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Kinetics of inflammatory cytokines in patients with scrub typhus receiving doxycycline treatment
Journal of Infection (2008) 56, 44e50
www.elsevierhealth.com/journals/jinf
Kinetics of inflammatory cytokines in patients with scrub typhus receiving doxycycline treatment Doo Ryeon Chung*, Young Seok Lee, Seung Soon Lee Division of Infectious Diseases, Department of Internal Medicine, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, 896 Pyeongchon-dong, Dongan-gu, Anyang-si, Gyeonggi-do 431-070, South Korea Accepted 19 September 2007 Available online 31 October 2007
KEYWORDS Scrub typhus; Orientia tsutsugamushi; Cytokines; TNF-a; Doxycycline
Summary Objectives: Animal models have suggested that various cytokines and chemokines play an important role in host defense against Orientia tsutsugamushi, however, human data are limited. We investigated the kinetics of inflammatory responses in patients with scrub typhus receiving doxycycline. Methods: Amongst patients being treated for scrub typhus, serially collected sera were tested for TNF-a, IFN-g, IL-2, IL-4, IL-5, and IL-10 by immunoassay. The mRNAs encoding TNF-a, IL-1b, IL-6, IL-8, IFN-g, IL-12, IL-2, IL-4, IL-5, and IL-10 were measured by semiquantitative reverse transcription-PCR. Results: The concentrations of TNF-a, IFN-g, and IL-10 of patients prior to doxycycline treatment were significantly higher than those of healthy volunteers. They decreased markedly within 24 h after starting doxycycline. The mRNAs for IL-1b, TNF-a, IL-6, IFN-g, and IL-10 were highly expressed. Expression of mRNAs for IL-1b, IFN-g, and IL-10 decreased at day 2e7 of doxycycline treatment. Conclusions: Inflammatory cytokines including TNF-a, IL-1b, and IL-6 are markedly upregulated in patients with scrub typhus. Doxycycline treatment rapidly reduces the production of these cytokines, corresponding to the early defervescence after the start of the treatment. The profiles of T cell-derived cytokines in patients with scrub typhus do not follow typical Th1 or Th2 patterns. ª 2007 The British Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction * Corresponding author. Present address: Division of Infectious Diseases, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Gangnam-gu, Seoul 135-710, South Korea. Tel.: þ82 2 3410 0323; fax: þ82 2 3410 0041. E-mail address: [email protected] (D.R. Chung).
Scrub typhus, which is an endemic rickettsial disease in Asian countries, is characterized by high fever of sudden onset, headache, myalgia, rash, and eschar at a mite bite site.1,2 Orientia tsutsugamushi, causing this disease, infects a kind of cells such as endothelial cells, macrophages, neutrophils,
0163-4453/$30 ª 2007 The British Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jinf.2007.09.009
Inflammatory cytokines in scrub typhus and lymphocytes. It proliferates in the cytoplasm, induces local inflammation, and then disseminates to whole body and provokes a disease.3 Scrub typhus shows variable clinical presentations in terms of severity from mild and self-limiting to fatal.4 Some cases manifest as severe diseases accompanying serious complications such as coma, meningoencephalitis, interstitial pneumonitis, acute respiratory distress syndrome, myocarditis, or septic shock.5e13 Such a diversity might be explained as the result of variable interactions between virulence factors of O. tsutsugamushi and immune response of the host. Animal experiments have revealed that O. tsutsugamushi strains exhibit different virulence according to their serotypes,14 however, even among the strains of the same serotype the pathogenicity of O. tsutsugamushi is also influenced by the host factors.15 That is, clinical manifestations of scrub typhus depend not only on the virulence factors of organisms, but also on the immune response of the infected persons. In fact, although activation of the immune system of the infected host is one of the host defense mechanism, exaggerated immune response itself can result in a serious clinical deterioration. Most studies on immunological response to infection by O. tsutsugamushi have been based on in vitro experiments or animal models. Macrophages and T lymphocytes have been suggested to play an important role in host defense to infection with O. tsutsugamushi, however, the role of host immune response in the pathogenesis of scrub typhus has not been clearly documented.4 Previous reports have suggested that various chemokines and cytokines are produced by the stimuli of O. tsutsugamushi and these play an important role in the pathogenesis of this acute febrile illness.16e20 However, there have been a limited number of reports on human data.21,22 A report from Thailand showed elevated levels of IFN-g and IFNg-inducing cytokines in 28 patients with scrub typhus and suggested a strong involvement of the cell-mediated immune response in host defense against O. tsutsugamushi.22 In order to further understand the pathogenesis of scrub typhus, it is essential to clearly delineate the immune response in the patients infected by O. tsutsugamushi and determine the role of this immune response in the disease manifestations. We investigated the kinetics of inflammatory and T cell responses in the patients with scrub typhus before and after doxycycline treatment in order to determine the role of host immune response in the pathogenesis of scrub typhus.
Materials and methods Patients Patients with the diagnosis of scrub typhus were prospectively enrolled at the Hallym University Sacred Heart Hospital in Anyang-si, Korea, during the period from September to December 2003. Patients with a characteristic eschar, fever, and maculopapular rash, who had IgG antibody titer greater than 1:80 to O. tsutsugamushi or 4-fold rising antibody titer in the repeated serum, were included in this study. Patients younger than 18 years or patients who had already received doxycycline treatment before
45 screening for this study were excluded. Demographic and clinical information including daily peak body temperatures were analyzed. Venous blood was collected from the enrolled patients at four time points including before doxycycline was given, at day 1, day 2e3, and day 4e7. Sera were separated and stored at 80 C until analyzed. For isolation and purification of RNA, we collected the blood in RNA-stabilizing tubes (PAXgene Blood RNA Tubes; Qiagen, Inc., USA). Total RNA was extracted using blood RNA isolation kit (Qiagen, Inc., USA), and stored at 80 C. Eight healthy volunteers provided their serum as control. All these procedures were in accordance with the ethical standards of the hospital and with the Helsinki Declaration. Informed consent was obtained from the patients.
Measurement of cytokine concentration in serum Serum concentrations of TNF-a, IFN-g, IL-2, IL-4, IL-5, and IL-10 were determined by a solid phase sandwich ELISA using monoclonal antibodies (BioSource, Inc., USA) in accordance with the manufacturer’s protocols. The concentration of each cytokine was measured in duplicate.
Semiquantitative analysis for cytokine mRNA expression in blood Expression of mRNA transcripts encoding TNF-a, IL-1b, IL-6, IL-8, IFN-g, IL-12, IL-2, IL-4, IL-5, and IL-10 was determined by semiquantitative reverse transcription-PCR (RT-PCR) method in total RNA samples extracted from patients’ blood. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was tested as a control housekeeping gene for semiquantitative analysis. Reverse transcription and amplification of cDNA was done using cDNA amplification kit (Invitrogen, Inc., USA) according to the instructions of the manufacturer. Multiplex PCR for the genes encoding various cytokines as above was performed using a commercial kit (BioSource, Inc., USA). Mixed primer sets in the kit were used. PCR products were separated by electrophoresis on a 2% agarose gel and stained with ethidium bromide. Gels were viewed and photographed under UV light illumination. Digital gel photographs of the stained PCR products were taken under UV exposure by using digital camera (Nikon Coolpix 990). Cytokine cDNA bands were determined as the integrated area (pixels) of the band intensities by densitometry analysis with ImageJ version 1.33 (National Institutes of Health, USA). The numerical values for cytokine cDNA band intensities were presented as ratios relative to band intensities of GAPDH.
Statistical analysis Comparisons of continuous variables were made by using Student’s t-test or ManneWhitney U-test. Statistical analysis was performed with use of SPSS Software Package Release 11.0 (SPSS Inc., USA). We considered differences significant at P < 0.05.
46
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Results Demographic and clinical characteristics of the patients A total of 22 patients were enrolled (Table 1). All the patients received oral doxycycline for 7 days. The average of daily peak body temperature in the patients decreased from 38.5 C (S.D. 0.9 C) to 37.5 C (S.D. 0.8 C) within 24 h after the first dose of doxycycline (P < 0.001) (Fig. 1). Two patients (9.1%) had septic shock, and meningoencephalitis was complicated in two patients (9.1%). Five patients (22.7%) had bilateral pneumonia. Elevation of serum LDH and CRP was observed in most patients (Table 1). Severe cases complicated with shock, meningoencephalitis, or pneumonia showed a higher median level of CRP compared with non-severe cases (111.5 vs. 33.8; P Z 0.040, ManneWhitney U-test). There was no significant difference in white blood cell (WBC) count, platelet, and lactate dehydrogenase (LDH) level between two groups. All the patients were cured without any long-term complications.
Cytokine levels in serum following doxycycline treatment
Table 1 Demographic and clinical characteristics of the patients (n Z 22) with scrub typhus Age in years Female gender, number (%) Days from onset Shock, number (%) Pneumonitis, number (%) Meningoencephalitis, number (%) Laboratory findings WBC (/mm3) Platelet (/mm3) ALT (IU/L) Bilirubin (mg/dl) Albumin (mg/dl) LDH (IU/L) Creatinine (g/dl) CRP (mg/dl)
U-test). However, IFN-g and IL-10 showed no significant difference (Table 2).
Expression of mRNA encoding cytokines in blood following doxycycline treatment
The concentrations of TNF-a, IFN-g, and IL-10 of the patients prior to doxycycline treatment were significantly higher than those of healthy volunteers (Fig. 2). These levels decreased markedly within 24 h after starting doxycycline treatment, although they were still higher than those of volunteers until day 3. The concentrations of IL-2 and IL-4 did not show any significant change before and after the doxycycline treatment. IL-5 was not detectable in both the patients and volunteers. The median TNF-a level of severe cases complicated with shock, meningoencephalitis, or pneumonitis was significantly higher than that of non-severe cases (96.2 vs. 20.1; P Z 0.003, ManneWhitney
Characteristics
Figure 1 Change of peak body temperature in patients with scrub typhus after the first dose of doxycycline (*P < 0.001 compared to peak temperature prior to start of doxycycline; n Z 22).
Mean (S.D.) 58 12 7.2 2 5 2 7827.3 125.6 84.8 0.9 3.2 821.5 1.0 70.4
(18) (54.5) (3.7) (9.1) (22.7) (9.1) (4164.7) (67.6) (61.2) (1.4) (0.5) (244.5) (0.2) (54.6)
Semiquantitative analyses for the expression of mRNA encoding inflammatory cytokines and Th1/Th2 cytokines for the patients with scrub typhus were performed (Fig. 3). Relatively high expression of mRNAs for IL-1b, TNF-a, IL-6, IFN-g, and IL-10 was observed before receiving doxycycline treatment (Fig. 4). IL-1b mRNA expression decreased significantly during the period of day 2e7 after the first dose of doxycycline treatment (P Z 0.001). Expression of TNF-a mRNA at day 2e7 was slightly lower compared to those at day 0 or day 1. IL-6 mRNA expression increased until day 1, however, its expression at day 2e7 was significantly lower than that before treatment (P Z 0.016). IFNg and IL-10 also showed higher mRNA expression at day 1 (P Z 0.004 and P Z 0.035, respectively), however, their expression decreased back at day 2e7. Expression of mRNA encoding IL-2 or IL-4 was very low although IL-4 mRNA expression at day 1 was transiently higher compared to that at day 0.
Discussion This study shows that the production of inflammatory cytokines such as TNF-a, IL-1b, and IL-6 is increased in the patients with scrub typhus. Interestingly, the increased production of these inflammatory cytokines decreased rapidly within 1 or 2 days after starting doxycycline treatment. We have also found that profiles of T cellderived cytokines in these patients do not follow typical Th1 or Th2 patterns. IFN-g, one of the Th1 subset cytokines, showed a high level before antibiotic treatment, and its level has markedly declined following the start of doxycycline treatment. IL-2, another classic Th1 subset T cell-derived cytokine, however, did not show any significant change in these patients. IL-10, one of the Th2 subset
Inflammatory cytokines in scrub typhus
47
Figure 2 Time kinetics of the cytokines in serum of the patients with scrub typhus after doxycycline treatment. Cytokines were measured in duplicate for each patient. (A) TNF-a, (B) IFN-g, (C) IL-2, (D) IL-10, (E) IL-4 (n Z 22).
T cell-derived cytokines, showed higher concentration compared to those of healthy volunteers, and it has rapidly decreased within 24 h after the first dose of doxycycline. IL-4 and IL-5 did not show any significant change in their concentrations or mRNA expression in the blood of the patients regardless of doxycycline treatment. Cytokine profile featured by increased production or upregulation of TNF-a, IL-1b, and IL-6 was found to be a major inflammatory response in the patients with scrub typhus before receiving adequate antibiotic treatment. This explains well that high fever is a chief complaint in
most patients with scrub typhus, because these cytokines play an important role in the generation of fever in humans. In addition, rapid reduction of TNF-a, IFN-g, and IL-10 production following the start of doxycycline treatment was in accordance with the decrease of peak temperature in these patients in the aspect of time. In fact, a rapid defervescence in response to doxycycline treatment in the patients with scrub typhus is a characteristic finding to support the clinical diagnosis of this disease. Our result is contradictory with the previous report from Japan that has revealed higher levels of TNF-a in the patients in
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Table 2 Comparison of cytokine levels between severe cases with shock, meningoencephalitis, or pneumonitis and non-severe cases Cytokines Median (range) Severe (n Z 6) TNF-a IFN-g IL-10 a
Non-severe (n Z 16) P a
96.2 (21.6e336.8) 20.1 (0e134.3) 10.1 (0e36.2) 3.25 (0.3e31.4) 17.6 (3.7e95.8) 6.25 (1.7e17.4)
0.003 0.641 0.098
ManneWhitney U-test.
convalescent stage than in acute stage of scrub typhus.21 TNF-a is an important cytokine mediating variable cellmediated immune responses to infection and plays an essential role in host defenses to intracellular pathogens. Previous studies have showed that heat-stable molecule of O. tsutsugamushi is responsible for the induction of TNF-a production.18
A few studies have suggested a role of specific T lymphocytes, especially Th1 cells, in host defense to O. tsutsugamushi,23,24 although there have been very limited data in humans. In our study, the patterns of cytokine production or expression in the patients with scrub typhus prior to doxycycline treatment were not typical for either Th1-type or Th2-type immune response. This can be a result of overlapped Th2 response following after inflammatory reaction and Th1 immune response elicited by O. tsutsugamushi infecting the host. Other possibility is that IFN-g might have been mainly produced by other inflammatory cells such as macrophages or natural killer cells rather than by T cells. Our finding that IL-2 production or expression was not significant in the patients with scrub typhus supports it. Furthermore, the importance of macrophages in the pathogenesis of scrub typhus has already been addressed through previous in vitro studies that revealed increased production of TNF-a and IL-6 as well as increased mRNA expression for various chemokines in murine macrophage cell line.17,18 It has been suggested that inhibitory cytokines
Figure 3 Expression of cytokine mRNA from the blood of a patient with scrub typhus. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the control for the semiquantitative measurement.
Inflammatory cytokines in scrub typhus
49
Figure 4 Time kinetics of cytokine-encoding mRNA expression in the blood of the patients with scrub typhus (*P < 0.05 compared to data at day 0; n Z 20).
including IL-10 and TGF-b are induced by O. tsutsugamushi to inhibit the production of the inflammatory cytokines in order to survive inside hostile macrophages.16,25 Rapid decline in IL-10 production after doxycycline treatment in our study is thought to have been followed after decrease of inflammatory cytokines including TNF-a and IFN-g. Limitation in our study is that time trends of mRNA expression do not exactly match with those of cytokine concentrations in the blood. Variability in the data of mRNA seems to be attributable to in vitro induction of mRNA expression.26 Although we tried to minimize such technical bias by collecting the blood into the RNA-stabilizing tubes, we expect that we could not be free from it. We performed a semiquantitative RT-PCR to measure mRNA expression
instead of quantitative RT-PCR. Semiquantitative method also limited precise comparison of mRNA expression between different time points. Another limitation is that we did not measure mRNA expression in healthy volunteers, so we could not compare mRNA expression between patients and healthy volunteers. To our knowledge, this is the first study to investigate time kinetics of cytokines in the patients with scrub typhus. Especially, we tried to explain the mechanism of rapid defervescence after starting doxycycline treatment by the change of cytokine production in the patients. We found that the production of inflammatory cytokines such as TNF-a, IL-1b, and IL-6 is increased in the patients with scrub typhus and their rapid decline coincides with early defervescence
50 following doxycycline treatment. Besides, profiles of T cellderived cytokines do not follow typical Th1 or Th2 patterns.
Conflict of interest The authors have no conflicts of interest.
Acknowledgments We thank Sun Hee Ahn for her excellent technical assistance. Financial support: This work was supported by Korea Research Foundation Grant (KRF 2003-003-E00101).
References 1. Allen AC, Spitz S. A comparative study of the pathology of scrub typhus (tsutsugamushi disease) and other rickettsial diseases. Am J Pathol 1945;21:603e61. 2. Burnett JW. Rickettsioses: a review for the dermatologist. J Am Acad Dermatol 1980;2:359e73. 3. Rikihisa Y, Ito S. Intracellular localization of Rickettsia tsutsugamushi in polymorphonuclear leukocytes. J Exp Med 1979; 150:703e8. 4. Seong SY, Choi MS, Kim IS. Orientia tsutsugamushi infection: overview and immune responses. Microbes Infect 2001;3:11e21. 5. Tsay RW, Chang FY. Serious complications in scrub typhus. J Microbiol Immunol Infect 1998;31:240e4. 6. Pai H, Sohn S, Seong Y, Kee S, Chang WH, Choe KW. Central nervous system involvement in patients with scrub typhus. Clin Infect Dis 1997;24:436e40. 7. Fang CT, Ferng WF, Hwang JJ, Yu CJ, Chen YC, Wang MH, et al. Life-threatening scrub typhus with meningoencephalitis and acute respiratory distress syndrome. J Formos Med Assoc 1997; 96:213e6. 8. Song SW, Kim KT, Ku YM, Park SH, Kim YS, Lee DG, et al. Clinical role of interstitial pneumonia in patients with scrub typhus: a possible marker of disease severity. J Korean Med Sci 2004; 19:668e73. 9. Tsay RW, Chang FY. Acute respiratory distress syndrome in scrub typhus. Q J Med 2002;95:126e8. 10. Watt G, Parola P. Scrub typhus and tropical rickettsioses. Curr Opin Infect Dis 2003;16:429e36. 11. Park JS, Jee YK, Lee KY, Kim KY, Myong NH, Seo PW. Acute respiratory distress syndrome associated with scrub typhus: diffuse alveolar damage without pulmonary vasculitis. J Korean Med Sci 2000;15:343e5. 12. Ben RJ, Feng NH, Ku CS. Meningoencephalitis, myocarditis and disseminated intravascular coagulation in a patient with scrub typhus. J Microbiol Immunol Infect 1999;32:57e62.
D.R. Chung et al. 13. Thap LC, Supanaranond W, Treeprasertsuk S, Kitvatanachai S, Chinprasatsak S, Phonrat B. Septic shock secondary to scrub typhus: characteristics and complications. Southeast Asian J Trop Med Public Health 2002;33:780e6. 14. Hanson B. Comparative susceptibility to mouse interferons of Rickettsia tsutsugamushi strains with different virulence in mice and of Rickettsia rickettsii. Infect Immun 1991;59: 4134e41. 15. Groves MG, Kelly DJ. Characterization of factors determining Rickettsia tsutsugamushi pathogenicity for mice. Infect Immun 1989;57:1476e82. 16. Koh YS, Yun JH, Seong SY, Choi MS, Kim IS. Chemokine and cytokine production during Orientia tsutsugamushi infection in mice. Microb Pathog 2004;36:51e7. 17. Cho NH, Seong SY, Huh MS, Han TH, Koh YS, Choi MS, et al. Expression of chemokine genes in murine macrophages infected with Orientia tsutsugamushi. Infect Immun 2000;68: 594e602. 18. Kim MK, Kang JS. Orientia tsutsugamushi suppresses the production of inflammatory cytokines induced by its own heatstable component in murine macrophages. Microb Pathog 2001;31:145e50. 19. Cho NH, Seong SY, Choi MS, Kim IS. Expression of chemokine genes in human dermal microvascular endothelial cell lines infected with Orientia tsutsugamushi. Infect Immun 2001;69: 1265e72. 20. Yun JH, Koh YS, Lee KH, Hyun JW, Choi YJ, Jang WJ, et al. Chemokine and cytokine production in susceptible C3H/HeN mice and resistant BALB/c mice during Orientia tsutsugamushi infection. Microbiol Immunol 2005;49:551e7. 21. Iwasaki H, Takada N, Nakamura T, Ueda T. Increased levels of macrophage colony-stimulating factor, gamma interferon, and tumor necrosis factor alpha in sera of patients with Orientia tsutsugamushi infection. J Clin Microbiol 1997;35:3320e2. 22. Chierakul W, de Fost M, Suputtamongkol Y, Limpaiboon R, Dondorp A, White NJ, et al. Differential expression of interferon-gamma and interferon-gamma-inducing cytokines in Thai patients with scrub typhus or leptospirosis. Clin Immunol 2004;113:140e4. 23. Jerrells TR, Osterman JV. Host defenses in experimental scrub typhus: delayed-type hypersensitivity responses of inbred mice. Infect Immun 1982;35:117e23. 24. Seong SY, Huh MS, Jang WJ, Park SG, Kim JG, Woo SG, et al. Induction of homologous immune response to Rickettsia tsutsugamushi Boryong with a partial 56-kilodalton recombinant antigen fused with the maltose-binding protein MBP-Bor56. Infect Immun 1997;65:1541e5. 25. Kim MJ, Kim MK, Kang JS. Orientia tsutsugamushi inhibits tumor necrosis factor alpha production by inducing interleukin 10 secretion in murine macrophages. Microb Pathog 2006;40: 1e7. 26. Ha ¨rtel C, Bein G, Mu ¨ller-Steinhardt M, Klu ¨ter H. Ex vivo induction of cytokine mRNA expression in human blood samples. J Immunol Methods 2001;249:63e71.
www.elsevierhealth.com/journals/jinf
Kinetics of inflammatory cytokines in patients with scrub typhus receiving doxycycline treatment Doo Ryeon Chung*, Young Seok Lee, Seung Soon Lee Division of Infectious Diseases, Department of Internal Medicine, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, 896 Pyeongchon-dong, Dongan-gu, Anyang-si, Gyeonggi-do 431-070, South Korea Accepted 19 September 2007 Available online 31 October 2007
KEYWORDS Scrub typhus; Orientia tsutsugamushi; Cytokines; TNF-a; Doxycycline
Summary Objectives: Animal models have suggested that various cytokines and chemokines play an important role in host defense against Orientia tsutsugamushi, however, human data are limited. We investigated the kinetics of inflammatory responses in patients with scrub typhus receiving doxycycline. Methods: Amongst patients being treated for scrub typhus, serially collected sera were tested for TNF-a, IFN-g, IL-2, IL-4, IL-5, and IL-10 by immunoassay. The mRNAs encoding TNF-a, IL-1b, IL-6, IL-8, IFN-g, IL-12, IL-2, IL-4, IL-5, and IL-10 were measured by semiquantitative reverse transcription-PCR. Results: The concentrations of TNF-a, IFN-g, and IL-10 of patients prior to doxycycline treatment were significantly higher than those of healthy volunteers. They decreased markedly within 24 h after starting doxycycline. The mRNAs for IL-1b, TNF-a, IL-6, IFN-g, and IL-10 were highly expressed. Expression of mRNAs for IL-1b, IFN-g, and IL-10 decreased at day 2e7 of doxycycline treatment. Conclusions: Inflammatory cytokines including TNF-a, IL-1b, and IL-6 are markedly upregulated in patients with scrub typhus. Doxycycline treatment rapidly reduces the production of these cytokines, corresponding to the early defervescence after the start of the treatment. The profiles of T cell-derived cytokines in patients with scrub typhus do not follow typical Th1 or Th2 patterns. ª 2007 The British Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction * Corresponding author. Present address: Division of Infectious Diseases, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Gangnam-gu, Seoul 135-710, South Korea. Tel.: þ82 2 3410 0323; fax: þ82 2 3410 0041. E-mail address: [email protected] (D.R. Chung).
Scrub typhus, which is an endemic rickettsial disease in Asian countries, is characterized by high fever of sudden onset, headache, myalgia, rash, and eschar at a mite bite site.1,2 Orientia tsutsugamushi, causing this disease, infects a kind of cells such as endothelial cells, macrophages, neutrophils,
0163-4453/$30 ª 2007 The British Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jinf.2007.09.009
Inflammatory cytokines in scrub typhus and lymphocytes. It proliferates in the cytoplasm, induces local inflammation, and then disseminates to whole body and provokes a disease.3 Scrub typhus shows variable clinical presentations in terms of severity from mild and self-limiting to fatal.4 Some cases manifest as severe diseases accompanying serious complications such as coma, meningoencephalitis, interstitial pneumonitis, acute respiratory distress syndrome, myocarditis, or septic shock.5e13 Such a diversity might be explained as the result of variable interactions between virulence factors of O. tsutsugamushi and immune response of the host. Animal experiments have revealed that O. tsutsugamushi strains exhibit different virulence according to their serotypes,14 however, even among the strains of the same serotype the pathogenicity of O. tsutsugamushi is also influenced by the host factors.15 That is, clinical manifestations of scrub typhus depend not only on the virulence factors of organisms, but also on the immune response of the infected persons. In fact, although activation of the immune system of the infected host is one of the host defense mechanism, exaggerated immune response itself can result in a serious clinical deterioration. Most studies on immunological response to infection by O. tsutsugamushi have been based on in vitro experiments or animal models. Macrophages and T lymphocytes have been suggested to play an important role in host defense to infection with O. tsutsugamushi, however, the role of host immune response in the pathogenesis of scrub typhus has not been clearly documented.4 Previous reports have suggested that various chemokines and cytokines are produced by the stimuli of O. tsutsugamushi and these play an important role in the pathogenesis of this acute febrile illness.16e20 However, there have been a limited number of reports on human data.21,22 A report from Thailand showed elevated levels of IFN-g and IFNg-inducing cytokines in 28 patients with scrub typhus and suggested a strong involvement of the cell-mediated immune response in host defense against O. tsutsugamushi.22 In order to further understand the pathogenesis of scrub typhus, it is essential to clearly delineate the immune response in the patients infected by O. tsutsugamushi and determine the role of this immune response in the disease manifestations. We investigated the kinetics of inflammatory and T cell responses in the patients with scrub typhus before and after doxycycline treatment in order to determine the role of host immune response in the pathogenesis of scrub typhus.
Materials and methods Patients Patients with the diagnosis of scrub typhus were prospectively enrolled at the Hallym University Sacred Heart Hospital in Anyang-si, Korea, during the period from September to December 2003. Patients with a characteristic eschar, fever, and maculopapular rash, who had IgG antibody titer greater than 1:80 to O. tsutsugamushi or 4-fold rising antibody titer in the repeated serum, were included in this study. Patients younger than 18 years or patients who had already received doxycycline treatment before
45 screening for this study were excluded. Demographic and clinical information including daily peak body temperatures were analyzed. Venous blood was collected from the enrolled patients at four time points including before doxycycline was given, at day 1, day 2e3, and day 4e7. Sera were separated and stored at 80 C until analyzed. For isolation and purification of RNA, we collected the blood in RNA-stabilizing tubes (PAXgene Blood RNA Tubes; Qiagen, Inc., USA). Total RNA was extracted using blood RNA isolation kit (Qiagen, Inc., USA), and stored at 80 C. Eight healthy volunteers provided their serum as control. All these procedures were in accordance with the ethical standards of the hospital and with the Helsinki Declaration. Informed consent was obtained from the patients.
Measurement of cytokine concentration in serum Serum concentrations of TNF-a, IFN-g, IL-2, IL-4, IL-5, and IL-10 were determined by a solid phase sandwich ELISA using monoclonal antibodies (BioSource, Inc., USA) in accordance with the manufacturer’s protocols. The concentration of each cytokine was measured in duplicate.
Semiquantitative analysis for cytokine mRNA expression in blood Expression of mRNA transcripts encoding TNF-a, IL-1b, IL-6, IL-8, IFN-g, IL-12, IL-2, IL-4, IL-5, and IL-10 was determined by semiquantitative reverse transcription-PCR (RT-PCR) method in total RNA samples extracted from patients’ blood. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was tested as a control housekeeping gene for semiquantitative analysis. Reverse transcription and amplification of cDNA was done using cDNA amplification kit (Invitrogen, Inc., USA) according to the instructions of the manufacturer. Multiplex PCR for the genes encoding various cytokines as above was performed using a commercial kit (BioSource, Inc., USA). Mixed primer sets in the kit were used. PCR products were separated by electrophoresis on a 2% agarose gel and stained with ethidium bromide. Gels were viewed and photographed under UV light illumination. Digital gel photographs of the stained PCR products were taken under UV exposure by using digital camera (Nikon Coolpix 990). Cytokine cDNA bands were determined as the integrated area (pixels) of the band intensities by densitometry analysis with ImageJ version 1.33 (National Institutes of Health, USA). The numerical values for cytokine cDNA band intensities were presented as ratios relative to band intensities of GAPDH.
Statistical analysis Comparisons of continuous variables were made by using Student’s t-test or ManneWhitney U-test. Statistical analysis was performed with use of SPSS Software Package Release 11.0 (SPSS Inc., USA). We considered differences significant at P < 0.05.
46
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Results Demographic and clinical characteristics of the patients A total of 22 patients were enrolled (Table 1). All the patients received oral doxycycline for 7 days. The average of daily peak body temperature in the patients decreased from 38.5 C (S.D. 0.9 C) to 37.5 C (S.D. 0.8 C) within 24 h after the first dose of doxycycline (P < 0.001) (Fig. 1). Two patients (9.1%) had septic shock, and meningoencephalitis was complicated in two patients (9.1%). Five patients (22.7%) had bilateral pneumonia. Elevation of serum LDH and CRP was observed in most patients (Table 1). Severe cases complicated with shock, meningoencephalitis, or pneumonia showed a higher median level of CRP compared with non-severe cases (111.5 vs. 33.8; P Z 0.040, ManneWhitney U-test). There was no significant difference in white blood cell (WBC) count, platelet, and lactate dehydrogenase (LDH) level between two groups. All the patients were cured without any long-term complications.
Cytokine levels in serum following doxycycline treatment
Table 1 Demographic and clinical characteristics of the patients (n Z 22) with scrub typhus Age in years Female gender, number (%) Days from onset Shock, number (%) Pneumonitis, number (%) Meningoencephalitis, number (%) Laboratory findings WBC (/mm3) Platelet (/mm3) ALT (IU/L) Bilirubin (mg/dl) Albumin (mg/dl) LDH (IU/L) Creatinine (g/dl) CRP (mg/dl)
U-test). However, IFN-g and IL-10 showed no significant difference (Table 2).
Expression of mRNA encoding cytokines in blood following doxycycline treatment
The concentrations of TNF-a, IFN-g, and IL-10 of the patients prior to doxycycline treatment were significantly higher than those of healthy volunteers (Fig. 2). These levels decreased markedly within 24 h after starting doxycycline treatment, although they were still higher than those of volunteers until day 3. The concentrations of IL-2 and IL-4 did not show any significant change before and after the doxycycline treatment. IL-5 was not detectable in both the patients and volunteers. The median TNF-a level of severe cases complicated with shock, meningoencephalitis, or pneumonitis was significantly higher than that of non-severe cases (96.2 vs. 20.1; P Z 0.003, ManneWhitney
Characteristics
Figure 1 Change of peak body temperature in patients with scrub typhus after the first dose of doxycycline (*P < 0.001 compared to peak temperature prior to start of doxycycline; n Z 22).
Mean (S.D.) 58 12 7.2 2 5 2 7827.3 125.6 84.8 0.9 3.2 821.5 1.0 70.4
(18) (54.5) (3.7) (9.1) (22.7) (9.1) (4164.7) (67.6) (61.2) (1.4) (0.5) (244.5) (0.2) (54.6)
Semiquantitative analyses for the expression of mRNA encoding inflammatory cytokines and Th1/Th2 cytokines for the patients with scrub typhus were performed (Fig. 3). Relatively high expression of mRNAs for IL-1b, TNF-a, IL-6, IFN-g, and IL-10 was observed before receiving doxycycline treatment (Fig. 4). IL-1b mRNA expression decreased significantly during the period of day 2e7 after the first dose of doxycycline treatment (P Z 0.001). Expression of TNF-a mRNA at day 2e7 was slightly lower compared to those at day 0 or day 1. IL-6 mRNA expression increased until day 1, however, its expression at day 2e7 was significantly lower than that before treatment (P Z 0.016). IFNg and IL-10 also showed higher mRNA expression at day 1 (P Z 0.004 and P Z 0.035, respectively), however, their expression decreased back at day 2e7. Expression of mRNA encoding IL-2 or IL-4 was very low although IL-4 mRNA expression at day 1 was transiently higher compared to that at day 0.
Discussion This study shows that the production of inflammatory cytokines such as TNF-a, IL-1b, and IL-6 is increased in the patients with scrub typhus. Interestingly, the increased production of these inflammatory cytokines decreased rapidly within 1 or 2 days after starting doxycycline treatment. We have also found that profiles of T cellderived cytokines in these patients do not follow typical Th1 or Th2 patterns. IFN-g, one of the Th1 subset cytokines, showed a high level before antibiotic treatment, and its level has markedly declined following the start of doxycycline treatment. IL-2, another classic Th1 subset T cell-derived cytokine, however, did not show any significant change in these patients. IL-10, one of the Th2 subset
Inflammatory cytokines in scrub typhus
47
Figure 2 Time kinetics of the cytokines in serum of the patients with scrub typhus after doxycycline treatment. Cytokines were measured in duplicate for each patient. (A) TNF-a, (B) IFN-g, (C) IL-2, (D) IL-10, (E) IL-4 (n Z 22).
T cell-derived cytokines, showed higher concentration compared to those of healthy volunteers, and it has rapidly decreased within 24 h after the first dose of doxycycline. IL-4 and IL-5 did not show any significant change in their concentrations or mRNA expression in the blood of the patients regardless of doxycycline treatment. Cytokine profile featured by increased production or upregulation of TNF-a, IL-1b, and IL-6 was found to be a major inflammatory response in the patients with scrub typhus before receiving adequate antibiotic treatment. This explains well that high fever is a chief complaint in
most patients with scrub typhus, because these cytokines play an important role in the generation of fever in humans. In addition, rapid reduction of TNF-a, IFN-g, and IL-10 production following the start of doxycycline treatment was in accordance with the decrease of peak temperature in these patients in the aspect of time. In fact, a rapid defervescence in response to doxycycline treatment in the patients with scrub typhus is a characteristic finding to support the clinical diagnosis of this disease. Our result is contradictory with the previous report from Japan that has revealed higher levels of TNF-a in the patients in
48
D.R. Chung et al.
Table 2 Comparison of cytokine levels between severe cases with shock, meningoencephalitis, or pneumonitis and non-severe cases Cytokines Median (range) Severe (n Z 6) TNF-a IFN-g IL-10 a
Non-severe (n Z 16) P a
96.2 (21.6e336.8) 20.1 (0e134.3) 10.1 (0e36.2) 3.25 (0.3e31.4) 17.6 (3.7e95.8) 6.25 (1.7e17.4)
0.003 0.641 0.098
ManneWhitney U-test.
convalescent stage than in acute stage of scrub typhus.21 TNF-a is an important cytokine mediating variable cellmediated immune responses to infection and plays an essential role in host defenses to intracellular pathogens. Previous studies have showed that heat-stable molecule of O. tsutsugamushi is responsible for the induction of TNF-a production.18
A few studies have suggested a role of specific T lymphocytes, especially Th1 cells, in host defense to O. tsutsugamushi,23,24 although there have been very limited data in humans. In our study, the patterns of cytokine production or expression in the patients with scrub typhus prior to doxycycline treatment were not typical for either Th1-type or Th2-type immune response. This can be a result of overlapped Th2 response following after inflammatory reaction and Th1 immune response elicited by O. tsutsugamushi infecting the host. Other possibility is that IFN-g might have been mainly produced by other inflammatory cells such as macrophages or natural killer cells rather than by T cells. Our finding that IL-2 production or expression was not significant in the patients with scrub typhus supports it. Furthermore, the importance of macrophages in the pathogenesis of scrub typhus has already been addressed through previous in vitro studies that revealed increased production of TNF-a and IL-6 as well as increased mRNA expression for various chemokines in murine macrophage cell line.17,18 It has been suggested that inhibitory cytokines
Figure 3 Expression of cytokine mRNA from the blood of a patient with scrub typhus. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the control for the semiquantitative measurement.
Inflammatory cytokines in scrub typhus
49
Figure 4 Time kinetics of cytokine-encoding mRNA expression in the blood of the patients with scrub typhus (*P < 0.05 compared to data at day 0; n Z 20).
including IL-10 and TGF-b are induced by O. tsutsugamushi to inhibit the production of the inflammatory cytokines in order to survive inside hostile macrophages.16,25 Rapid decline in IL-10 production after doxycycline treatment in our study is thought to have been followed after decrease of inflammatory cytokines including TNF-a and IFN-g. Limitation in our study is that time trends of mRNA expression do not exactly match with those of cytokine concentrations in the blood. Variability in the data of mRNA seems to be attributable to in vitro induction of mRNA expression.26 Although we tried to minimize such technical bias by collecting the blood into the RNA-stabilizing tubes, we expect that we could not be free from it. We performed a semiquantitative RT-PCR to measure mRNA expression
instead of quantitative RT-PCR. Semiquantitative method also limited precise comparison of mRNA expression between different time points. Another limitation is that we did not measure mRNA expression in healthy volunteers, so we could not compare mRNA expression between patients and healthy volunteers. To our knowledge, this is the first study to investigate time kinetics of cytokines in the patients with scrub typhus. Especially, we tried to explain the mechanism of rapid defervescence after starting doxycycline treatment by the change of cytokine production in the patients. We found that the production of inflammatory cytokines such as TNF-a, IL-1b, and IL-6 is increased in the patients with scrub typhus and their rapid decline coincides with early defervescence
50 following doxycycline treatment. Besides, profiles of T cellderived cytokines do not follow typical Th1 or Th2 patterns.
Conflict of interest The authors have no conflicts of interest.
Acknowledgments We thank Sun Hee Ahn for her excellent technical assistance. Financial support: This work was supported by Korea Research Foundation Grant (KRF 2003-003-E00101).
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