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Original Article Immunology/Infection| Volume 136, ISSUE 7, P1398-1407, July 2016

Cross-Regulation of Proinflammatory Cytokines by Interleukin-10 and miR-155 in Orientia tsutsugamushi-Infected Human Macrophages Prevents Cytokine Storm

Open ArchivePublished:February 24, 2016DOI:https://doi.org/10.1016/j.jid.2015.11.034
      Scrub typhus is caused by the obligate intracellular bacterium Orientia tsutsugamushi. Macrophages are host cells for its replication and clearance. Severe complications in patients are mainly caused by a cytokine storm resulting from overproduction of proinflammatory cytokines; nevertheless, the molecular mechanism for the occurrence remains obscure. Herein, we investigate the interactive regulation of cytokines and micro-RNA (miR) in human macrophages infected with low and high doses of O. tsutsugamushi. During low dose infection, macrophages produce high levels of IL-10 through extracellular signal-regulated kinase activation, which inhibits proinflammatory cytokine production and facilitates pathogen replication. Increasing levels of pathogen results in reduced levels of IL-10, and macrophages begin to generate high levels of proinflammatory cytokines through NF-κB activation. However, during a high dose infection, macrophages produce high levels of miR-155 to slow the proinflammatory response. The extracellular signal-regulated kinase/IL-10 axis suppresses the NF-κB/tumor necrosis factor alpha axis via activation of signal transducer and activator of transcription 3. Both IL-10 and miR-155 inhibit the NF-κB signaling pathway. Furthermore, IL-10 is a potent inhibitor of miR-155. Patients susceptible to a cytokine storm, peripheral blood mononuclear cells showed significantly lower IL-10 and miR-155 responses to O. tsutsugamushi challenge. Thus, IL-10 and miR-155 operate inhibitory mechanisms to achieve a proper defense mechanism and prevent a cytokine storm.

      Abbreviations:

      ERK (extracellular signal-regulated kinases), LPS (lipopolysaccharide), miR (micro-RNA), O. tsutsugamushi (Orientia tsutaugamushi), PBMC (peripheral blood mononuclear cell), STAT3 (signal transducer and activator of transcription 3), TLR (toll-like receptor), TNF-α (tumor necrosis factor alpha)

      Introduction

      Scrub typhus is caused by the obligate intracellular bacterium Orientia tsutsugamushi (O. tsutsugamushi), which is transmitted to humans via the bite of infected mite vectors. Scrub typhus is an important travel-associated zoonosis (
      • Jensenius M.
      • Fournier P.E.
      • Raoult D.
      Rickettsioses and the international traveler.
      ). It is endemic across much of Asia and the Western Pacific region (
      • Kelly D.J.
      • Fuerst P.A.
      • Ching W.M.
      • Richards A.L.
      Scrub typhus: the geographic distribution of phenotypic and genotypic variants of Orientia tsutsugamushi.
      ,
      • Seong S.Y.
      • Choi M.S.
      • Kim I.S.
      Orientia tsutsugamushi infection: overview and immune responses.
      ). In Taiwan, most of the genotypes of O. tsutsugamushi belong to the Karp, Kawasaki, and Kuroki strains, and are closely related to strains from Thailand, Japan, and Korea. TW-1, one of the Karp strains, is the most common (33.6%) and highly virulent strain found in Taiwan (
      • Lu H.Y.
      • Tsai K.H.
      • Yu S.K.
      • Cheng C.H.
      • Yang J.S.
      • Su C.L.
      Phylogenetic analysis of 56-kDa type-specific antigen gene of Orientia tsutsugamushi isolates in Taiwan.
      ).
      In human skin, the primary targets of O. tsutsugamushi are macrophages; however, it can also infect monocytes, neutrophils, and endothelial cells (
      • Moron C.G.
      • Popov V.L.
      • Feng H.M.
      • Wear D.
      • Walker D.H.
      Identification of the target cells of Orientia tsutsugamushi in human cases of scrub typhus.
      ,
      • Seong S.Y.
      • Choi M.S.
      • Kim I.S.
      Orientia tsutsugamushi infection: overview and immune responses.
      ,
      • Walsh D.S.
      • Myint K.S.
      • Kantipong P.
      • Jongsakul K.
      • Watt G.
      Orientia tsutsugamushi in peripheral white blood cells of patients with acute scrub typhus.
      ). The outer membrane protein TSA56 of O. tsutsugamushi is mainly responsible for the adhesion and/or invasion of this pathogen into mammalian cells (
      • Lin C.C.
      • Chou C.H.
      • Lin T.C.
      • Yang M.C.
      • Cho C.L.
      • Chang C.H.
      • et al.
      Molecular characterization of three major outer membrane proteins, TSA56, TSA47 and TSA22, in Orientia tsutsugamushi.
      ). Within 2 hours of the initial phagocytic uptake into a membrane-bound endosome, O. tsutsugamushi escapes from the endosome and replicates in the host cytosol. Inflammation is initiated by O. tsutsugamushi-infected macrophages and endothelial cells in the dermis, and the pathogen spreads via monocytes into the systemic circulation. Mononuclear cells containing O. tsutsugamushi have been identified in the peripheral blood smears of patients (
      • Seong S.Y.
      • Choi M.S.
      • Kim I.S.
      Orientia tsutsugamushi infection: overview and immune responses.
      ,
      • Walsh D.S.
      • Myint K.S.
      • Kantipong P.
      • Jongsakul K.
      • Watt G.
      Orientia tsutsugamushi in peripheral white blood cells of patients with acute scrub typhus.
      ), and in a mouse model macrophages were found to play an important role in the early host defense against O. tsutsugamushi infection. Tumor necrosis factor alpha (TNF-α) is a well-characterized proinflammatory cytokine released primarily from monocytes and macrophages on invasion by O. tsutsugamushi (
      • Cho N.H.
      • Seong S.Y.
      • Huh M.S.
      • Han T.H.
      • Koh Y.S.
      • Choi M.S.
      • et al.
      Expression of chemokine genes in murine macrophages infected with Orientia tsutsugamushi.
      ), and the production of chemokines and cytokines has been associated with susceptibility during O. tsutsugamushi infection (
      • Yun J.H.
      • Koh Y.S.
      • Lee K.H.
      • Hyun J.W.
      • Choi Y.J.
      • Jang W.J.
      • et al.
      Chemokine and cytokine production in susceptible C3H/HeN mice and resistant BALB/c mice during Orientia tsutsugamushi infection.
      ).
      Clinically, scrub typhus is usually self-limiting; however, serious complications may develop including adult respiratory distress syndrome, acute renal failure, acute hepatic failure, and multiple organ dysfunction syndrome (
      • Lee B.J.
      • Chen C.Y.
      • Hu S.Y.
      • Tsan Y.T.
      • Lin T.C.
      • Wang L.M.
      Otalgia and eschar in the external auditory canal in scrub typhus complicated by acute respiratory distress syndrome and multiple organ failure.
      ,
      • Tsay R.W.
      • Chang F.Y.
      Serious complications in scrub typhus.
      ,
      • Wang C.C.
      • Liu S.F.
      • Liu J.W.
      • Chung Y.H.
      • Su M.C.
      • Lin M.C.
      Acute respiratory distress syndrome in scrub typhus.
      ). The pathogenesis of adult respiratory distress syndrome and multiple organ dysfunction syndrome results from a cytokine storm, which is an inflammatory response flaring out of control (
      • Tisoncik J.R.
      • Korth M.J.
      • Simmons C.P.
      • Farrar J.
      • Martin T.R.
      • Katze M.G.
      Into the eye of the cytokine storm.
      ,
      • Wang H.
      • Ma S.
      The cytokine storm and factors determining the sequence and severity of organ dysfunction in multiple organ dysfunction syndrome.
      ). Increased levels of TNF-α, IL-1β, IL-6, IFN-γ, IL-10, and IL-12p40 have been reported in patients with scrub typhus, and serum TNF-α concentration has been significantly correlated with the severity of disease in the acute phase. In addition, bacterial DNA concentration has been positively correlated with the level of IL-10, however, not with IFN-γ, TNF-α, or IL-1β (
      • Chung D.R.
      • Lee Y.S.
      • Lee S.S.
      Kinetics of inflammatory cytokines in patients with scrub typhus receiving doxycycline treatment.
      ,
      • Iwasaki H.
      • Mizoguchi J.
      • Takada N.
      • Tai K.
      • Ikegaya S.
      • Ueda T.
      Correlation between the concentrations of tumor necrosis factor-alpha and the severity of disease in patients infected with Orientia tsutsugamushi.
      ,
      • Kramme S.
      • An le V.
      • Khoa N.D.
      • Trin le V.
      • Tannich E.
      • Rybniker J.
      • et al.
      Orientia tsutsugamushi bacteremia and cytokine levels in Vietnamese scrub typhus patients.
      ). The control mechanisms of the extent and strength of cytokine production and the pathogenesis of clinical manifestations from a mild to severe course are unclear (
      • Paris D.H.
      • Shelite T.R.
      • Day N.P.
      • Walker D.H.
      Unresolved problems related to scrub typhus: a seriously neglected life-threatening disease.
      ).
      The innate immune response provides a critical defense against pathogens by detecting microbial ligands through pattern recognition receptors such as the toll-like receptors (TLRs) that are expressed at high levels on macrophages (
      • Kawai T.
      • Akira S.
      The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors.
      ). The exact sensing molecules for O. tsutsugamushi have yet to be clarified, although single nucleotide polymorphisms in TLR-2 and TLR-4 have been reported in patients susceptible to scrub typhus (
      • Janardhanan J.
      • Joseph Martin S.
      • Astrup E.
      • Veeramanikandan R.
      • Aukrust P.
      • Abraham O.C.
      • et al.
      Single-nucleotide polymorphisms in Toll-like receptor (TLR)-2, TLR4 and heat shock protein 70 genes and susceptibility to scrub typhus.
      ). Several micro-RNAs (miRs) have been shown to be upregulated in response to TLR ligands, and many directly target components of the TLR signaling system. miR-146a, miR-21, and miR-155 were shown to become induced in response to lipopolysaccharide (LPS, a TLR4 ligand) treatment in monocytes (
      • O'Connell R.M.
      • Taganov K.D.
      • Boldin M.P.
      • Cheng G.
      • Baltimore D.
      MicroRNA-155 is induced during the macrophage inflammatory response.
      ,
      • Quinn S.R.
      • O'Neill L.A.
      A trio of microRNAs that control Toll-like receptor signalling.
      ,
      • Sheedy F.J.
      • Palsson-McDermott E.
      • Hennessy E.J.
      • Martin C.
      • O'Leary J.J.
      • Ruan Q.
      • et al.
      Negative regulation of TLR4 via targeting of the proinflammatory tumor suppressor PDCD4 by the microRNA miR-21.
      ,
      • Taganov K.D.
      • Boldin M.P.
      • Chang K.J.
      • Baltimore D.
      NF-κB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses.
      ). Because O. tsutsugamushi has been demonstrated to lack LPS in its cell envelop (
      • Amano K.
      • Tamura A.
      • Ohashi N.
      • Urakami H.
      • Kaya S.
      • Fukushi K.
      Deficiency of peptidoglycan and lipopolysaccharide components in Rickettsia tsutsugamushi.
      ,
      • Nakayama K.
      • Yamashita A.
      • Kurokawa K.
      • Morimoto T.
      • Ogawa M.
      • Fukuhara M.
      • et al.
      The whole-genome sequencing of the obligate intracellular bacterium Orientia tsutsugamushi revealed massive gene amplification during reductive genome evolution.
      ), studies investigating O. tsutsugamushi-induced miRs are warranted.
      The purpose of this study was to investigate the mechanisms of cytokine homeostasis in human macrophages infected with O. tsutsugamushi. We report together IL-10 and miR-155, which orchestrated a sophisticated inhibitory mechanism to achieve a balanced defense without damaging the host.

      Results

      Survival of infected human THP-1-induced macrophages and replication of the O. tsutsugamushi increased in parallel with IL-10 production

      Human THP-1-induced macrophages were used as the experimental model for the present study. To confirm the ability of O. tsutsugamushi to infect these cells, we traced CellTracker Red-labeled (Molecular Probes) O. tsutsugamushi by confocal microscopy. The pathogens were visible as red dots in the cytoplasm vacuoles after 90 minutes of infection (Figure 1a, detail shown in Supplementary Figure S1 online). The survival rate and cytokine production in the macrophages infected with different doses of O. tsutsugamushi (method for quantitation in Supplementary Section S2.3 online) were further investigated. At a dose of 1,000 pathogens per cell, only 40% of the macrophages survived, whereas at a dose of less than 20 pathogens per cell, more than 80% of the macrophages survived for more than 24 hours (Figure 1b). At a dose of less than 20 pathogens per cell, TNF-α production was suppressed compared with the controls. However, at a dose of 2 pathogens per cell, IL-10 production was increased by up to 5-fold compared with the controls. A reciprocal rise and fall in the levels of IL-10 and TNF-α were closely correlated to a low to high dose of the pathogen (Figure 1c). High TNF-α production was associated with low survival of the macrophages, whereas high IL-10 production was associated with a high survival rate (Figure 1b and c). For the following studies, we defined a low infection dose as 2 pathogens per cell for the highest IL-10 production, and a high infection dose as 100 pathogens per cell for high TNF-α production with acceptable cell survival for at least 48 hours (at a dose of 1,000 pathogens per cell, most cells died by 48 hours, data not shown). To examine whether O. tsutsugamushi could replicate in macrophages at a low infection dose, we cultured infected macrophages for 3 days and analyzed the DNA of the pathogens by reverse transcription polymerase chain reaction. The data showed that the pathogens could replicate in the macrophages with a 7-fold increase in the amount of DNA (Figure 1d). Thus, a low infection dose induced the macrophages to produce high levels of IL-10, which was associated with pathogen replication and macrophage survival, whereas a high infection dose induced the macrophages to produce high levels of TNF-α, which was associated with a high level of cell death. The rise and fall of IL-10 and TNF-α level implied a counteracting regulation between their production.
      Figure 1
      Figure 1Identification 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.

      Activation of the extracellular signal-regulated kinase (ERK)-IL-10 axis and NF-κB-TNF-α axis in type I and type II response in low and high dose of O. tsutsugamushi infections

      We further investigated the production profiles of proinflammatory cytokines including TNF-α, IL-1β, IL-6, and IL-12p70, and anti-inflammatory cytokines including IL-10 and INF-γ during O. tsutsugamushi infection over time. We found that at low dose infection, IL-10 steadily increased to 3- to 4-fold during the first 12 hours and to more than 10-fold from 24 to 48 hours; the increase in IL-10 was associated with a decrease in TNF-α, IL-1β, and IL-6, a condition we defined as a type I response (Figure 2a). During high dose infection, IL-10 steadily increased by 3- to 4-fold during the first 12 hours but reached a plateau thereafter, and the production of TNF-α, IL-1β, and IL-6 steadily increased from the beginning up to 24 to 48 hours, a condition we defined as a type II response (Figure 2b). Comparing type I and type II responses, we observed an increased production of IL-10 by 10-fold with complete suppression of TNF-α at 48 hours in type I, whereas IL-10 increased by a maximum of 4-fold at 48 hours but TNF-α steadily increased by 2- to 3-fold up to 48 hours in type II (Figure 2c). Of note, there was a correlation between a type I response and increasing pathogen replication, and a correlation between a type II response and increasing cell death (Figures 1 and 2c).
      Figure 2
      Figure 2Type 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.
      ERK, NF-κB, and signal transducer and activator of transcription 3 (STAT3) are key molecules that mediate innate immune responses elicited by O. tsutsugamushi infection (
      • Iwasaki H.
      • Mizoguchi J.
      • Takada N.
      • Tai K.
      • Ikegaya S.
      • Ueda T.
      Correlation between the concentrations of tumor necrosis factor-alpha and the severity of disease in patients infected with Orientia tsutsugamushi.
      ,
      • Kawai T.
      • Akira S.
      The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors.
      ,
      • Paris D.H.
      • Shelite T.R.
      • Day N.P.
      • Walker D.H.
      Unresolved problems related to scrub typhus: a seriously neglected life-threatening disease.
      ). To examine the cellular signaling governing the type I and type II responses, we performed western blot analysis of the phosphorylated activation of these proteins. The results showed that ERK was activated in a time-dependent manner during a low dose infection, but was stably expressed during a high dose infection (Figure 2d). These ERK activation profiles were correlated with the production profiles of IL-10 in the type I and type II responses (Figure 2a and b). NF-κB is the key transcription factor in the synthesis of TNF-α, IL-1β, and IL-6 (
      • Taganov K.D.
      • Boldin M.P.
      • Chang K.J.
      • Baltimore D.
      NF-κB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses.
      ). During a low dose infection, the activation of NF-κB was suppressed, whereas during a high dose infection it was activated in a time-dependent manner (Figure 2d). These NF-κB activation profiles were correlated with the production profiles of TNF-α, IL-1β, and IL-6 in the type I and type II responses (Figure 2a and b).
      We next investigated whether the negative regulatory loop between IL-10 and TNF-α was involved with the negative regulation of NF-κB by STAT3. In western blot analysis, STAT3 was activated in a time-dependent manner during a low dose infection. In contrast, it was transiently activated at 4 hours and inactivated thereafter during a high dose infection (Figure 2d). The activation profiles of STAT3 in low and high dose infections were correlated with the production profiles of IL-10, and inversely correlated with the activation profiles of NF-κB and the production profiles of TNF-α (Figure 2c and d). Consistent with these findings, in experiments with immunofluorescence staining, the intensity of p-NF-κB staining decreased in parallel with the increased intensity of p-STAT3 during a low dose infection (Figure 2e). Thus, a type I response was characterized by the activation of the ERK-IL-10 axis, and type II by the activation of the NF-κB-TNF-α axis. STAT3 may act as a negative feedback control to link these two axes.

      IL-10 played a pivotal role in the type I or type II response of macrophages, independently of the infection dose

      To investigate whether IL-10 could protect O. tsutsugamushi-infected macrophages from cell death, we treated the macrophages with IL-10 (20 ng/ml) 4 hours before high dose infection. An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay demonstrated that IL-10 treatment significantly increased the survival rate of macrophages compared with the controls (Figure 3a). We further found that IL-10 treatment completely suppressed the production of TNF-α, IL-1β, and IL-6, even during a high dose infection (Figure 3b).
      Figure 3
      Figure 3IL-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.
      To confirm whether O. tsutsugamushi-induced IL-10 production was via the ERK pathway and to investigate the connection between ERK and STAT3 activation, we pretreated macrophages with 50 μM of PD98059, which is an inhibitor of ERK. Western blotting revealed that phosphorylation of ERK and STAT3 was completely inactivated (Figure 3c). Thus, O. tsutsugamushi-infected macrophages produced IL-10 mainly via the ERK pathway. Moreover, STAT3 activation could be regulated by IL-10. Because of a lack of IL-10 production to initiate the IL-10 receptor signaling pathway, STAT3 was unable to activate an anti-inflammatory response.
      We then investigated whether endogenous IL-10 plays a pivotal role in regulating the proinflammatory response. During a low dose infection, macrophages pretreated with PD98059 showed no IL-10 production; however, TNF-α, IL-1β, and IL-6 were produced to a similar level of a type II response (Figure 3d). Thus, with impairment of an IL-10 response, macrophages are prone to produce excessive proinflammatory cytokines even under a small amount of pathogen challenge. We then treated macrophages with IL-10 via inhibiting the ERK pathway, and found that STAT3 was stimulated without endogenous IL-10 production.

      miR-155 and miR-146a, but not miR-21, were induced in macrophages during O. tsutsugamushi infection in a dose-dependent manner

      We next evaluated three inflammation-associated miRs, miR-146a, miR-155, and miR-21, during O. tsutsugamushi infection. miR-155 and miR-146a were specifically responsive to O. tsutsugamushi, with miR-155 being upregulated up to 10-fold and 4-fold at 24 hours during high and low dose infections, respectively (Figure 4a). miR-146a was upregulated up to 7-fold and 3-fold during high and low dose infections, respectively (Figure 4b). Both reached peak levels at 24 hours. miR-155 sustained peak levels longer, maintaining an 8-fold increase until 48 hours, whereas miR-146 declined faster, displaying a 4-fold increase at 48 hours. miR-21 showed no response during infection. Thus, miR-155 and miR-146 were specific miRs produced by microphages during O. tsutsugamushi infection.
      Figure 4
      Figure 4Micro-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.

      miR-155, but not miR-146a, was negatively regulated by IL-10

      Because IL-10 has been shown to have an inhibitory effect on the LPS-induced expression of miR-155 (
      • Elton T.S.
      • Selemon H.
      • Elton S.M.
      • Parinandi N.L.
      Regulation of the MIR155 host gene in physiological and pathological processes.
      ), we examined whether IL-10 could regulate miR-155 during an O. tsutsugamushi infection, in which LPS is not present. Macrophages pretreated with IL-10 (20 ng/ml) for 4 hours almost completely inhibited the induction of miR-155 (Figure 4d). However, IL-10 did not inhibit the induction of miR-146a (Figure 4e). Because the IL-10 peak level in our experimental model was below 100 pg/ml (0.1 ng/ml) (Figure 2a), we further examined the inhibitory effect of IL-10 on miR-155 using a physiological dose from 20 to 0.005 ng/ml. Surprisingly, IL-10 achieved the same strong inhibitory effect (Figure 4f). Thus, the induction of miR-155 in macrophages during O. tsutsugamushi infection could be negatively regulated by a very low dose of IL-10 approximately 0.005 to 0.1 ng/ml.

      Inhibition of miR-155 caused massive TNF-α production

      After transfection of anti-miR-155, miR-155 production during a high dose infection was effectively blocked (Figure 5a). We measured the cytokines harvested from the supernatant of the same plates, and without miR-155 the TNF-α production was increased to 4-fold, compared with 2.5-fold with miR-155 (Figure 5b). The intensity of p-NF-κB staining was enhanced in the nuclei; however, the intensity of p-ERK staining was attenuated (Figure 5e). Transfection of anti-miR-146 effectively reduced its induction during a high dose infection (Figure 5c). However, cytokine assays of the supernatant of the same plates revealed no significant change in TNF-α production in the presence or absence of anti-miR-146 (Figure 5d). Thus, blocking miR-155 during a high dose infection caused increased TNF-α production, whereas blocking miR-146 did not change the cytokine production profiles.
      Figure 5
      Figure 5Inhibition 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.

      Scrub typhus patients who recovered from a cytokine storm showed defective production of IL-10 and miR-155

      In this retrospective study, a total of seven severe cases, who recovered from cytokine storm, and six mild cases, who recovered spontaneously, were enrolled. The age and gender were matched between the two groups (age: 61.1 ± 9.6 years vs. 54.3 ± 6.1 years, Student’s t-test, P = 0.5757; gender: Fisher’s exact test, P = 1.000). The freshly isolated peripheral blood mononuclear cells (PBMCs) from patients were infected with the TW-1 strain for 24 hours. Cytokine assays with ELISA showed the production of TNF-α, IL-1β, IL-6, and IL-10 from the PBMCs of these patients, but not IL-12p70 or IFN-γ. miR assays with RT-PCR showed the induction of miR-155 and miR-146a, but not miR-21 in the cells. The severe group showed a significantly lower production of IL-10 but higher production of TNF-α, IL-1β, and IL-6, compared with the mild group (Figure 6a). In contrast, the induction of miR-155 in the severe group was significantly lower than in the mild group (Figure 6b).
      Figure 6
      Figure 6IL-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.

      Discussion

      Previous studies investigating O. tsutsugamushi-induced immune responses have mainly used the Boryong strain (a low virulence strain) to infect murine macrophage J774A.1 with lethal doses of pathogen (
      • Cho B.A.
      • Cho N.H.
      • Seong S.Y.
      • Choi M.S.
      • Kim I.S.
      Intracellular invasion by Orientia tsutsugamushi is mediated by integrin signaling and actin cytoskeleton rearrangements.
      ,
      • Cho K.-A.
      • Jun Y.H.
      • Suh J.W.
      • Kang J.S.
      • Choi H.J.
      • Woo S.Y.
      Orientia tsutsugamushi induced endothelial cell activation via the NOD1-IL-32 pathway.
      ,
      • Kim M.J.
      • Kim M.K.
      • Kang J.S.
      Orientia tsutsugamushi inhibits tumor necrosis factor α production by inducing interleukin 10 secretion in murine macrophages.
      ,
      • Koo J.E.
      • Hong H.J.
      • Dearth A.
      • Kobayashi K.S.
      • Koh Y.S.
      Intracellular invasion of Orientia tsutsugamushi activates inflammasome in ASC-dependent manner.
      ,
      • Yun J.H.
      • Koo J.E.
      • Koh Y.S.
      Mitogen-activated protein kinases are involved in tumor necrosis factor alpha production in macrophages infected with Orientia tsutsugamushi.
      ). To elucidate a human innate immune response to O. tsutsugamushi infection, we studied human THP-1-induced macrophages infected with low and high doses of the TW-1 strain and, in addition, challenged PBMCs of patients with scrub typhus with pathogens. The cytokine and miR response patterns to O. tsutsugamushi were similar in both conditions, indicating the validity of the THP-1 experimental model for studying the mechanisms of human innate immunity in scrub typhus. Herein, we propose how IL-10 and miR-155 coordinately regulate the macrophage response to obligate intracellular pathogens to control infection while preventing a cytokine burst (Figure 6c and d).
      The production of IL-10, rather than TNF-α, is the major response of normal human macrophages to low dose of intracellular pathogens. Although the cellular receptor for O. tsutsugamushi is still unknown, MyD88- and TRIF-dependent TLR signals as well as non-TLR signals (CD40 ligation) have been reported to mediate the macrophage production of IL-10 (
      • Boonstra A.
      • Rajsbaum R.
      • Holman M.
      • Marques R.
      • Asselin-Paturel C.
      • Pereira J.P.
      • et al.
      Macrophages and myeloid dendritic cells, but not plasmacytoid dendritic cells, produce IL-10 in response to MyD88- and TRIF-dependent TLR signals, and TLR-independent signals.
      ), which in turn suppresses proinflammatory activity of macrophages expressing the highest levels of IL-10 receptor (IL-10R). The STAT3 transcription factor is essential in many types of cells for the anti-inflammatory activity of IL-10/IL-10R (
      • Levy D.E.
      • Lee C.K.
      What does Stat3 do?.
      ,
      • Murray P.J.
      Understanding and exploiting the endogenous interleukin-10/STAT3-mediated anti-inflammatory response.
      ). STAT3 has been reported to inhibit NF-κB via suppressor of cytokine signaling 3 (
      • Hutchins A.P.
      • Diez D.
      • Miranda-Saavedra D.
      The IL-10/STAT3-mediated anti-inflammatory response: recent developments and future challenges.
      ,
      • Yoshimura A.
      • Naka T.
      • Kubo M.
      SOCS proteins, cytokine signalling and immune regulation.
      ). Macrophages with a high ERK expression can induce a high level of IL-10 production, suggesting that the ERK pathway is the major signaling cascade involved in IL-10 expression (
      • Saraiva M.
      • O'Garra A.
      The regulation of IL-10 production by immune cells.
      ). Consistent with these findings, our ERK inhibitor studies with PD98059 indicated that ERK activity is indeed required for O. tsutsugamushi-induced IL-10 production in macrophages. Moreover, the expression of IL-10 was correlated with the activation of STAT3, and inversely correlated with the activation NF-κB.
      It has been proposed that IL-10 increases host susceptibility to numerous intracellular microorganisms such as Mycobacterium tuberculosis, Mycobacterium leprae, and Leishmania major, and is involved in the evolution of infectious diseases such as Q fever, and in the persistence of M. tuberculosis (
      • Honstettre A.
      • Imbert G.
      • Ghigo E.
      • Gouriet F.
      • Capo C.
      • Raoult D.
      • et al.
      Dysregulation of cytokines in acute Q fever: role of interleukin-10 and tumor necrosis factor in chronic evolution of Q fever.
      ). On the other hand, IL-10 may protect hosts from exaggerated inflammatory reactions and tissue injuries secondary to infections (
      • Mege J.L.
      • Meghari S.
      • Honstettre A.
      • Capo C.
      • Raoult D.
      The two faces of interleukin 10 in human infectious diseases.
      ). Based on our results, IL-10 is induced in the early phase of infection and functions to downregulate proinflammatory cytokines, but rapidly declines when the number of pathogens increase (high dose infection). Whereas, because of its anti-inflammatory role, persistent high level expression of IL-10 may facilitate intracellular bacteria proliferation and increase the host susceptibility to infection, impairment of IL-10 production will compromise the control of proinflammatory cytokines and damage the host.
      In addition to the regulatory role of IL-10, recent studies demonstrated that miRs could also operate negative feedback mechanisms to achieve homeostasis in the innate immune system (
      • Marques-Rocha J.L.
      • Samblas M.
      • Milagro F.I.
      • Bressan J.
      • Martinez J.A.
      • Marti A.
      Noncoding RNAs, cytokines, and inflammation-related diseases.
      ,
      • Rossato M.
      • Curtale G.
      • Tamassia N.
      • Castellucci M.
      • Mori L.
      • Gasperini S.
      • et al.
      IL-10-induced microRNA-187 negatively regulates TNF-alpha, IL-6, and IL-12p40 production in TLR4-stimulated monocytes.
      ). miR-146b and miR-187 are IL-10-dependent miR that plays differential roles in IL-10-mediated suppression of proinflammatory cytokines in TLR4-stimulated monocytes (
      • Curtale G.
      • Mirolo M.
      • Renzi T.A.
      • Rossato M.
      • Bazzoni F.
      • Locati M.
      Negative regulation of Toll-like receptor 4 signaling by IL-10-dependent microRNA-146b.
      ,
      • Rossato M.
      • Curtale G.
      • Tamassia N.
      • Castellucci M.
      • Mori L.
      • Gasperini S.
      • et al.
      IL-10-induced microRNA-187 negatively regulates TNF-alpha, IL-6, and IL-12p40 production in TLR4-stimulated monocytes.
      ). However, the induction of some other miRs may not be IL-10 dependent, such as miR-146a and miR-155 (
      • Curtale G.
      • Mirolo M.
      • Renzi T.A.
      • Rossato M.
      • Bazzoni F.
      • Locati M.
      Negative regulation of Toll-like receptor 4 signaling by IL-10-dependent microRNA-146b.
      ). LPS treatment can induce miR-146a, miR-155, and miR-21 in monocytes (
      • O'Connell R.M.
      • Taganov K.D.
      • Boldin M.P.
      • Cheng G.
      • Baltimore D.
      MicroRNA-155 is induced during the macrophage inflammatory response.
      ,
      • Sheedy F.J.
      • Palsson-McDermott E.
      • Hennessy E.J.
      • Martin C.
      • O'Leary J.J.
      • Ruan Q.
      • et al.
      Negative regulation of TLR4 via targeting of the proinflammatory tumor suppressor PDCD4 by the microRNA miR-21.
      ,
      • Taganov K.D.
      • Boldin M.P.
      • Chang K.J.
      • Baltimore D.
      NF-κB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses.
      ). Among them, only miR-155 can be inhibited by IL-10 (
      • McCoy C.E.
      • Sheedy F.J.
      • Qualls J.E.
      • Doyle S.L.
      • Quinn S.R.
      • Murray P.J.
      • et al.
      IL-10 inhibits miR-155 induction by Toll-like receptors.
      ). These findings indicate that miRs are fine-tuners of IL-10-centered anti-inflammatory mechanisms in the innate immunity system (
      • O'Neill L.A.
      • Sheedy F.J.
      • McCoy C.E.
      MicroRNAs: the fine-tuners of Toll-like receptor signalling.
      ).
      Here we examined the cross-regulation of IL-10 and miRs in the infectious disease scrub typhus. Our studies identified that live O. tsutsugamushi (without LPS) can induce miR-146a and miR-155, but no miR-21 in THP-1-induced macrophages and human PBMCs. This finding suggests that O. tsutsugamushi bears pathogen-associated molecular pattern molecules capable of eliciting both LPS-like and different responses in monocytes, yet their precise nature remains to be elucidated.
      miR-146a is thought to regulate tumor necrosis factor receptor-associated family 6 and interleukin-1 receptor-associated kinase 1, two upstream signaling components within the TLR4 pathway (
      • Taganov K.D.
      • Boldin M.P.
      • Chang K.J.
      • Baltimore D.
      NF-κB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses.
      ). The major role of miR-155 in infection and immunity against infection is to fine tune the NF-κB pathway by targeting TAB2 or IKKε (
      • Ceppi M.
      • Pereira P.M.
      • Dunand-Sauthier I.
      • Barras E.
      • Reith W.
      • Santos M.A.
      • et al.
      MicroRNA-155 modulates the interleukin-1 signaling pathway in activated human monocyte-derived dendritic cells.
      ,
      • Lu F.
      • Weidmer A.
      • Liu C.G.
      • Volinia S.
      • Croce C.M.
      • Lieberman P.M.
      Epstein-Barr virus-induced miR-155 attenuates NF-kappaB signaling and stabilizes latent virus persistence.
      ,
      • Ma X.
      • Becker Buscaglia L.E.
      • Barker J.R.
      • Li Y.
      MicroRNAs in NF-κB signaling.
      ). In our study, blocking the expression of miR-146a did not change the cytokine expression pattern in response to O. tsutsugamushi, and in contrast to the blocking of miR-155, its expression could not be inhibited by IL-10. These results are in line with a previous report indicating that miR-155 and miR-146a play a differential role in the regulation of the NF-κB pathway (
      • Schulte L.N.
      • Westermann A.J.
      • Vogel J.
      Differential activation and functional specialization of miR-146 and miR-155 in innate immune sensing.
      ).
      miR-155 was noted to assume a critical role in physiological and pathological processes such as inflammation, immunity, and infections (
      • Elton T.S.
      • Selemon H.
      • Elton S.M.
      • Parinandi N.L.
      Regulation of the MIR155 host gene in physiological and pathological processes.
      ), targeting targets at least 140 genes and regulatory proteins of inflammation (
      • Nielsen C.B.
      • Shomron N.
      • Sandberg R.
      • Hornstein E.
      • Kitzman J.
      • Burge C.B.
      Determinants of targeting by endogenous and exogenous microRNAs and siRNAs.
      ). A very unique character of miR-155 among many other immunity-associated miRs is its inhibition by IL-10 (20 ng/ml) in LPS-stimulated bone marrow-derived macrophages and human peripheral blood monocytes (
      • McCoy C.E.
      • Sheedy F.J.
      • Qualls J.E.
      • Doyle S.L.
      • Quinn S.R.
      • Murray P.J.
      • et al.
      IL-10 inhibits miR-155 induction by Toll-like receptors.
      ). Here we further demonstrated that IL-10 is a very strong inhibitor on miR-155 induced by O. tsutsugamushi, working at physiological concentrations of IL-10 (0.005– 0.1 ng/ml), in a dose-dependent manner.
      We examined seven patients with scrub typhus who had recovered from the cytokine storm. Whereas this number of cases is limited, their PBMCs showed consistently lower production of IL-10 and miR-155 during the O. tsutsugamushi challenge. These findings corroborate our in vitro data, underscoring the important regulatory function of IL-10 and miR-155 in O. tsutsugamushi infection, a mechanism of tuning NF-κB activation to provide protection and to prevent cytokine overproduction. Our study also calls for considering IL-10 and miR155 expression as possible biomarkers for predicting the outcome of O. tsutsugamushi infection, and possibly as targets of immunomodulation for preventing infection-associated cytokine storm.

      Materials and Methods

      Human THP-1-induced macrophages infected with O. tsutsugamushi

      The TW-1 strain of O. tsutsugamushi was obtained from the Taiwan Centers for Disease Control. The methods for TW-1 culture, isolation, quantification (see Supplementary Figure S2 online), and identification (see Supplementary Figure S3 online) are shown in Supplementary Section S2. The THP-1 cells were induced to differentiate into macrophages as previously described (
      • Tsuchiya S.
      • Kobayashi Y.
      • Goto Y.
      • Okumura H.
      • Nakae S.
      • Konno T.
      • et al.
      Induction of maturation in cultured human monocytic leukemia cells by a phorbol diester.
      ) and infected with various doses of TW-1 to find the optimal infection dose to achieve replication and cell death (Supplementary Section S3 online). For the following experiments, a high infection dose was defined as one macrophage infected by 100 pathogens, and a low dose infection was defined as one macrophage infected by 2 pathogens.

      Cell viability, cytokine assay, western blot, and immunofluorescence staining of O. tsutsugamushi-infected macrophages; tracing of O. tsutsugamushi

      Details are shown in Supplementary Section S1 online.

      IL-10 exogenous treatment

      Macrophages were treated with recombinant human IL-10 (Millipore, Billerica, MA) with various doses 8 hours before infection. To inhibit IL-10 production, the macrophages were treated with PD98059 50 μM (Cell Signaling, Danvers, MA) 4 hours before infection or cotreated with PD98059 and IL-10 for 24 hours.

      miR assay and transfection of anti-miR

      Total RNA was extracted by Trizol (Invitrogen, Carlsbad, CA), and the miRs were converted to cDNA using stem-loop hairpin reverse transcription primers (ABI, Applied Biosystems, Carlsbad, CA). Assays of miR-146a (4395274), miR-155 (4395459), miR-21 (4427975), and U6 snRNA (4395470) were performed on an ABI 7500 system according to the manufacturer’s instructions. For miR inhibition, cells were treated with Turbofect (Thermo Scientific, Waltham, MA) transfection reagent with anti-miR-146a (AM13059, Ambion, Carlsbad, CA) or anti-miR-155 (AM12634, Ambion), or nonspecific anti-miR inhibitor as a negative control. After 24 hours of inhibition, the cells were infected with pathogens and examined as previously described.

      Patients and ethics statements

      The charts of patients with scrub typhus diagnosed from 2006 to 2011 were reviewed. The patients who suffered from a cytokine storm during the acute phase of scrub typhus and finally recovered were defined as the severe group. The patients with mild symptoms and signs who spontaneously recovered were defined as the mild group. The enrolled patients had no history of diabetes mellitus, renal disease, liver disease, or cancer, and all signed informed consent for blood sampling. Ethical approval was given by the Institutional Review Board of Buddhist Tzu Chi General Hospital (IRB102-53) and Kaohsiung Medical University Hospital (OT-101-104).

      PBMC isolation and O. tsutsugamushi challenge

      Fifty milliliters of whole blood were collected from each patient. Leukocytes were isolated using a Ficoll-Paque PLUS system (GE Healthcare, Chicago, IL). CD14+ monocytes were isolated using BD IMag-CD14 Magnetic Particles (BD, Franklin Lakes, NJ), and cultured with RPMI 1640 (Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA) overnight and infected with pathogens (100:1). The cytokines and miRs were assayed as previously described.

      Statistics

      Statistical analysis was performed using SAS 9.3 (Cary, NC) software. Student’s t-test or analysis of variance was used for continuous variables, and the chi-square or Fisher’s exact test was used for nominal variables. A difference was considered to be significant when the P-value was 0.05 or less.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      We thank Dr Pei-Yun Shu at Taiwan Center of Disease Control for providing the O. tsutsugamushi TW-1 strain. We appreciate the Center for Research Resources and Development at Kaohsiung Medical University for providing the Olympus confocal microscope and fluorescence microscope. This work was supported by grants from the National Science Council of Taiwan (NSC97-2745-B-037-004- and NSC-101-2314-B-037-26-) and from Buddhist Tzu Chi General Hospital (TCRD102-34).

      Supplementary Material

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