240 Cytokine Release by Lipopolysaccharide-Stimulated Whole Blood from Patients with Typhoid Fever Deborah House,1,4 Nguyen T Chinh,3,5 Tran T Hien,3 Christopher P Parry,2,4 Nguyen T Ly,5 To S Diep,3 John Wain,1,4 Sarah Dunstan,4 Nicholas J White,2,4 Gordon Dougan,1 and Jeremy J Farrar2,4 Centre for Molecular Microbiology and Infection, Imperial College of Science, Technology, and Medicine, London, and 2Department of Tropical Medicine, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom; 3Cho Quan Hospital and 4Wellcome Trust Clinical Research Unit, Centre for Tropical Diseases, and 5Department of Infectious Diseases, Faculty of Medicine, University of Medicine and Pharmacy, Ho Chi Minh City, Vietnam The ex vivo cytokine response to lipopolysaccharide (LPS) of whole blood from patients with typhoid fever was investigated Tumor necrosis factor (TNF)–a release by LPS-stimulated blood was found to be lower during acute typhoid fever than after a course of antimicrobial therapy (P р 001) Ex vivo interleukin (IL)–1b, but not IL-1 receptor antagonist, release was also depressed during the acute stage of typhoid fever Low ex vivo production of TNF-a was associated with delayed recovery No association was found between the TNFA-308 promoter polymorphism and LPS-induced TNF-a release, either during an active infection or after treatment In acute typhoid fever, the ability of peripheral blood leukocytes to release proinflammatory cytokines in response to an inflammatory stimulus is depressed, and this may contribute to delayed recovery following antibiotic treatment Typhoid fever is a human systemic illness caused by an infection with the gram-negative bacterium Salmonella enterica subspecies serotype Typhi The symptoms of typhoid fever are diverse and include fever, abdominal pain or discomfort, myalgia, and headache [1, 2] In the absence of antimicrobial therapy, the signs and symptoms worsen progressively over 2–3 weeks and may culminate in potentially lethal ileal perforation or hemorrhage Patients treated with an appropriate antibiotic recover, usually, within week [3] Studies with Salmonella Typhimurium, which causes a typhoidlike illness in susceptible (NrampϪ/Ϫ) mice, have shown that the proinflammatory cytokine tumor necrosis factor (TNF)–a is involved in both the clearance of Salmonella during a primary infection and the protective immune response against a secondary challenge [4–7] There is increased production of proinflammatory cytokines (TNF-a, interleukin [IL]–1b, and IL-6) and cytokine antagonists (IL-1 receptor antagonist [IL-1ra] and soluble TNF-a receptor) in some patients with typhoid fever [8–10], and associations between circulating cytokine levels and both Received 19 December 2001; revised 27 March 2002; electronically published July 2002 Informed consent was obtained from the individuals admitted into the study, which was approved by the Ethical and Scientific Committee of the Centre for Tropical Diseases, Ho Chi Minh City, Vietnam Financial support: Wellcome Trust United Kingdom (programme grant 035783) Reprints or correspondence: Dr Deborah House, Centre for Molecular Microbiology and Infection, Imperial College of Science, Technology, and Medicine, Exhibition Rd., South Kensington, London SW7 2AZ, United Kingdom (d.house@ic.ac.uk) The Journal of Infectious Diseases 2002; 186:240–5 ᭧ 2002 by the Infectious Diseases Society of America All rights reserved 0022-1899/2002/18602-0013$15.00 disease severity [10] and response to treatment [8] have been documented Of interest, the ability of circulating leukocytes to secrete proinflammatory cytokines in response to lipopolysaccharide (LPS) is reduced in persons with naturally acquired Salmonella Typhi infection [9] Similarly, the proliferative response of spleen cells to polygenic stimuli is reduced in mice infected with Salmonella Typhimurium [11–14] Using an ex vivo whole blood stimulation assay, we investigated TNF-a release in LPS-stimulated blood from patients with typhoid fever TNF-a levels were determined both during the active infection and following a course of antibiotic treatment and were compared with those of LPS-stimulated blood from healthy control subjects The relationship between TNFa secretion and both response to treatment and the genetic background of the host was investigated Patients and Methods Patients and isolates The study was conducted at the Centre for Tropical Diseases, an infectious diseases referral hospital in Ho Chi Minh City, Vietnam Venous blood was obtained from patients with nonsevere, blood culture–confirmed typhoid fever who were enrolled into treatment studies between 1997 and 1999 [15] The antibiotic susceptibility pattern of the Salmonella Typhi isolates was determined by using National Committee for Clinical Laboratory Standards guidelines [16] The following treatment regimens were used in the study: ofloxacin, 200 mg twice a day for days (n p 27 patients); pefloxacin, 0.4 g twice a day for days (n p 10 patients); or azithromycin, g once a day for days (n p 23 patients) All treatments were given orally Ex vivo whole blood stimulation Venous blood was collected from patients with typhoid fever on separate occasions: imme- JID 2002;186 (15 July) Cytokine Release by LPS-Stimulated Blood in Typhoid Fever diately before the start of antimicrobial therapy (day 1) and on the fourth and seventh day after the start of treatment Venous blood was collected on a single occasion from a group of healthy adult control subjects who were working in the hospital at the time of the study (n p 18) Blood samples were collected into heparin tubes between and 10 A.M and were kept at ambient temperature until processed (within h of collection) Aliquots of whole blood (1 mL) were placed in tubes containing either mg/mL of Escherichia coli LPS (O55:H5; Sigma) diluted in endotoxin-free water or an equivalent volume of diluent (negative control) and were incubated at 37ЊC for 24 h Following stimulation, the blood samples were centrifuged to pellet the cells (2000 rpm, 10 min), and the plasma was harvested and stored at рϪ20ЊC until assayed Cytokine detection by ELISAs Plasma cytokine levels were determined by sandwich ELISA, using cytokine-specific mouse monoclonal antibodies and recombinant human cytokines purchased from Pharmingen (TNF-a) and Cambridge Bioscience (IL1b and IL-1ra) ELISAs were optimized according to the manufacturers’ instructions All procedures were performed at ambient temperature, unless otherwise stated All other ELISA reagents were purchased from Sigma Ninety-six-well flat-bottomed microtiter plates (Nunc Maxisorb) were coated overnight at 4ЊC with 50 mL of capture antibody (1 mg/mL) in 0.1 M NaHCO3 (pH 8.2) The plates were washed times with 0.95% NaCl containing 0.05% Tween 20 Vacant binding sites on the wells were blocked for h with 100 mL of PBS containing 3% bovine serum albumin (BSA; 3% BSA/PBS) The plates were washed a second time, and 50 mL of the appropriate standard or plasma sample diluted in 3% BSA/PBS was added to the appropriate wells (blank wells contained diluent alone) The plates were incubated for a further h The plates were then washed, and 50 mL of biotin-conjugated detecting antibody (1 mg/mL) was added to each well, after which the plates were incubated for hour The plates were washed, and 50 mL of avidin–alkaline phosphatase conjugate diluted 1:4000 in Tris-buffered saline containing 0.1% BSA was added to each well for 30 Following a final wash, 50 mL of p-nitrophenyphosphate (1 mg/mL) was added to each well, and the plates were incubated in the dark for 45–60 The absorbance at 405 nm (reference filter, 450 nm) was determined, using an automated plate reader (BioRad) Plasma samples were diluted 1:1 for the TNF-a ELISA and 1: for the IL-1b and IL-1ra ELISAs A series of standards was included on each plate Both standards and plasma samples were assayed in duplicate, and the mean absorbance was calculated The concentration of cytokine in the samples was determined by interpolation All samples from an individual patient were assayed on the same plate, and ELISAs were performed in a single run to minimize interassay variation Net LPS-induced cytokine secretion was taken as the concentration of the cytokine in the plasma from the LPS-stimulated blood minus the concentration in the plasma of the negative control (spontaneous release) TNFA-308 genotyping The polymorphism in the promoter region of TNFA-308 was genotyped by polymerase chain reaction (PCR) restriction fragment–length polymorphism, using the following primer pairs: 5′ AG CAA TAG GTT TTG AGG GCG AT 3′ and 5′ TCC TCC CTG CTC CGA TTC CG 3′ In total, ∼100 ng of genomic DNA was added to a 12.5-mL PCR reaction containing 30 ng of each primer, 0.25 mM of each dNTP, 1.5 mM of 241 MgCl2, and 0.15 U of Biotaq polymerase (Bioline) PCR cycling conditions were as follows: 94ЊC for min, 30 cycles of 94ЊC for min, 60ЊC for min, and 72ЊC for min, and 72ЊC for once The undigested TNFA-308 PCR product was 107 bp (allele 2, A), and after digestion with 10 U of NcoI, fragments of 87 bp and 20 bp were generated (allele 1, G) DNA fragments were separated on 4% agarose gel Allelic frequencies were determined by the formula n/2N, where n equals the number of times a particular allele is represented and N equals the number of individuals tested (i.e., 2N p the total no of chromosomes) Statistics Statistical analyses were performed with the computer package SPSS for Windows (SPSS Benelux) The following nonparametric tests were used: the Wilcoxon signed-rank test, Mann-Whitney U test, and x2 test with Yates’s correction Results Patients The aim of the study was to assess cytokine release in LPS-stimulated blood from patients with typhoid fever Sixty adults with culture-confirmed typhoid fever were recruited (34 men and 26 women), with a median (interquartile range [IQR]) age of 25 (19–32) years The median (IQR) length of illness was 10 (7.25–14) days All patients were febrile on admission to the study, with a median (IQR) admission temperature of 40ЊC (39.8ЊC–40.5ЊC) The median (IQR) fever clearance time (i.e., time from start of controlled antibiotic treatment to when oral temperature fell below 37.5ЊC and remained so for у24 h) was 114 (84–150) h Nineteen patients were still febrile after completion of antibiotic treatment (day 7) Of these, 12 patients (63%) were subsequently shown to be infected with nalidixic acid–resistant (NAR) Salmonella Typhi isolates Fortyfour (56%) of the Salmonella Typhi isolates were multiply drug resistant, with MIC values 512 mg/L for ampicillin, cotrimoxazole (trimethoprim ϩ sulphamethoxamole), and chloramphenicol (table 1) Whole blood was also collected from healthy adult control subjects (n p 18 ; men and 11 women) who were working in the hospital at the time of the study Their median (IQR) age was 37 (29–44) years Table Treatment regimen for patients with typhoid fever in relation to the drug susceptibility pattern of the infecting Salmonella serotype Typhi isolate Drug sensitivity pattern of a Salmonella Typhi isolate Fully sensitive MDR, NAS MDR, NAR Non-MDR, NAR Not determined a No of patients treated with No of patients Ofloxacin 11 22 22 11 11 0 b c Pefloxacin Azithromycin 3 1 d Fully sensitive, sensitive to ampicillin, cotrimoxazole, choramphenicol, and nalidixic acid; MDR, resistant to ampicillin, cotrimoxazol, and chloramphenicol; NAR, resistant to nalidixic acid; NAS, sensitive to nalidixic acid b Dose, 200 mg orally times per day for days (n p 27) c Dose, 0.4 g orally times per day for days (n p 10) d Dose, g orally once per day for days (n p 23) 242 House et al Ex vivo cytokine release in LPS-stimulated whole blood from patients with typhoid fever The spontaneous release of TNFa, IL-1b, and IL-1ra was similar on each of the study days (P 05, Wilcoxon signed-rank test; data not shown) Plasma levels of the cytokines were elevated in whole blood stimulated with LPS for 24 h, both blood taken from patients with typhoid fever and blood from healthy control subjects Levels of LPSinduced TNF-a (figure 1A) and IL-1b (figure 1B) release in blood collected from patients with typhoid fever before starting antibiotic therapy (day 1) were decreased significantly, compared with those seen in blood collected during or following treatment (P р 002, Wilcoxon signed-rank test) or in blood collected from the control group (P р 001, Mann-Whitney U test) LPS-induced IL-1ra release in LPS-stimulated blood collected from the patients with typhoid fever was similar on the study days (P 05, Wilcoxon signed-ranked test) and was similar to that of the control group (figure 1C; P 05, MannWhitney U test) Ex vivo TNF-a release and response to treatment Previous studies of patients with bacterial infections have shown an association between ex vivo cytokine secretion and disease severity or outcome [9, 17, 18], and TNF-a has been shown to be important in the control of bacterial growth in the murine model of typhoid fever [4, 7] We were, therefore, interested in the relationship between LPS-induced TNF-a release and response to antimicrobial therapy, as assessed by fever clearance time, for the patients with typhoid fever Using the lower 95% confidence interval of the ex vivo TNF-a response to LPS of blood from the control group as a cutoff, we grouped the patients with typhoid fever as having either a “normal” (у7200 pg/mL; n p 16) or a “low” (!7200 pg/mL; n p 44) ex vivo TNF-a response to LPS on admission to the study (i.e., on day 1) Since fever clearance time is dependent on the type of antimicrobial therapy [15], the data were stratified according to antibiotic treatment (fluoroquinolone [ofloxacin or pefloxacin] or azithromycin; table 1) Of the 37 patients with typhoid fever treated with fluoroquinolones, 24 were in the low response group and 13 in the normal response group These groups were similar in regard to length of illness and total white blood cell counts (table 2), both of which might have affected the ex vivo cytokine response The patients with typhoid fever in the normal response group had a shorter fever clearance time than patients in the low response group (median [IQR], 60 [45–90] h and 144 [103–232] h, respectively; P ! 001, Mann-Whitney U test; figure 2) This did not appear to be due to differences in antimicrobial therapy or in the sensitivity of the infecting isolate to quinolones, since the proportion of patients treated with ofloxacin or infected with NAR Salmonella Typhi was similar for the groups (table 2) Of the patients in the normal response group (n p 13), only one (7%) was febrile following days of antimicrobial therapy, compared with 12 (50%) of the 24 patients in the low response group Of interest, almost all (17/18) of the patients in the low response group had plasma JID 2002;186 (15 July) Figure Tumor necrosis factor (TNF)–a (top), interleukin (IL)–1b (center), and IL-1 receptor antagonist (IL-1ra) (bottom) release in lipopolysaccharide (LPS)–stimulated whole blood collected from patients with typhoid fever and from healthy control subjects (HC) Blood was collected from the patients with typhoid fever before (day 1) and after (day 4) and (day 7) days of antibiotic treatment Bars indicate median ע95% confidence interval *P р 001, vs day (Wilcoxon signed-rank test) and healthy control subjects (Mann-Whitney U test) JID 2002;186 (15 July) Cytokine Release by LPS-Stimulated Blood in Typhoid Fever Table Details of patients with typhoid fever who had a “normal” (у7200 pg/mL) or “low” (!7200 pg/mL) day ex vivo tumor necrosis factor (TNF)–a response to lipopolysaccharide (LPS) (patients treated with fluoroquinolones only) Patient characteristic Median (IQR) age, years Median (IQR) length of a illness, days Male:female ratio Ofloxacin:pefloxacin ratio c NAR :NAS ratio Median (IQR) white blood cell count, 103 cells/mL TNF-a release Normal (n p 13) Low (n p 24) 28 (19.3–32) 24 (19–30) 11 (6.5–13) 11:2 9:4 4:7 10 (7.3–13.5) b 12:12 18:6 11:13 (5–9.2) 6.7 (5–8.6) NOTE Patients were grouped on the basis of TNF-a release in LPS-stimulated blood taken before the start of antibiotic therapy (“normal” response or “low” response) The cutoff of 7200 pg/mL was the lower 95% confidence interval of TNF-a release in LPS-stimulated blood from healthy control subjects IQR, interquartile range a Duration of fever before start of treatment b P p 04, vs normal response group (x2 test with Yates’s correction) c Ratio of nalidixic acid–resistant (NAR) to nalidixic acid–sensitive (NAS) isolates Two isolates were not typed in the normal response group C reactive protein (CRP) levels у96 mg/mL, compared with only of patients in the normal response group It was not possible to perform similar analyses on the patients treated with azithromycin, as there were only patients in the normal response group Ex vivo TNF-a secretion and TNFA-308 polymorphism An earlier study demonstrated an association between TNFA-308 polymorphism and susceptibility to typhoid fever [19] To determine whether there was an association between this promoter polymorphism and the magnitude of the ex vivo TNF-a response to LPS, patients with typhoid fever were typed for the TNFA308 promoter polymorphism The allelic frequencies were 0.89 for allele (G) and 0.11 for allele (A), which are similar to those reported in the earlier study for persons from this region of Vietnam [19] Of the 59 patients genotyped, 45 were G/G homozygotes and 14 were G/A heterozygotes No difference in TNF-a release in LPS-stimulated blood taken from homozygotes and heterozygotes was seen before, during, or following treatment (P 05, Mann-Whitney U test; table 3) 243 this study, the ex vivo cytokine response of peripheral blood leukocytes from patients with typhoid fever was investigated We chose to use a whole blood stimulation assay rather than the more traditional cell isolation methods, because there is no depletion of specific cell populations, and the cells are maintained in an environment similar to that found in vivo The simple methods minimize both the risk of contamination and the risk of nonspecific activation of cells, particularly monocytes Our results show that TNF-a release in LPS-stimulated whole blood is reduced during a systemic infection with Salmonella Typhi IL-1b release was also reduced, which shows that this phenomenon extends to other proinflammatory cytokines The release of both TNF-a and IL-1b normalized following treatment, suggesting an acute disease-specific effect This supports the findings of a study from Indonesia [9] The mechanism(s) responsible for the reduced release of proinflammatory cytokines during infection is not known but may include a decreased number of circulating leukocytes and/or changes in the expression of LPS receptors on the surface of circulating leukocytes Alterations in the absolute number and proportion of different populations of circulating white blood cells have been documented in patients with typhoid fever [1, 21, 22]; however, most patients enrolled in the study reported here had total white blood cell counts within the normal range on admission Wyant et al [23] have shown that the number of peripheral blood mononuclear cells (PBMC) expressing the LPS receptor CD14 is reduced following incubation of the cells in vitro with purified flagella from Salmonella Typhi The same Discussion Cytokines play a major role in both pathogenesis and control of systemic Salmonella infections In typhoid fever, particular TNF-a haplotypes have been associated with both protection and susceptibility [19], and TNF-a has been shown to be an important component of the host immune response in the murine model of typhoid fever [4–7] Although TNF-a is important in the host immune response, this cytokine has also been shown to mediate much of the pathology associated with gramnegative bacterial infections [20], and an association between high plasma TNF-a levels and severity of disease has been described in children infected with Salmonella Typhi [10] In Figure Kaplan-Meir plot of fever clearance time for patients with typhoid fever treated with fluoroquinolones Patients were grouped on the basis of tumor necrosis factor (TNF)–a release in lipopolysaccharide (LPS)–stimulated blood taken before the start of antibiotic therapy: “normal” response (у7200 pg/mL [dashed line]; n p 13) or “low” response (!7200 pg/mL [solid line]; n p 24 ) The cutoff between normal and low response was the lower 95% confidence interval of TNF-a release in LPS-stimulated blood from healthy control subjects 244 House et al JID 2002;186 (15 July) Table Tumor necrosis factor (TNF)–a release in lipopolysaccharide-stimulated whole blood on treatment days 1, 4, and in relation to TNFA-308 genotype Genotype Homozygote (G/G) Heterozygote (G/A) NOTE Median (IQR) TNF-a release, pg/mL No of patients Day Day Day 45 14 2990 (865–9981) 2296 (1053–6250) 5970 (1563–10,654) 7940 (4050–9650) 7522 (4264–11,000) 8254 (6427–11,357) IQR, interquartile range group also found that PBMC coincubated with Salmonella Typhi or Salmonella Typhi antigens (flagella and LPS) have a reduced response to antigens and mitogens [24] Whether this phenomenon occurs in vivo during an infection with Salmonella Typhi and is responsible for the reduced release of proinflammatory cytokines reported here remains to be demonstrated IL-1ra release over 24 h was enhanced by LPS and was similar on all study days and similar to the release seen with blood from healthy control subjects, which suggests that the low TNFa and IL-1b responses were not due to an inability to secrete cytokines or to respond to LPS However, it is possible that the major cellular source of the cytokines differed It is also possible that soluble factors that inhibit or suppress ex vivo LPS-induced TNF-a release are present in the plasma of patients with typhoid fever, as has been reported for mycobacterial infections [17] Both IL-6 and IL-1ra have been shown to inhibit LPS-induced IL-1b and TNF-a, but not IL-1ra, release in vitro [25, 26], and both of these cytokines can be elevated in the blood of persons infected with Salmonella Typhi [8–10] In this study, we demonstrated an association between LPSinduced TNF-a release and fever clearance time in typhoid fever patients treated with fluoroquinolones Patients with a low ex vivo TNF-a response before the start of treatment had a longer fever clearance time than patients with a TNF-a response within or above the normal range (as defined by the range of responses seen with blood taken from healthy control subjects) The fever clearance time of patients treated with fluoroquinolones is dependent, in part, on the susceptibility of the infecting isolate to quinolones (measured best by susceptibility to NA [NAS]) [3] However, infection with NAR organisms did not account for the differences in the fever clearance time observed, since the proportion of NAR isolates was similar for the groups, and the difference in fever clearance time was still apparent when the analyses were performed on patients infected with either NAS or NAR Salmonella Typhi (data not shown) TNF-a has been shown to be important in the clearance of bacteria from the liver and spleen in murine enteric fever, and it is possible that a reduced TNF-a response impairs the host’s ability to clear an infection with Salmonella Typhi, resulting in a longer time to recovery Alternatively, the depressed release of TNF-a in response to LPS may result from having a more severe infection, possibly via the greater activation of anti-inflammatory pathways (e.g., activation of the hypothalamic-pituitary-adrenal axis) There are lines of evidence that support this latter hypothesis First, the ex vivo cytokine response to LPS was restored following treatment; and second, high serum CRP levels (у96 mg/L) were more common in patients with typhoid fever with a low ex vivo TNF-a response The suppression of peripheral proinflammatory cytokine release in response to inflammatory stimuli during an infection may actually be beneficial to the host, since overproduction of inflammatory cytokines, such as TNF-a, and the subsequent induction of other inflammatory mediators could result in hypotension, shock, and even death [20] The amount of TNF-a released in response to an inflammatory stimulus is dependent, in part, on the genetic background of the host A recent study of host genetic factors demonstrated an association between the TNFA*2-308 DRB1*0301 HLA class II haplotype and susceptibility to typhoid fever [19] The allelic frequencies of the TNFA-308 promoter polymorphism in the patients with typhoid fever in the present study were similar to those reported in this earlier study; however, no association between the magnitude of the TNF-a response to LPS and the TNFA-308 genotype was found The role of the TNFA-308 promoter polymorphism in the host’s TNF-a response remains unclear Some groups have shown the rare allele (TNF-2) to be a stronger transcriptional activator than the more common TNF-1 allele [27], resulting in higher TNF-a secretion in response to LPS in vitro [28, 29], whereas other groups have found no difference in transcription rates [30] or in TNF-a secretion [31] between the TNFA-308 alleles In this study, we have demonstrated that the release of TNFa by LPS-stimulated peripheral blood leukocytes is reduced in patients infected with Salmonella Typhi The depressed release of this cytokine is not due to an inability to release cytokines or to respond to LPS, since release of IL-1ra was not depressed during the infection, and the TNF-a response was restored following a course of antimicrobial therapy Patients in whom TNF-a release was below the normal range before treatment took longer to recover than patients in whom release was within or above the normal range Thus, TNF-a release by LPS-stimulated whole blood may predict response to treatment in patients infected with Salmonella Typhi Acknowledgments We wish to acknowledge the assistance of the following persons: N T T Quyen, P T Doan, and S Jones We thank the officials of the Centre for Tropical Diseases, Ho Chi Minh City, Vietnam, for facilitating these studies JID 2002;186 (15 July) Cytokine Release by LPS-Stimulated Blood in Typhoid Fever References Stuart BM, Pullen RL Typhoid: clinical analyses of three hundred and sixty cases Arch Intern Med 1946; 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(bottom) release in lipopolysaccharide (LPS)–stimulated whole blood collected from patients with typhoid fever and from healthy control subjects (HC) Blood was collected from the patients with typhoid. .. not shown) Plasma levels of the cytokines were elevated in whole blood stimulated with LPS for 24 h, both blood taken from patients with typhoid fever and blood from healthy control subjects Levels... correction Results Patients The aim of the study was to assess cytokine release in LPS-stimulated blood from patients with typhoid fever Sixty adults with culture-confirmed typhoid fever were recruited