The authors have declared that no competing interests exist.
Conceived and designed the experiments: CPS BW. Performed the experiments: THTN THQN TTV. Analyzed the data: THTN THQN TTV CPS. Contributed reagents/materials/analysis tools: THTD VNT JF KLP TLH SSW MW MLH BW CPS. Wrote the paper: THTN BW CPS.
Dysregulated immune responses may contribute to the clinical complications that occur in some patients with dengue.
In Vietnamese pediatric dengue cases randomized to early prednisolone therapy, 81 gene-transcripts (0.2% of the 47,231 evaluated) were differentially abundant in whole-blood between high-dose (2 mg/kg) prednisolone and placebo-treated patients two days after commencing therapy. Prominent among the 81 transcripts were those associated with T and NK cell cytolytic functions. Additionally, prednisolone therapy was not associated with changes in plasma cytokine levels.
The inability of prednisolone treatment to markedly attenuate the host immune response is instructive for planning future therapeutic strategies for dengue.
Dengue is an acute, mosquito-borne febrile illness and around 390 million cases occur annually in more than 100 countries. A host pro-inflammatory immune response is widely believed to contribute to the clinical complications that occur in some patients with dengue. Synthetic glucocorticoids, which are immunomodulatory agents commonly used in medicine, have been suggested as a therapy for dengue. We recently performed a randomized, controlled trial of early oral glucocorticoid therapy in 225 dengue cases in Vietnam, comparing a three day regimen of high (2 mg/kg) or low (0.5 mg/kg) dose prednisolone with placebo. Here, we report on immunological changes occurring during prednisolone therapy with a view to understanding the lack of clinical benefit by glucocorticoid therapy and to guide future intervention strategies for dengue. In whole-blood gene expression arrays we found 81 transcripts from 64 genes differentially abundant between high-dose prednisolone and placebo treated patients. Prominent were the genes associated with T and NK cell cytolytic functions. These results are a reminder that the mechanisms causally behind some of the complications of dengue (e.g. altered capillary permeability) are very poorly understood and represent a major knowledge gap in our understanding of disease pathogenesis that also undermines attempts to improve clinical management.
Dengue is an acute, mosquito-borne illness caused by any of the four types of dengue virus (DENV1-4). There are an estimated 390 million symptomatic and asymptomatic infections per year
Synthetic glucocorticoids are frequently employed as adjunctive therapy in disease states where the host immune response is thought to be a significant contributor to disease pathogenesis. We recently performed a randomized, controlled trial of early oral prednisolone therapy in 225 confirmed pediatric dengue cases
A randomized, placebo-controlled double-blind trial assessing the safety of early oral corticosteroid therapy in dengue patients was conducted at the Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam between August 2009 and January 2011 as approval from the Ethical Committee of the Ministry of Health of Vietnam (2407/QĐ-BYT) and the Oxford Tropical Research Ethics Committee (OxTREC 33-08) and has been reported elsewhere
Eleven cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IFNγ, TNFα and IP10) were quantified using a multiplex biometric immunoassay following the instructions of the manufacturer (Bio-Plex Precision Pro Assays, Human cytokine 10-Plex, Bio-Rad Inc., USA). The limits of detection were as follows: 0.23 (IL-1β), 0.84 (IL-2), 0.14 (IL-4), 1.5 (IL-5), 1.23 (IL-6), 0.96 (IL-10), 0.2 (IL-12p70), 1.19 (IL-13), 0.34 (IFN-γ), 0.14 (TNF-α) and 10 (IP10), all pg/ml.
The gene expression microarray assay, and the procedures for normalization and analysis of the microarray data were conducted as described elsewhere
A fluidigm system (Fluidigm Corp., USA) was used for realtime PCR (RT-PCR) validation of those differentially abundant transcripts identified in the gene expression microarray, using samples from the whole patient cohort and following the manufacturer's instructions. The delta Ct value for each gene was calculated by subtracting the Ct value of the gene of interest from the Ct value of 18S, the house-keeping gene.
All group comparisons of microarray data were based on ANOVA with correction for multiple testing with the Benjamini-Hochberg method, as implemented in the GeneSpring Software (Silicon Genetics). A fold change of 1.5 was defined as the cut-off for screening significant entities. We used multivariable linear regression modeling for all comparisons of PCR results, expressed as delta Ct values, between the treatment arms. For the overall comparison across treatment arms, a linear trend test was used. In view of the likely evolution of gene expression during the illness episode, and the known associations of many of the genes of interest to immune parameters, we adjusted for day of illness and the absolute neutrophil and lymphocyte counts. Additionally we adjusted for the pre-treatment value when examining within-patient changes in delta Ct over time. P values for testing of multiple PCR results were corrected using the Benjamini-Hochberg method. The relative expression ratios of the genes between the treatment arms were estimated using the delta delta Ct formula: R = 2−ΔΔCt with corresponding 95% confidence intervals based on the bootstrap. Genes with significantly different relative expression ratios between treatment arms (i.e. adjusted p<0.05 and 95%CI not including 1) were considered up or down regulated as appropriate.
Multivariable linear regression was used for all comparisons of log-transformed values of cytokine concentrations adjusting for day of illness and pre-treatment values and linear trend tests performed as described above. All the analyses were corrected for multiple tests using the Benjamini-Hochberg method. All analyses other than those pertaining to the microarray data were performed using R, version R2.13.2 (R Foundation for Statistical Computing, Vienna, Austria).
The GEO accession number for microarray data is GSE40165
Baseline characteristics for the first 123 dengue patients consecutively enrolled in the trial who formed the discovery cohort are described in
Shown is the ratio in abundance of transcripts measured by microarray and by RT-PCR for the 67 genes that distinguished high-dose prednisolone treated patients from placebo treated patients. Where results from multiple probes in the microarray were available for 1 gene, we used the mean result. Genes have been grouped and annotated according to their recognized biological functions. Median (IQR) times from a) the start of treatment and b) the most recent dose, to the time of the microarray sample were similar; 43 (42–43) hours and 23 (22–24) hours for the high-dose prednisolone recipients, and 43 (42–44) hours and 22 (22–23) hours for the placebo recipients.
Microarray population | RT-PCR population | |||||
(N = 123 |
(N = 223 |
|||||
Placebo | Low-dose | High-dose | Placebo | Low-dose | High-dose | |
(N = 40) | (N = 42) | (N = 41) | (N = 75) | (N = 74) | (N = 74) | |
11 (28) | 11 (26) | 16 (39) | 19 (25) | 21 (28) | 24 (32) | |
13 (11–14) | 11 (10–14) | 12 (10–13) | 13 (12–15) | 12 (11–14) | 12 (10–14) | |
0 (-) | 0 (-) | 1 (2) | 0 (-) | 1 (1) | 1 (1) | |
17 (42) | 14 (33) | 16 (39) | 28 (37) | 23 (31) | 29 (39) | |
23 (58) | 28 (67) | 24 (59) | 47 (63) | 50 (68) | 44 (59) | |
−5 (−6, −4) | −4 (−6, −4) | −5 (−6, −4) | −5 (−6, −4) | −4 (−6, −4) | −4 (−6, −4) | |
27 (68) | 27 (64) | 29 (71) | 41 (55) | 46 (62) | 49 (66) | |
11 (28) | 8 (19) | 3 (7) | 29 (39) | 17 (23) | 11 (15) | |
2 (5) | 5 (12) | 6 (15) | 4 (5) | 9 (12) | 10 (14) | |
0 (-) | 2 (5) | 3 (7) | 1 (1) | 2 (3) | 4 (5) | |
3.1 (2.5–4.3) | 4.0 (3.0–5.9) | 4.2 (2.7–5.8) | 3.8 (2.7–5.0) | 3.7 |
4.2 |
|
2.2 (1.3–3.1) | 2.7 (1.8–4.1) | 2.8 (2.0–3.6) | 2.6 (1.7–3.5) | 2.5 |
2.9 |
|
0.7 (0.5–0.9) | 0.7 (0.5–1.0) | 0.7 (0.5–1.0) | 0.7 (0.6–0.9) | 0.7 |
0.7 |
Continuous variables are summarized as median (IQR), categorical variables are summarized as number and frequency (%).
358 whole-blood RNA samples from the first 123 consecutively enrolled patients were included in the microarray experiment. 11 follow-up samples were missing.
600 samples from 223 patients (including the123 patients that were sampled for the microarray) were included in the RT-PCR validation study. (15 pre-treatment, 23 post-treatment and 31 follow-up samples were either missing or of poor quality and were therefore not included).
There was one missing value.
Fever day (median (IQR) refers to the day of enrolment relative to the day of defervescence, defined as fever day 0.
Concentrations of 11 cytokines and chemokines were measured in 636 serial plasma samples from 222 patients at the three time-points. Although cytokine/chemokine concentrations were within the detectable range in 98% of samples tested, their levels were not significantly elevated during the acute phase of illness compared to follow-up, and there were no significant differences between treatment groups 2 days after starting therapy (
Cytokine | Plasma concentration 2 days after starting therapy | Trend test |
|||
High-dose prednisolone | Low-dose prednisolone | Placebo | Multiplicative effect | Adjusted P | |
(Fever day |
(Fever day = −2 (−4, −2)) | (Fever day = −3 (−4, −2)) | (95%CI) | ||
IL-1β | 11.3 | 12.5 | 13.1 | 0.90 | 0.24 |
(8.5–15.1) | (10.7–16.3) | (10.2–18.3) | (0.75–1.07) | ||
IL-2 | 50.6 | 54.6 | 50.9 | 0.89 | 0.31 |
(37.6–66.6) | (40.2–69.5) | (38.4–74.6) | (0.72–1.11) | ||
IL-4 | 13.4 | 14.1 | 13.3 | 0.91 | 0.29 |
(10.2–17.2) | (10.2–16.9) | (10.0–19.0) | (0.77–1.08) | ||
IL-5 | 79 | 79 | 79.3 | 0.91 | 0.42 |
(62.6–101.1) | (63.3–92.0) | (64.6–96.4) | (0.73–1.14) | ||
IL-6 | 132.9 | 125.7 | 140.1 | 0.98 | 0.83 |
(93.3–183) | (106.4–162.4) | (102.3–199.6) | (0.80–1.20) | ||
IL-10 | 119.4 | 118.2 | 126.7 | 0.98 | 0.65 |
(80.8–166) | (96.7–152.6) | (98.4–167.4) | (0.92–1.05) | ||
IL-12p70 | 24 | 23.1 | 23.2 | 0.97 | 0.70 |
(16.0–32.4) | (16.0–30.1) | (15.8–30.2) | (0.82–1.14) | ||
IL-13 | 26.4 | 23.6 | 25.6 | 0.94 | 0.42 |
(17.3–40.1) | (17.1–40.1) | (18.5–36.1) | (0.80–1.10) | ||
IFN-γ | 56.5 | 58.1 | 62.2 | 0.97 | 0.48 |
(41.0–96.0) | (42.4–88.5) | (41.8–85.3) | (0.87–1.06) | ||
TNFα | 13.2 | 12.7 | 14.5 | 0.95 | 0.56 |
(10.6–17.6) | (10.2–16.2) | (10.0–18.1) | (0.780–1.13) | ||
IP-10 | 8398 | 8097.5 | 8099 | 1.06 | 0.14 |
(5,099.2–11,506.5) | (4,998.9–11,424.4) | (4,701.8–10,630.3) | (0.98–1.14) |
All data are presented as median (IQR) values.
Fever day (median (IQR) refers to the day of sampling relative to the day of defervescence, defined as fever day 0.
Trend test using multivariable linear regression of log transformed values adjusted for pre-treatment value and day of illness at enrolment. The effect corresponds to the estimated multiplicative difference between low-dose vs. placebo or high-dose vs. low-dose, respectively, which were assumed to be identical as a linear dose-response effect was estimated.
The current study was linked to a randomized controlled trial of early prednisolone therapy for dengue that demonstrated the safety of prednisolone but did not provide evidence of improved clinical or laboratory outcomes for patients
Dysregulated immune responses are widely believed to contribute to the pathogenesis of severe dengue
Changes in the whole-blood host gene expression profile occurred in patients that had received 2 mg/kg prednisolone (but not 0.5 mg/kg) compared to placebo-treated patients. This is to our knowledge the first ex vivo and “global’ investigation of the effect of prednisolone on the host immune response during an infectious disease. A striking finding, that was independent of the blood lymphocyte count, was the prednisolone-associated under-abundance of transcripts representing granzyme B (GZMB), granzyme H (GZMH), granulysin (GNLY), perforin (PRF1), Ksp37 (FGFBP2) and cathepsin W (CTSW), each of which is associated with the secretory and cytolytic activities of T and NK cells. Taken together, the diminished abundance of transcripts encoding T and NK effector proteins might suggest impaired anti-viral cytolytic responses during high-dose prednisolone therapy. However prolonged viremia levels were not observed in prednisolone-treated patients
Transcripts from neutrophils, independent of the absolute neutrophil count, were more abundant in high-dose prednisolone treated patients. This included three neutrophil markers (IL1R2, S100A12 and ORM1), all known to be corticosteroid induced
There are limitations to our study. We did not measure complement activation yet there is good evidence that complement is consumed and split products generated in the course of dengue and that this might be important to pathogenesis
(DOC)
We gratefully acknowledge the support of the patients and their families, as well as the doctors and nurses at the Hospital for Tropical Diseases for their help with the conduct of this study. We would like to thank Ling Ling and Ahmad Nazri Mohamed Naim (GIS, Singapore) for their help with the microarray and RT-PCR validation experiments.