoE patients. The quantitative real-time PCR analysis indicated that mRNA expression for several iNKT cell-related genes such as cell surface molecule, TCR and their subcomponents, CD1d, Vα24, Vβ11, CXCR6, and chemokine CXCL16 were significantly induced in the esophageal biopsies of EoE patients compared with comparison control normal subjects (Figures 3a–f). The details of primers used to analyze the mRNA levels of iNKT cell-associated genes are provided in Supplementary Table 1.


Invariant natural killer T-cell neutralization is a possible novel therapy for human eosinophilic esophagitis.

Rayapudi M, Rajavelu P, Zhu X, Kaul A, Niranjan R, Dynda S, Mishra A, Mattner J, Zaidi A, Dutt P, Mishra A - Clin Transl Immunology (2014)

Analysis of iNKT cell-specific genes in human esophageal biopsies. The mRNA levels of iNKT cell surface molecule, TCR and T-cell components like CD1d (a), Vα24 (b), Vβ11 (c), Jα18, (d), CXCR6 (e) and chemokine CXCL16 (f) were examined by performing quantitative real-time PCR analysis. Each data point represents an individual patient (n=12–15 normal and 24–28 in EoE). The mRNA expression was normalized to GAPDH and expressed as relative expression. Statistical significance was calculated using the Mann–Whitney test. P-values for each experiment are provided in the figure (a–f).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232063&req=5

fig3: Analysis of iNKT cell-specific genes in human esophageal biopsies. The mRNA levels of iNKT cell surface molecule, TCR and T-cell components like CD1d (a), Vα24 (b), Vβ11 (c), Jα18, (d), CXCR6 (e) and chemokine CXCL16 (f) were examined by performing quantitative real-time PCR analysis. Each data point represents an individual patient (n=12–15 normal and 24–28 in EoE). The mRNA expression was normalized to GAPDH and expressed as relative expression. Statistical significance was calculated using the Mann–Whitney test. P-values for each experiment are provided in the figure (a–f).
Mentions: IL-15 is known as a growth and survival factor for iNKT cells,37, 38, 39 and we recently reported that IL-15 mRNA and protein levels are increased and correlate with esophageal eosinophilia in patients with EoE.27 In order to further explore the relationship of IL-15 and iNKT cells in the pathogenesis of human EoE, we performed immunofluorescence staining on frozen proximal and distal esophageal biopsy sections of normal subjects and EoE patients, using an iNKT cell-specific receptor anti-hVα24Jα18 antibody (eBioscience, San Diego, CA, USA). Our analysis indicated that induced numbers of iNKT cell receptor, anti-hVα24Jα18, positive cells are accumulated in the esophageal mucosa of human EoE (Figure 1A). In contrast, none to few anti-hVα24Jα18-iNKT-positive cells were detected in esophageal biopsies of normal subjects (Figure 1B). The specificity of the staining was demonstrated by the lack of positive staining of isotype-matched control antibodies on the EoE patient biopsy sections (Figure 1C). Further, anti-hVα24Jα18-positive iNKT cells were quantified in the esophageal biopsies of normal subjects and EoE patients. The analysis indicated that ∼20±5 iNKT cells (Vα24Jα18+) accumulated per high power field (hpf) in the esophageal biopsies of EoE patients compared with average <1 iNKT cells/hpf (Figure 1D). In addition, we examined the number of circulating iNKT cells in normal subjects (comparison control individual), EoE and non-EoE/CE patients by staining total blood cells with anti-CD3 and anti-hVα24Jα18 antibodies followed by flow cytometry analysis, and gated populations are shown in Figures 2a–d. The absolute number as well as percent of iNKT cells was decreased in the blood of EoE patients compared with normal subjects or non-EoE/CE patients (Figures 2e and f). The decrease of iNKT cells was confirmed by examining the mRNA level of iNKT cell specific T-cell components in the blood of normal and EoE patients. The levels of Vβ11 and Vα24 were found significantly reduced in EoE patients compared with normal (comparison control) subjects (Figures 2g and h). All EoE patients used in the analyses have active EoE (>15 eosinophil/hpf) without any steroids or other treatments. The decreased number of iNKT cells in the blood of human EoE patients may be due to the induced chemotaxis of iNKT cells in the esophagus. Notably, our flow cytometric analysis of comparison control normal subjects exhibit a large inter-donor variation of iNKT cell percent as well as in absolute cell number counts that suggests iNKT cell response varies from individual to individual even at healthy state. However, in the earlier studies investigators indicated that iNKT cells are rarely present in normal individuals.14 To further confirm the anatomical relationship of iNKT cells with esophageal epithelial cells, we measured mRNA levels of iNKT cell-specific genes in the esophageal biopsies from normal subjects and EoE patients. The quantitative real-time PCR analysis indicated that mRNA expression for several iNKT cell-related genes such as cell surface molecule, TCR and their subcomponents, CD1d, Vα24, Vβ11, CXCR6, and chemokine CXCL16 were significantly induced in the esophageal biopsies of EoE patients compared with comparison control normal subjects (Figures 3a–f). The details of primers used to analyze the mRNA levels of iNKT cell-associated genes are provided in Supplementary Table 1.

Bottom Line: Herein, we show that iNKT cell number, their receptor subcomponents Vα24 and Vβ11 expression, and associated chemokine CXCL16 levels (or expression) are induced significantly in EoE patients compared with normal individuals.Furthermore, we show that anti-mCD1d- and anti-hVα24Jα18-neutralizing antibody treatment protects allergen-induced experimental EoE.Taken together, we have shown first time that iNKT cells have a critical pathogenic role in human and experimental EoE. iNKT cell neutralization by humanized anti-CD1d and anti-Vα24Jα18 antibodies might be a novel and potential therapy for human EoE.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cincinnati , Cincinnati, OH, USA.

ABSTRACT
Eosinophilic esophagitis (EoE) is a recently recognized inflammatory disorder that needs a potential therapeutic strategy. We earlier showed that iNKT cell-deficient mice are protected from allergen-induced EoE. Therefore, we now tested the hypothesis that iNKT cells are induced in the human EoE and is a novel possible target for the treatment of human EoE. Accordingly, we examine number of iNKT cells and eosinophils and expression of iNKT-associated cell surface receptors and chemokines by performing immunofluorescence, qPCR and ELISA in the esophageal biopsies and blood samples of normal subjects (comparison control) and EoE patients. Herein, we show that iNKT cell number, their receptor subcomponents Vα24 and Vβ11 expression, and associated chemokine CXCL16 levels (or expression) are induced significantly in EoE patients compared with normal individuals. In addition, we show that CXCL16 levels (or expression) correlate with the mRNA levels of Vα24 receptor but not well with esophageal eosinophilia in human EoE. Of note, we show that in vivo activation of iNKT cells is sufficient to induce EoE in mice. Furthermore, we show that anti-mCD1d- and anti-hVα24Jα18-neutralizing antibody treatment protects allergen-induced experimental EoE. Taken together, we have shown first time that iNKT cells have a critical pathogenic role in human and experimental EoE. iNKT cell neutralization by humanized anti-CD1d and anti-Vα24Jα18 antibodies might be a novel and potential therapy for human EoE.

No MeSH data available.


Related in: MedlinePlus