Rickettsia conorii is a potent complement activator in vivo and combined inhibition of complement and CD14 is required for attenuation of the cytokine response ex vivo  K. Otterdal, A. Portillo, E. Astrup, J.K. Ludviksen, C. Schjalm, D. Raoult, J.P. Olano, B. Halvorsen, J.A. Oteo, P. Aukrust, T.E. Mollnes, P.H. Nilsson  Clinical Microbiology and Infection  Volume 22, Issue 8, Pages 734.e1-734.e6 (August 2016) DOI: 10.1016/j.cmi.2016.05.010 Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions

Fig. 1 Levels of complement activation markers in patients with Mediterranean spotted fever. Plasma levels for (a) C3-activation (C3bc), (b) alternative pathway C3-convertase formation (C3bBbP), (c) terminal pathway activation (sC5b-9) and (d) C1rs-C1INH are shown for 36 patients (33 for C1rs-C1INH) at admission, four patients at follow up (28–42 days after symptom onset) and for nine healthy controls. Normal population reference values for each marker are shown in the grey area between dotted lines. Data are given as means and 95% CI. Statistical differences between patients at admission were compared against controls and patients at follow up with the Kruskal–Wallis test; *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001, ns, not significant. Clinical Microbiology and Infection 2016 22, 734.e1-734.e6DOI: (10.1016/j.cmi.2016.05.010) Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions

Fig. 2 Levels of sCD14 in patients with Mediterranean spotted fever. Serum levels of soluble CD14 (sCD14) are shown for 36 patients at admission, 15 patients at follow up (28–42 days after symptom onset) and nine healthy controls. Data are presented on a logarithmic scale. Statistical differences between patients at admission were compared against controls and patients at follow up with the Kruskal–Wallis test; **p <0.01, ****p <0.0001. Clinical Microbiology and Infection 2016 22, 734.e1-734.e6DOI: (10.1016/j.cmi.2016.05.010) Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions

Fig. 3 Complement activation and cytokine response in human whole blood after incubation with Rickettsia conorii. (a) Rickettsia conorii was incubated for 2 h in whole blood and complement activation was measured as levels of sC5b-9. Data are presented as means and 95% CI (n=6). The effect of R. conorii was statistically tested against the buffer control using a paired t-test, **p <0.01. (b) Rickettsia conorii was incubated in whole blood for 4 h, levels of 27 cytokines, chemokines and growth factors were measured in plasma after incubation. Cytokines that showed a mean fold increase of two or more from three individual experiments in the presence of R. conorii (dark grey), compared with whole blood incubated without bacteria (light grey), are depicted with mean levels±standard deviation (n=3). Clinical Microbiology and Infection 2016 22, 734.e1-734.e6DOI: (10.1016/j.cmi.2016.05.010) Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions

Fig. 4 Effect of C3- and CD14-inhibition on pro-inflammatory cytokines in response to Rickettsia conorii incubated in human whole blood ex vivo. Incubation of R. conorii in the presence or absence of a complement C3 inhibitor (compstatin Cp40), an F(ab′)2-fragment blocking CD14, or a combination thereof. The inflammatory response was evaluated in a subset of pro-inflammatory cytokines represented by (a) interleukin-1β (IL-1β), (b) IL-6, (c) IL-8 and (d) tumour necrosis factor (TNF). Data are presented as means and 95% CI (n=6). Effect of R. conorii was statistically tested against the basal control using Student’s t-test (###p <0.001). Effect of respective inhibition was statistically tested against R. conorii using repeated measures one-way analysis of variance, *p <0.05, **p <0.01, ***p <0.001, ns, not significant. Clinical Microbiology and Infection 2016 22, 734.e1-734.e6DOI: (10.1016/j.cmi.2016.05.010) Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions