Live and heat-treated probiotics differently modulate IL10 mRNA stabilization and microRNA expression Audrey Demont, BS, Feriel Hacini-Rachinel, PhD,

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Live and heat-treated probiotics differently modulate IL10 mRNA stabilization and microRNA expression Audrey Demont, BS, Feriel Hacini-Rachinel, PhD, Rémi Doucet-Ladevèze, MS, Catherine Ngom-Bru, PhD, Annick Mercenier, PhD, Guénolée Prioult, PhD, Carine Blanchard, PhD Journal of Allergy and Clinical Immunology Volume 137, Issue 4, Pages 1264-1267.e10 (April 2016) DOI: 10.1016/j.jaci.2015.08.033 Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Cytokine expression in PBMCs stimulated with live or heat-treated L paracasei NCC 2461. A, IL-10 protein secreted in the supernatant. B, IL10 mRNA expression in PBMCs stimulated with live or heat- treated L paracasei NCC 2461. C, IL10 mRNA half-life in PBMCs. Fig 1, B and C, Experiments shown are means of triplicates and representative of 5 independent PBMC donors. D, The median of IL10 mRNA half-life is shown in 5 PBMC donors. Journal of Allergy and Clinical Immunology 2016 137, 1264-1267.e10DOI: (10.1016/j.jaci.2015.08.033) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Role of L paracasei NCC 2461 in the 3′UTR-mediated IL10 mRNA stabilization and in the differential microRNA expression. A, Effect of heat-treated L paracasei NCC2461 on IL10-3′UTR-dependent luciferase activity. B, Differential expression level of microRNA in PBMCs stimulated with live or heat-treated L paracasei NCC 2461. C, Expression level of miR-27a in PBMCs. D, Effect of miR-27a on the IL10-3′UTR-dependent luciferase activity. Journal of Allergy and Clinical Immunology 2016 137, 1264-1267.e10DOI: (10.1016/j.jaci.2015.08.033) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Intracellular IL-10 or IFN-γ cytokine expressions in PBMCs. Intracellular IL-10 or IFN-γ cytokine expressions were determined in CD3− CD3+ and CD14+ cell subpopulations. Geometric means of fluorescence intensity (MFI) are shown. *P < .05 compared with unstimulated in the corresponding population subset using the Mann-Whitney U test. Journal of Allergy and Clinical Immunology 2016 137, 1264-1267.e10DOI: (10.1016/j.jaci.2015.08.033) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 IFN-γ expression in PBMCs stimulated with live or heat-treated L paracasei NCC 2461. A, IFN-γ protein secreted in the supernatant. B, IFNG mRNA expression in PBMCs stimulated with live or heat-treated L paracasei NCC 2461. C, IFNG mRNA half-life in PBMCs. Experiments shown are means of triplicates and representative of 5 independent PBMC donors. Journal of Allergy and Clinical Immunology 2016 137, 1264-1267.e10DOI: (10.1016/j.jaci.2015.08.033) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Comparative expression of the microRNA targeting IL10 mRNA. Fold changes in microRNA expression in PBMCs stimulated with L paracasei (live or heat-treated) compared with unstimulated cells were plotted using Ingenuity Pathway Analysis. *Only 1 of the mircoRNAs is shown while several are in the list. Here, miR-27a is downregulated, miR-27b is upregulated. Only miR-27b upregulation is shown here. See Table E1 for exact fold changes of individual microRNAs. Journal of Allergy and Clinical Immunology 2016 137, 1264-1267.e10DOI: (10.1016/j.jaci.2015.08.033) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Number of sequences with at least 70% similarity to miR-27a and control sequence in the genome of 6 probiotic strains. A control sequence present in the pre–miR-27a sequence and the miR-27a sequence were compared with the chromosomes of 6 probiotic strains. The 2 sequences of 20-21 nucleotides were compared after reverse translation U->T against the genome of 6 strains using FUZZNUC (EMBOSS package) allowing for 4 to 6 mismatches (80% to 70% similarity). Journal of Allergy and Clinical Immunology 2016 137, 1264-1267.e10DOI: (10.1016/j.jaci.2015.08.033) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions