Transition of Serum Cytokine Concentration in Rickettsia Japonica Infection

Article Transition of Serum Cytokine Concentration in Rickettsia japonica Infection

Makoto Kondo, Yoshiaki Matsushima, Kento Mizutani, Shohei Iida, Koji Habe and Keiichi Yamanaka * Department of Dermatology, Mie University Graduate School of medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan; [email protected] (M.K.); [email protected] (Y.M.); [email protected] (K.M.); [email protected] (S.I.); [email protected] (K.H.) * Correspondence: [email protected]; Tel.: +81-59-231-5025; Fax: +81-59-231-5206

Received: 31 October 2020; Accepted: 8 December 2020; Published: 11 December 2020

Abstract: (1) Background. Rickettsia japonica (R. japonica) infection induces severe inﬂammation, and the disappearance of eosinophil in the acute stage is one of the phenomena. (2) Materials and Methods. In the current study, we measured the serum concentrations of Th1, Th2, and Th17 cytokines in the acute and recovery stages. (3) Results. In the acute phase, IL-6 and IFN-γ levels were elevated and we speculated that they played a role as a defense mechanism against R. japonica. The high concentration of IFN-γ suppressed the diﬀerentiation of eosinophil and induced apoptosis of eosinophil, leading to the disappearance of eosinophil. On day 7, IL-6 and IFN-γ concentrations were decreased, and Th2 cytokines such as IL-5 and IL-9 were slightly increased. On day 14, eosinophil count recovered to the normal level. The transition of serum cytokine concentration in R. japonica infection was presented. (4) Conclusions. IL-6 and IFN-γ seem to be critical cytokines as defense mechanism against R. japonica in the acute phase, and this may deeply connect to the decrease of eosinophil.

Keywords: Japanese spotted fever; Rickettsia japonica; IL-6; IFN-γ; eosinophil

1. Introduction Japanese spotted fever (JSF) caused by Rickettsia japonica (R. japonica) infection was first reported from Tokushima Prefecture in Japan in 1984 [1]. The typical symptom of JSF is characterized by diffuse erythema on the whole body, palmar erythema, headache, high fever, and chills after two to eight days following a tick bite [2,3]. Tetracycline is recommended to treat JSF. One of the severest complications is disseminated intravascular coagulation (DIC), low consciousness level, low blood pressure, multiple organ failure, and edema, which occurs if the disease progress is sudden or the diagnosis of JSF is delayed, and some fatal cases have been reported [2–4]. Therefore, the deterioration of symptoms following a JSF infection needs to be understood. R. japonica infection induces severe inflammation [2,5], and moreover the disappearance of eosinophil in the acute stage of JSF is one of the phenomena. In the current study, we measured the serum concentrations of Th1, Th2, and Th17 cytokines in the acute and recovery stages and discussed the possible immune mechanism in R. japonica infection.

2. Materials and Methods This study enrolled 7 patients (3 males and 4 females from 70 to 87 years old, living in the endemic area in Mie Prefecture, Japan) after obtaining written informed consent. The protocol was approved by the Institutional Review Board at Mie University (Tsu, Mie, Japan, permit number 3064). All patients were diagnosed as having JSF by performing PCR for R. japonica-speciﬁc DNA from blood and/or skin samples [6], or anti-R. japonica antibody titer of IgM > 80 in the acute stage, or elevation of IgM and/or IgG more than 4 times in the recovery stage compared to acute stage by indirect immunoﬂuorescence

Infect. Dis. Rep. 2020, 12, 127–131; doi:10.3390/idr12030023 www.mdpi.com/journal/idr Infect. Dis. Rep. 2020, 12, FOR PEER REVIEW 2 blood and/or skin samples [6], or anti-R. japonica antibody titer of IgM > 80 in the acute stage, or elevation of IgM and/or IgG more than 4 times in the recovery stage compared to acute stage by Infect. Dis. Rep. 2020 12 indirect immunofluorescence, assay (IFA). For this assay slides are coated by Vero cells cultured with128 R. japonica and incubated with the patient’s serum. If antibodies against R. japonica are present, the cellsassay fluoresc (IFA). Fore. this assay slides are coated by Vero cells cultured with R. japonica and incubated with the patient’sThe serum serum. samples If antibodies were collected against atR. the japonica first visitare (day present, 1, acute the cellsstage) fluoresce. and 14 days after the first visit (recoveryThe serum stage) samples and werethen stored collected at − at80 the °C firstbefore visit use. (day Serum 1, acute sample stage)s were and also 14 days collected after theon days first 3visit and (recovery7 for cases stage) 1–3. We and measured then stored cytoki at ne80 concentrationsC before use. including Serum samples Th1 (IFN- wereγ, IL-12p70, also collected TNF- onα, − ◦ IL-2,days IL-8), 3 and Th2 7 for (IL-4, cases IL 1–3.-5, IL-9, We measuredIL-33), and cytokine Th17 (IL-6, concentrations IL-17A, IL-17F, including IL-22). Th1 All (IFN-patientsγ, IL-12p70, received minocyclineTNF-α, IL-2, on IL-8), day Th21 and (IL-4, recovered IL-5, IL-9, on day IL-33), 14 with and no Th17 symptoms (IL-6, IL-17A, of JSF IL-17F,and no IL-22).abnormal All data. patients For casesreceived 1, 2, minocycline and 3, the above-mentioned on day 1 and recovered cytokines on day were 14 withmeasured no symptoms at 4 points: of JSF days and 1, no 3, abnormal7, and 14. data. The cytokineFor cases measurements 1, 2, and 3, the were above-mentioned performed with cytokines 15 µL were of serum measured for each at 4 points:sample days using 1, 3,a 7,panel and 14.kit (AimPlexThe cytokine Biosciences, measurements Pomona, were CA, performed USA) and withflow 15cytometerµL of serum analysis for with each a sample BD Accuri using C6 a device panel (Becton,kit (AimPlex Dickinson Biosciences, and Company, Pomona, CA,Franklin USA) Lake ands, flow NJ, cytometerUSA) according analysis to with the amanufacturer’s BD Accuri C6 instructions.device (Becton, We Dickinsonalso presented and Company,data of white Franklin blood Lakes, cell (WBC), NJ, USA) neutrophil, according and to eosinophil the manufacturer’s count, as wellinstructions. as C-reactive We alsoprotein presented (CRP). dataThe data of white were blood measured cell (WBC), at the laboratory neutrophil, in andour eosinophilhospital by count,using anas automated well as C-reactive indicator. protein (CRP). The data were measured at the laboratory in our hospital by using an automatedStatistical indicator.analysis was performed using PRISM software version 6 (GraphPad, San Diego, CA, USA).Statistical The Mann–Whitney analysis was U test performed was used using to compar PRISMe cytokine software concentrations version 6 (GraphPad, between Sandays Diego, 1 and 14.CA, D USA).ifferences The were Mann–Whitney considered Usignificant test was usedat p

Figure 1. In the acute phase, white blood cell (WBC) and neutrophil were within the normal range, Figure 1. In the acute phase, white blood cell (WBC) and neutrophil were within the normal range, and C-reactive protein (CRP) were increased, but decreased at day 14 by the treatment of minocycline. and C-reactive protein (CRP) were increased, but decreased at day 14 by the treatment of minocycline. The loss of eosinophil was detected in the acute stage but recovered to the normal range in all cases. The loss of eosinophil was detected in the acute stage but recovered to the normal range in all cases. Infect. Dis. Rep. 2020, 12 129 Infect. Dis. Rep. 2020, 12, FOR PEER REVIEW 3

3.2.3.2. Inflammatory Inﬂammatory Cytokine Among thethe 1313 cytokines cytokines that that we we measured, measured, IFN- IFN-γ andγ and IL-6 IL-6 levels levels were were increased increased in the inacute the acute stage andstage decreased and decreased clearly clearly after theafter treatment the treatment except exce forpt cases for cases 3 and 3 5.and Regarding 5. Regarding the otherthe other measured measured Th1, Th2,Th1, andTh2, Th17and Th17 cytokines, cytokines, Th1 cytokinesTh1 cytokines showed showed a slightly a slightly decreased decreased tendency tendency in most in most of the of cases. the Incases. contrast, In contrast, Th2 and Th2 Th17and Th17 cytokines cytokines were were unchanged unchanged for for two two data data points. points. In In casecase 3, the the counter counter cytokines againstagainst intracellularintracellular parasite, parasite, IL-6 IL-6 was was undetected undetected at daysat days 1 and 1 and 14, but14, otherbut other cytokines cytokines were detectedwere detected (Figure (Figure2). Statistical 2). Statistical analysis analysis was performed was performed for the for cytokine the cytokine concentration concentration between between days 1 anddays 14, 1 and and 14, signiﬁcance and significance was detected was detected in IFN- inγ andIFN- IL-6γ and levels IL-6 (levelsp = 0.0111). (p = 0.0111).

Figure 2. Cytokine concentration in the the acute acute and and reco recoveryvery stages stages is is show shown.n. The The increased increased IFN- IFN-γγ andand IL-6IL-6 levels were clearly decreased in in the the recovery recovery st stageage in in most most of of the the cases. cases. For For Th1, Th1, Th2, Th2, and and Th17 Th17 cytokines, exceptexcept forfor IFN-IFN-γγand and IL-6, IL-6, cytokine cytokine levels levels were were unchanged unchanged for for 2 data2 data points points (days (days 1 and 1 and 14). Case14). Case 3 was 3 was exceptional exceptional and and the cytokines,the cytokines, except except for IFN-for IFN-γ, IL-6,γ, IL-6, and and IL-12p70, IL-12p70, were were elevated elevated in thein recoverythe recovery stage. stage. Statistical Statistical analysis analysis was performedwas performed for the for cytokine the cytokine concentration concentration between between days 1 days and 14,1 and signiﬁcance14, and significance was detected was detected in IFN- γinand IFN- IL-6γ and levels IL-6 (* levelsp = 0.0111). (* p = 0.0111).

Cytokine concentration was was measured measured at at four four points points in in cases cases 1, 1, 2, 2, and and 3. 3. Among Among 13 13 cytokines, cytokines, elevated IFN-γ and IL-6 levels were clearly decreased over time (Figure 33).). Th1 cytokines including IL-12p70,IL-12p70, TNF-α,, IL-2, and and IL-8 IL-8 did did not not show show this this decrease. decrease. IL-5 IL-5 and and IL-9 IL-9 are are involved involved in in the the Th2 Th2 cytokine families, and there seems to be no no significan signiﬁcancece in in two two points points of of the the acute acute and and recovery recovery stages stages (Figure(Figure2 2).). However,However, thethe concentrationsconcentrations ofof thosethose cytokinescytokines increased on day 7 and then decreased in thethe recoveryrecovery stage close to the day 1 level. IL-17A/F IL-17A/F also also showed showed a a slight slight elevation elevation on on days days 3 3 and and 7 7 (Figure(Figure3 3).). Infect. Dis. Rep. 2020, 12 130 Infect. Dis. Rep. 2020, 12, FOR PEER REVIEW 4

FigureFigure 3. Cytokine concentrations were measured at 4 points in cases 1 1,, 2, and and 3. 3. IFN- IFN-γγ andand IL-6 IL-6 levels levels showedshowed aa peakpeak on day 1, and reducedreduced in a time-dependent manner. manner. IL IL-5-5 and and IL-9 IL-9 are are involved involved in in the the Th2Th2 cytokinecytokine families, families, and and the the concentrations concentrations of those of those cytokines cytokines increased increased on day on 7 and day then 7 and decreased then indecreased the recovery in the stage. recovery Th17 stage. families Th17 except families for IL-6 except showed for IL-6 a slight showed peak a on slight day 7.peak This on tendency day 7. This was nottendency noticed was for not 2 data noticed points for (days 2 data 1 points and 14). (days 1 and 14).

4.4. DiscussionDiscussion ThereThere hashas beenbeen a report of R. japonica infection focused focused on on inflammatory inflammatory cytokines. cytokines. In In R.R. japonica japonica infection,infection, IL-6, IFN-γ, TNF- α,, IL-8, IL-8, MCP-1a, MCP-1a, and and MIP-1 MIP-1ββ levelslevels were were elevated elevated according according to to the the previousprevious reportreport [[7].7]. TheThe firstfirst line of defense against Rickettsia infectioninfection is is phagocytosis phagocytosis by by macrophage, macrophage, but but RickettsiaRickettsia isis so-calledso-called macrophage-tropicmacrophage-tropic bacteria,bacteria, andand interactioninteraction occursoccurs duringduring thethe phagocytosis,phagocytosis, releasingreleasing IL-6IL- and6 and IFN- IFN-γ γfrom from macrophage macrophage as as an an immune immune protection. protection. RickettsiaRickettsia existing as an intracellular intracellular parasite parasite hashas thethe ability ability to infectto infect the endocapillarythe endocapillary cells. Althoughcells. Although IL-6 is the IL-6 key is cytokine the key for cytokine the differentiation for the ofdifferentiation Th17 cells, IL-6 of Th17 was releasedcells, IL-6 from was the released stimulated from macrophage-activated the stimulated macrophage-activated Th1 cells during Th1 the acutecells stageduring of theR. japonicaacute stageinfection, of R. japonica and Th1 infection, cells produced and Th1 IFN- cellsγ. IL-6produced together IFN- withγ. IL-6 IFN- togetherγ strongly with activate IFN- macrophageγ strongly activate and evoke macrophage differentiation and ofevoke naïve differentiation T cells into mature of naïve Th1 T cells cells in into order mature to attack Th1 pathogens cells in suchorder as toR. attack japonica pathogensfrom the such outside as R. [ 8japonica,9]. from the outside [8,9]. InIn RickettsiaRickettsiainfection, infection, a higha high level level of CRP of CRP was detected,was detected, but WBC but countWBC showedcount showed a slight elevation.a slight Aelevation. previous A report previous showed report that showed eosinophil that eosinophil counts were counts diminished were diminished similarly tosimilarly the current to the cases current [10]. Becausecases [10]. of theBecause low concentration of the low concentration on day 1 and theon increaseday 1 and on daythe 7increase of Th2 cytokine on day including7 of Th2 IL-4,cytokine IL-5, andincluding IL-33, IL-4, we speculated IL-5, and IL-33, that there we speculated may be suppressing that there may factors be onsuppressing eosinophil factors in R. japonicaon eosinophilinfection, in whichR. japonica is purposeful infection, which in the is acute purposeful stage. in Th2 the cytokines acute stage. have Th2 lower cytokines priority have because lower priority of nonecessity because ofof dinoff erentiationnecessity of todifferentiation eosinophil in to the eosinophil acute phase. in the The acute high phase. concentration The high ofconcentration IFN-γ suppresses of IFN- theγ disuppressesfferentiation the of differentiation eosinophil and of induceseosinophil apoptosis and indu ofces eosinophil apoptosis [11 of], leadingeosinophil to the [11], disappearance leading to the of eosinophil.disappearance Around of eosinophil. day 7, the Around number day of R. 7, japonica the numberis decreased, of R. japonica together is decreased, with the decrease together in with IL-6 the and IFN-decreaseγ concentrations, in IL-6 and IFN- andγ the concentrations, number of eosinophil and the number recovers of and eosinophil Th2 and Th17recovers cytokines and Th2 are and relatively Th17 increased.cytokines are In relatively the current increased. study, case In the 3 showed current astudy, high case concentration 3 showed a in high most concentration cytokines measured, in most althoughcytokines themeasured, patient did although not have the a specific patient past did history not suchhave asa immunodeficiency,specific past history severe such allergy, as orimmunodeficiency, clinical course associated severe allergy, with multiple or clinical organ course failure associated and but there with might multiple be another organ accidentalfailure and event. but Otherwise,there might the be innate another and accidental acquired immuneevent. Otherwis system maye, the react innate strongly and acquired against danger immune signals system in casemay 3. react strongly against danger signals in case 3. Infect. Dis. Rep. 2020, 12 131

5. Conclusions We reported the cytokine levels in patients infected by R. japonica before and after the treatment of minocycline. The current information would be beneﬁcial to understanding the immune reaction against R. japonica infection especially for four data points. IL-6 and IFN-γ seem to be critical cytokines as a defense mechanism against R. japonica in the acute phase, and this may be linked to the decrease in eosinophils.

Author Contributions: M.K. planned and investigated the study after obtaining informed consent from patients and wrote this article. Y.M., K.M., and S.I. analyzed the data. K.H. and K.Y. offered advice for the study and reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: The authors did not receive any financial support for the present study. Conflicts of Interest: The authors declare no conflict of interest.

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