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Modulation of mTOR Effector Phosphoproteins in Blood Basophils from Allergic Patients

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Abstract

The mammalian target of rapamycin (mTOR) pathway contributes to various immunoinflammatory processes. Yet, its potential involvement in basophil responses in allergy remains unclear. In this pilot study, we quantified two key mTOR effector phosphoproteins, the eukaryotic initiation factor 4E (peIF4E) and S6 ribosomal protein (pS6rp), in blood basophils from nut allergy patients (NA, N = 16) and healthy controls (HC, N = 13). Without stimulation in vitro, basophil peIF4E levels were higher in NA than HC subjects (P = 0.014). Stimulation with nut (offending) but not chicken / rice (non-offending) extract increased basophil peIF4E and pS6rp levels (+32%, P = 0.018, and +98%, P = 0.0026, respectively) in NA but not HC subjects, concomitant with increased surface levels of CD203c and CD63, both known to reflect basophil activation. Pre-treatment with the mTOR inhibitor rapamycin decreased pS6rp and CD203c responses in nut extract-stimulated basophils in NA subjects. Thus, basophil responses to offending allergens are associated with modulation of mTOR effector phosphoproteins.

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Abbreviations

EDTA:

ethylene diamine tetraacetic acid

eIF4E:

eukaryotic Initiation Factor 4E

HC:

healthy control

MFI:

median fluorescence intensity

mTOR:

mammalian target of rapamycin

NA:

nut allergy

PBS:

phosphate buffered saline

p:

phosphorylated

S6rp:

S6 ribosomal protein

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Acknowledgments

We thank our subjects for their participation; C. Crumpton and J. Van Dyke at the Stanford FACS Facility for technical support; Drs. K. Atkuri, E. Ghosn, A. Kumar, K. Mukai, P. Sadate-Ngatchou, and M Tsai, as well as C. McDonald-Hyman for critical advice; Dr. V. Saper for recruiting two subjects; and M. Miglianico and E. Hoyte for technical assistance.

Competing interest statement

The authors declare to hold no conflict of interest with the publication of the results included in this manuscript.

Funding

Stanford School of Medicine’s Dean Fellowship (YG), Stanford Morgridge and Gallo Fellowship (YG) and the Stanford Institute of Immunity, Transplantation and Infectious Diseases Seed Grant (KCN), Orsak Family Fund (KCN), the Skippy Frank Foundation (RT and YG), the Stanford School of Medicine SPARK/SPECTRUM program (YG and KN), and United States Public Health Service grants AI23990, AI070813 and CA72074 (SJG).

Author information

Authors and Affiliations

  1. Department of Genetics, Stanford University School of Medicine, Stanford, CA, 94305, USA

    Yael Gernez & Leonore A. Herzenberg

  2. Department of Pediatrics, Stanford University School of Medicine, 269 Campus Drive East, Rm 2115, CCSR Bldg, Stanford, CA, 94305, USA

    Rabindra Tirouvanziam, Neha Reshamwala, Grace Yu, Brittany C. Weldon & Kari C. Nadeau

  3. Emory+Children’s Center for Cystic Fibrosis Research, Department of Pediatrics, Emory University School of Medicine and Children’s Healthcare of Atlanta, 2015 Uppergate Drive, Atlanta, GA, 30322, USA

    Rabindra Tirouvanziam

  4. Department of Pathology, Stanford University School of Medicine, Stanford, CA, 94305, USA

    Stephen J. Galli

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  1. Yael Gernez
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  2. Rabindra Tirouvanziam
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Corresponding authors

Correspondence to Rabindra Tirouvanziam or Kari C. Nadeau.

Additional information

Yael Gernez and Rabindra Tirouvanziam contributed equally to studies presented here and share first authorship.

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Supplementary Material

(DOC 39 kb)

Supplementary Figure 1

Percent increase in elF4E and S6rp upon 10-minute stimulation with nut extract in blood basophils from NA patients. Data are presented for total (T) and phosphorylated (p) forms of eIF4E and S6rp proteins. Results are shown for each of these 4 markers as % increase (mean±SE; N = 2), calculated as: 100 x ([MFI for marker upon nut extract stimulation] - [MFI for marker without stimulation]) / [MFI for marker without stimulation]. (JPEG 88 kb)

Supplementary Figure 2

Surface CD203c and CD63 levels in blood basophils without stimulation and upon stimulation with nut (offending) or chicken / rice (non-offending) extracts. Shown are box plots (delimited by 25th and 75th percentiles, with median line inside and outside bars for 10th and 90th percentiles) for CD203c and CD63 levels in blood basophils from healthy control (HC, N = 12) and nut allergy (NA, N = 15) subjects. Each point represents an individual subject. The significance of differences between HC and NA were calculated with the Wilcoxon rank-sum test. NS for not significant. (JPEG 165 kb)

Supplementary Figure 3

Effect of 10-minute and 30-minute stimulation with nut extract on levels of effector phosphoproteins in blood basophils from nut allergy (NA) subjects. Shown are levels of pS6rp (left) and peIF4E (right) in basophils, comparing 10-minute stimulation (black histograms) to 30-minute stimulation (grey histograms). Data are presented as % response compared to the 10-minute timepoint (mean±SE, with data at the 10-minute timepoint set at 100, by definition; N = 3 patients). (JPEG 43 kb)

Supplementary Figure 4

Effect of rapamycin pre-treatment on blood basophil CD203c levels following nut allergen stimulation. Shown are box plots (delimited by 25th and 75th percentiles, with median line inside and outside bars marking 10th and 90th percentiles) for differential changes in CD203c in blood basophils from nut allergy (NA, N = 5) subjects, calculated as follows: differential = [MFI for CD203c upon nut allergen stimulation, without rapamycin pre-treatment] - [MFI for the same surface marker upon nut allergen stimulation, with 30-minute pre-treatment with 10 nM rapamycin]. Each point represents an individual subject. (JPEG 67 kb)

Supplementary Figure 5

Neutrophil and eosinophil phosphoprotein profiling in blood. a: Live neutrophils and eosinophils were selected as Live/Deadlo / CD3-/ CD16+ / CD20- / CD56- / CD66b+ and CD123- and CD123+ populations, respectively. b: peIF4E and pS6rp were then quantified in gated eosinophils and neutrophils. FMO: intracellular phosphoprotein “Fluorescence Minus One” control (see Methods section above for details). Shown here is one representative NA subject, whose neutrophils and eosinophils were studied after nut extract stimulation. Blood neutrophils and eosinophils from all HC (N = 13) and NA (N = 16) subjects were successfully gated using this strategy, in all conditions studied (under no stimulation or upon stimulation with either nut or chicken / rice extracts). (JPEG 463 kb)

Supplementary Figure 6

Phosphoprotein profiling in eosinophils without stimulation and upon stimulation with nut (offending) or chicken / rice (non-offending) extract. Shown are box plots (delimited by 25th and 75th percentiles, with median line inside and outside bars marking 10th and 90th percentiles) for levels of peIF4E (a) and pS6rp (b) in blood eosinophils from healthy control (HC, N = 13) and nut allergy (NA, N = 15) subjects. Each point represents an individual subject. The significance of differences between HC and NA were calculated with the Wilcoxon rank-sum test. NS for not significant. (JPEG 140 kb)

Supplementary Figure 7

Phosphoprotein profiling in neutrophils without stimulation and upon stimulation with nut (offending) or chicken / rice (non-offending) extract. Shown are box plots (delimited by 25th and 75th percentiles, with median line inside and outside bars marking 10th and 90th percentiles) for levels of peIF4E (a) and pS6rp (b) in blood neutrophils from healthy control (HC, N = 12) and nut allergy (NA, N = 16) subjects. Each point represents an individual subject. The significance of differences between HC and NA were calculated with the Wilcoxon rank-sum test. NS for not significant. (JPEG 177 kb)

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Gernez, Y., Tirouvanziam, R., Reshamwala, N. et al. Modulation of mTOR Effector Phosphoproteins in Blood Basophils from Allergic Patients. J Clin Immunol 32, 565–573 (2012). https://doi.org/10.1007/s10875-012-9651-x

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