Intact interferon-γ response against Coxiella burnetii by peripheral blood mononuclear cells in chronic Q fever  T. Schoffelen, J. Textoris, C.P. Bleeker-Rovers,

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Intact interferon-γ response against Coxiella burnetii by peripheral blood mononuclear cells in chronic Q fever  T. Schoffelen, J. Textoris, C.P. Bleeker-Rovers, A. Ben Amara, J.W.M. van der Meer, M.G. Netea, J.-L. Mege, M. van Deuren, E. van de Vosse  Clinical Microbiology and Infection  Volume 23, Issue 3, Pages 209.e9-209.e15 (March 2017) DOI: 10.1016/j.cmi.2016.11.008 Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions

Fig. 1 C. burnetii-induced IFN-γ production is increased in chronic Q fever patients compared with healthy individuals. IFN-γ was measured in supernatants of PBMCs stimulated for 48 hours with inactivated C. burnetii Nine Mile (106 bacteria/mL), C. burnetii 3262 (106 bacteria/mL), C. burnetii Henzerling (100 ng/mL) (all phase I) and Candida albicans (105 conidia/mL). Values are expressed as mean ± standard error. p-Values are calculated using Mann-Whitney U test. **p <0.01, ***p <0.001. Clinical Microbiology and Infection 2017 23, 209.e9-209.e15DOI: (10.1016/j.cmi.2016.11.008) Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions

Fig. 2 Distinct transcriptional profile of C. burnetii-stimulated PBMCs of chronic Q fever patients. PBMCs of six chronic Q fever patients (P) and four healthy volunteers (HV) were either not stimulated (NS), or stimulated with heat-inactivated C. burnetii Nine Mile (107 bacteria/mL) (Cb), or stimulated with E. coli LPS (10 ng/mL) (LPS) for 8 hours. RNA was extracted and a microarray was performed. (a) Graphical representation of the samples based on the correspondence analysis of the modulated probes. The samples are coloured according to the group (patients versus healthy volunteers) and stimulus (not stimulated, C. burnetii or LPS). (b) Heatmap representation of the modulated probes in stimulated PBMCs compared with unstimulated PBMCs. The probes are shown in the rows and the samples in the columns. The expression levels are colour-coded from blue to red. Below the columns, samples are colour-coded as in A. Clinical Microbiology and Infection 2017 23, 209.e9-209.e15DOI: (10.1016/j.cmi.2016.11.008) Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions

Fig. 3 Chronic Q fever patients show increased relative activation of the ‘response to interferon-gamma’ pathway (Gene Ontology GO-term GO:0034341). PBMCs of four healthy volunteers and six chronic Q fever patients were either not stimulated, stimulated with E. coli LPS (10 ng/mL), or stimulated with heat-inactivated C. burnetii Nine Mile (107 bacteria/mL) for 8 hours. RNA was extracted and a microarray was performed. The relative activation of the ‘response to interferon-gamma’ pathway was based on median standardized gene expression of the 54 probes in the microarray that corresponded to 42 genes mapped in GO:0034341 and were significantly modulated in stimulated PBMCs (|FC|>2 and p <0.01) (see Table S1 for a list of these 54 probes). The horizontal lines present the median, the boxes present the interquartile range (IQR), and the whiskers present 1.5*IQR. Outliers (outside the whiskers) are plotted as dots. C. burnetii-stimulated PBMCs of patients showed significantly higher relative activation than those of healthy individuals (Wilcoxon test; p <0.001), whereas there was no significant difference in relative activation on E. coli LPS stimulation between patients' and healthy individuals (p >0.001). Clinical Microbiology and Infection 2017 23, 209.e9-209.e15DOI: (10.1016/j.cmi.2016.11.008) Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions

Fig. 4 C. burnetii-induced neopterin is significantly increased in chronic Q fever patients. (a) Neopterin was measured in serum of chronic Q fever patients (n=21) and healthy individuals (n=11). (b) Neopterin was measured in supernatant of PBMCs of patients (n=8) and of healthy individuals (n=4) after stimulation for 48 hours with inactivated C. burnetii Nine Mile (NM) phase I (107 bacteria/mL), C. burnetii NM phase I (106 bacteria/mL), and C. albicans (105 bacteria/mL). Medians of healthy individuals and patients are compared by Mann-Whitney U test. ** p <0.01. Clinical Microbiology and Infection 2017 23, 209.e9-209.e15DOI: (10.1016/j.cmi.2016.11.008) Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions