Cross-Regulation of Proinflammatory Cytokines by Interleukin-10 and miR-155 in Orientia tsutsugamushi-Infected Human Macrophages Prevents Cytokine Storm  Ming-Hsien Tsai, Chung-Hsing Chang, Rong-Kung Tsai, Yi-Ren Hong, Tsung-Hsien Chuang, Kan-Tang Fan, Chi-Wen Peng, Ching-Ying Wu, Wen-Li Hsu, Lih-Shinn Wang, Li-Kuang Chen, Hsin-Su Yu  Journal of Investigative Dermatology  Volume 136, Issue 7, Pages 1398-1407 (July 2016) DOI: 10.1016/j.jid.2015.11.034 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Identification of O. tsutsugamushi in human THP-1-induced macrophages and the effect of the infection dose of O. tsutsugamushi on the survival and IL-10/TNF-α production of macrophages. (a) Identification of O. tsutsugamushi labeled by CellTracker Red CMTPX (red dot indicated by the white arrow) in THP-1-induced macrophages using a CellTracker green fluorescent probe. Scale bar = 20 μm (b) Survival of the macrophages was determined by an MTT assay at 24 hours after infection. The macrophages were infected with different doses of pathogen as indicated. (c) Production of IL-10 and TNF-α was analyzed with ELISA at 24 hours after infection. (d) Pathogen DNA quantitated by RT-PCR of O. tsutsugamushi demonstrated replication in macrophages at a low dose infection. All results were representative of three replicates. *P < 0.05. **P < 0.01. ***P < 0.001. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; O. tsutsugamushi, Orientia tsutaugamushi; THP-1, human acute monocytic leukemia cell line; TNF-α, tumor necrosis factor alpha. Journal of Investigative Dermatology 2016 136, 1398-1407DOI: (10.1016/j.jid.2015.11.034) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Type I and type II responses of macrophages with low and high doses of O. tsutsugamushi infection. (a) Type I immune response. Macrophages were infected with a low dose of pathogen. (b) Type II immune response. Macrophages were infected with a high dose of pathogen. The production of different cytokines was measured with ELISA. (c) Comparison of IL-10 and TNF-α production in type I (red line) and type II (blue line) immune responses from the data shown in (a) and (b). (d) Western blot analysis for the activation of ERK and NF-κB in the type I and type II immune responses. (e) Immunofluorescence staining of p-STAT3 and p-NF-κB in macrophages infected with a low dose of pathogen. Scale bar = 30 μm. All results were representative of three replicates. **P < 0.01. ***P < 0.001. ERK, extracellular signal-regulated kinases; O. tsutsugamushi, Orientia tsutaugamushi; STAT3, signal transducer and activator of transcription 3; TNF-α, tumor necrosis factor alpha. Journal of Investigative Dermatology 2016 136, 1398-1407DOI: (10.1016/j.jid.2015.11.034) Copyright © 2016 The Authors Terms and Conditions

Figure 3 IL-10 played a pivotal role in type I and type II responses of macrophages during O. tsutsugamushi infection. (a) Macrophages were treated with IL-10 (20 ng/ml) 4 hours before O. tsutsugamushi challenge. Cell survival was analyzed with MTT assays. (b) Macrophages were infected with a high dose of pathogen. Cytokine production was measured with ELISA. (c) Macrophages were treated with ERK inhibitor (PD98059) (4–48 hours) or cotreated with PD98059 and IL-10 (24 hours) or cotreated with IL-10 and a STAT3 inhibitor (24 hours). Phosphorylation of ERK, NF-κB, and STAT3 was examined by western blot. (d) Macrophages were treated with PD98059 and then infected with a low dose of pathogen for different periods of time. Production of cytokines was measured with ELISA. All results were representative of three replicates. *P < 0.05. ***P < 0.001. ERK, extracellular signal-regulated kinases; O. tsutsugamushi, Orientia tsutaugamushi; STAT3, signal transducer and activator of transcription 3. Journal of Investigative Dermatology 2016 136, 1398-1407DOI: (10.1016/j.jid.2015.11.034) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Micro-RNA response of macrophages during O. tsutsugamushi infection. Macrophages were infected with low and high doses of pathogen as indicated. Expressions of (a) miR-155, (b) miR-146a, and (c) miR-21 were analyzed with RT-PCR. Macrophages were treated with and/or without IL-10 and then infected with a high dose of pathogen as indicated. Expressions of (d) miR-155 and (e) miR-146a were analyzed with RT-PCR. (f) Macrophages were treated with different doses of IL-10 from 20 to 0.005 ng/ml and then infected with a high dose of pathogen as indicated to mimic the physiological dose. The expression of miR-155 was determined with RT-PCR. All results were representative of three replicates. *P < 0.05. ***P < 0.001. miR, micro-RNA; O. tsutsugamushi, Orientia tsutaugamushi. Journal of Investigative Dermatology 2016 136, 1398-1407DOI: (10.1016/j.jid.2015.11.034) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Inhibition of miR-155 caused high TNF-α and impaired IL-10 production during high dose infection. Macrophages were treated with or without anti-miR-155 and infected with a high dose of pathogen. (a) Expressions of miR-155 were analyzed with RT-PCR. (b) Production of IL-10 and TNF-α was measured with ELISA. Macrophages were treated with or without anti-miR-146 and infected with a high dose of pathogen. (c) Expressions of miR-146 were analyzed with RT-PCR. (d) Production of IL-10 and TNF-α was measured with ELISA. (e) Immunofluorescence staining of p-NF-κB and p-ERK in macrophages treated with and/or without anti-miR-155 and infected with a high dose of pathogen. All results were representative of three replicates. ERK, extracellular signal-regulated kinases; miR, micro-RNA; TNF-α, tumor necrosis factor alpha. Journal of Investigative Dermatology 2016 136, 1398-1407DOI: (10.1016/j.jid.2015.11.034) Copyright © 2016 The Authors Terms and Conditions

Figure 6 IL-10 and miR-155 production in O. tsutsugamushi-infected peripheral blood mononuclear cells (PBMCs) from patients with scrub typhus. PBMCs isolated from mild and severe scrub typhus cases were infected with the pathogen. (a) Cytokine production was measured with ELISA. (b) Expressions of miRs were determined with RT-PCR. The basal level was set as one-fold. All results were representative of three replicates. **P < 0.01. ***P < 0.001. (c, d) Schematic summary of the cross-regulation of proinflammatory cytokine production by IL-10 and miR-155 to prevent cytokine storm. During low dose infection, macrophages produce high levels of IL-10 and low levels of miR-155. These activate STAT3 and inhibit the TNF-α-NF-κB axis, as a way to prevent cytokine storm, yet facilitate O. tsutsugamushi proliferation. While the infection progresses, miR-155 expression is suppressed by increasing levels of IL-10, relieving the micro-RNA repression of the TNF-α-NF-κB axis, and allowing sufficient NF-κB activation for producing the proinflammatory cytokines required for pathogen immunity. During high dose infection, macrophages express high levels of miR-155 simultaneously with low levels of IL-10, keeping NF-κB in check to prevent a cytokine storm. Impaired regulation of either IL-10 or miR-155 expression may compromise the host either due to excessive pathogen proliferation or to cytokine storm. miR, micro-RNA; O. tsutsugamushi, Orientia tsutaugamushi; STAT3, signal transducer and activator of transcription 3; TNF-α, tumor necrosis factor alpha. Journal of Investigative Dermatology 2016 136, 1398-1407DOI: (10.1016/j.jid.2015.11.034) Copyright © 2016 The Authors Terms and Conditions