Human CD40 ligand deficiency dysregulates the macrophage transcriptome causing functional defects that are improved by exogenous IFN-γ Otavio Cabral-Marques,

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Presentation transcript:

Human CD40 ligand deficiency dysregulates the macrophage transcriptome causing functional defects that are improved by exogenous IFN-γ Otavio Cabral-Marques, PhD, Rodrigo Nalio Ramos, PhD, Lena F. Schimke, MD, Taj Ali Khan, PhD, Eduardo Pinheiro Amaral, PhD, Caio César Barbosa Bomfim, MSc, Osvaldo Reis Junior, MSc, Tabata Takahashi França, MSc, Christina Arslanian, BSc, Joanna Darck Carola Correia Lima, MSc, Cristina Worm Weber, MD, Janaíra Fernandes Ferreira, MD, Fabiola Scancetti Tavares, MD, Jing Sun, MD, Maria Regina D'Imperio Lima, PhD, Marília Seelaender, PhD, Vera Lucia Garcia Calich, PhD, José Alexandre Marzagão Barbuto, MD, PhD, Beatriz Tavares Costa-Carvalho, MD, PhD, Gabriela Riemekasten, MD, Gisela Seminario, MD, Liliana Bezrodnik, MD, Luigi Notarangelo, MD, Troy R. Torgerson, MD, PhD, Hans D. Ochs, MD, Antonio Condino-Neto, MD, PhD Journal of Allergy and Clinical Immunology Volume 139, Issue 3, Pages 900-912.e7 (March 2017) DOI: 10.1016/j.jaci.2016.07.018 Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 rhIFN-γ, but not sCD40L, improves the defective fungicidal activity and oxidative burst of MDMs from CD40L-deficient patients. A, Cell morphology (top) was assessed by means of phase contrast (Axio Vert.A1) after 5 days in the presence of M-CSF. Flow cytometric analysis (bottom) was used to characterize expression of CD14, HLA-DR, CD64, CD163, and CD86 on the surfaces of MDMs. B, After challenging MDMs with P brasiliensis, fungicidal activity was assessed by determining CFU values. Before assay, MDMs were untreated (−) or treated with (+) sCD40L (500 ng/mL) or rhIFN-γ (100 U/mL) for 48 hours. CFU values (as percentages of control values) were determined in relation to the CFU number of untreated MDMs from healthy control subjects. The results in scatter plots and raw data in CFU per milliliter are shown in Fig E2. C, MDMs remained untreated or were cultured for 48 hours in the presence of sCD40L (500 ng/mL) or rhIFN-γ (100 U/mL); the respiratory burst of MDMs was induced by PMA (90 mmol/L). Cells were analyzed with the luminol-enhanced chemiluminescence assay, and values are expressed as relative light units (RLU). A significant difference is denoted as follows: *P ≤ .05 (n = 6 patients and 6 control subjects), Mann-Whitney test. NS, Not significant. Journal of Allergy and Clinical Immunology 2017 139, 900-912.e7DOI: (10.1016/j.jaci.2016.07.018) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Impaired cytokine production by macrophages from CD40L-deficient patients. Patients' macrophages secrete abnormal levels of IL-6, TNF-α, IL-1β, macrophage inflammatory protein 1β [MIP-1β], IP-10, and G-CSF in response to M tuberculosis and P brasiliensis. After 48 hours of sCD40L (500 ng/mL) or rhIFN-γ (100 U/mL) treatment, cytokine production by patients' macrophages achieved a pattern similar to that observed in healthy control subjects. Significant differences are denoted as follows: *P ≤ .05 (n = 6 patients and 6 control subjects), Mann-Whitney test. Journal of Allergy and Clinical Immunology 2017 139, 900-912.e7DOI: (10.1016/j.jaci.2016.07.018) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Defective control of the proliferation of M tuberculosis by MDMs from CD40L-deficient patients is improved by rhIFN-γ. A, Macrophages were challenged with M tuberculosis (H37Rv strain), and the phagocytosis index was determined on day 0 based on CFU counts. B, The bacterial proliferation index on day 6 was determined based on the ratio of CFU numbers on day 6 to CFU numbers on day 0. Data were normalized according to the average mycobacterial growth of untreated MDMs from healthy control subjects. Significant differences are denoted as follows: *P ≤ .05 (n = 6 patients and 6 control subjects), Mann-Whitney test. NS, Not significant. Journal of Allergy and Clinical Immunology 2017 139, 900-912.e7DOI: (10.1016/j.jaci.2016.07.018) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Hierarchical cluster analysis showing MDM gene signature in patients with CD40L deficiency and the subsequent effects of IFN-γ. A, RNA from MDMs was sequenced, and the transcripts per million (TPM) values are represented on a log2 scale, where green shows low expression and red shows high expression. The results of untreated (left panel) and rhIFN-γ–treated (right panel) cells are shown in the heat map. B, Interaction networks for the DEGs in CD40L deficiency are shown. Networks are shown as predicted by GeneMania and visualized with Cytoscape. Journal of Allergy and Clinical Immunology 2017 139, 900-912.e7DOI: (10.1016/j.jaci.2016.07.018) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Heat map of genes not dysregulated in patients with CD40L deficiency but affected by rhIFN-γ treatment. Results for untreated and rhIFN-γ–treated cells are shown in the heat map. RNA from MDMs were sequenced, and the transcripts per million (TPM) values are represented on a log2 scale, where green shows low expression and red shows high expression. Genes significantly upregulated or downregulated are listed. Journal of Allergy and Clinical Immunology 2017 139, 900-912.e7DOI: (10.1016/j.jaci.2016.07.018) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Effect of rhIFN-γ on expression of CD40, TLRs, and CLRs by macrophages. Representative histograms (A) and graphics (B) showing CD40, TLR (TLR1, TLR2, and TLR4), and CLR (dectin-1, dectin-3, mannose receptor or MR, DEC-205, and CD209) expression, as analyzed by means of cytometry. MDMs were analyzed after 5 days in the presence of M-CFS, followed by 2 additional days in the presence or absence of rhIFN-γ. No significant differences in MDMs from patients versus healthy subjects were observed. *P ≤ .05 (n = 6 patients and 6 control subjects), Mann-Whitney test. NS, Not significant; MFI, Mean fluorescence intensity. Journal of Allergy and Clinical Immunology 2017 139, 900-912.e7DOI: (10.1016/j.jaci.2016.07.018) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 rhIFN-γ, but not sCD40L, improves the defective fungicidal activity seen in CD40L-deficient patients. A, After challenging MDMs with P brasiliensis, fungicidal activity was assessed by determining CFU values. Before assay, MDMs were either untreated (−) or treated with (+) sCD40L (500 ng/mL) or rhIFN-γ (100 U/mL) for 48 hours. CFU values (percentage of control values) were determined in relation to the CFU number of untreated MDMs from healthy control subjects. B, Raw data in CFU/mL are also shown. Significant differences are denoted as follows: *P ≤ .05 (n = 6 patients and 6 control subjects), Mann-Whitney test. Journal of Allergy and Clinical Immunology 2017 139, 900-912.e7DOI: (10.1016/j.jaci.2016.07.018) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 M-CSF does not induce CD40L expression on monocytes/macrophages. Expression of CD40L, CD69, and CD163 on MDMs was analyzed by means of flow cytometry on each day of macrophage differentiation. No expression of CD40L and CD69 on MDMs was observed. On the other hand, CD163 expression was increased in the presence of M-CSF. MFI, Mean fluorescence intensity. Journal of Allergy and Clinical Immunology 2017 139, 900-912.e7DOI: (10.1016/j.jaci.2016.07.018) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 sCD40L increases the capacity of the promyelocytic HL-60 cells to control M tuberculosis proliferation. Promyelocytic HL-60 cells, which express CD40 (left panel), were cultivated in the absence presence of sCD40L (500 ng/mL), and their ability to control M tuberculosis proliferation was analyzed by CFU (right panel). *P ≤ .05 (n = 6 patients and 6 control subjects), Mann-Whitney test. NS, Not significant. Journal of Allergy and Clinical Immunology 2017 139, 900-912.e7DOI: (10.1016/j.jaci.2016.07.018) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Normal expression of CD86, CD206, and CD163 on macrophages from CD40L-deficient patients. MDMs were cultured in the presence of IL-4 or IFN-γ alone or in concert with M tuberculosis. Expression of CD86 (A), CD163 (B), and CD206 (C) was analyzed by using flow cytometry, and data were represented as mean fluorescence intensity (MFI). *P ≤ .05 (n = 6 patients and 6 control subjects), Mann-Whitney test. NS, Not significant. Journal of Allergy and Clinical Immunology 2017 139, 900-912.e7DOI: (10.1016/j.jaci.2016.07.018) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 The IFN-γ/IL-12 axis is normally activated by rhIFN-γ and rhIL-12. A, PBMCs were activated for 48 hours in the presence of rhIFN-γ (100 U/mL) to induce IL-12 production. B, Cells were stimulated with rhIL-12 (10 ng/mL) for 48 hours to analyze IFN-γ production. Supernatants were harvested and analyzed by means of ELISA. Journal of Allergy and Clinical Immunology 2017 139, 900-912.e7DOI: (10.1016/j.jaci.2016.07.018) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions