Alternative complement pathway deregulation is correlated with dengue severity

Abstract

Background: The complement system, a key component that links the innate and adaptive immune responses, has three pathways: the classical, lectin, and alternative pathways. In the present study, we have analyzed the levels of various complement components in blood samples from dengue fever (DF) and dengue hemorrhagic fever (DHF) patients and found that the level of complement activation is associated with disease severity.

Methods and results: Patients with DHF had lower levels of complement factor 3 (C3; p = 0.002) and increased levels of C3a, C4a and C5a (p<0.0001) when compared to those with the less severe form, DF. There were no significant differences between DF and DHF patients in the levels of C1q, immunocomplexes (CIC-CIq) and CRP. However, small but statistically significant differences were detected in the levels of MBL. In contrast, the levels of two regulatory proteins of the alternative pathway varied widely between DF and DHF patients: DHF patients had higher levels of factor D (p = 0.01), which cleaves factor B to yield the active (C3bBb) C3 convertase, and lower levels of factor H (p = 0.03), which inactivates the (C3bBb) C3 convertase, than did DF patients. When we considered the levels of factors D and H together as an indicator of (C3bBb) C3 convertase regulation, we found that the plasma levels of these regulatory proteins in DHF patients favored the formation of the (C3bBb) C3 convertase, whereas its formation was inhibited in DF patients (p<0.0001).

Conclusion: The data suggest that an imbalance in the levels of regulatory factors D and H is associated with an abnormal regulation of complement activity in DHF patients.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Levels of complement factor 3 (C3); plotted values are nonlog for ease of comprehension.

(A) Plasma C3 levels in dengue-infected individuals during the acute phase (3–8 days after the onset of symptoms) and in healthy subjects. (B) Paired C3 levels of DHF patients during the acute and convalescent phases of dengue infection. The mean of C3 levels for acute and convalescence were 1.0+0.5 and 1.4+0.3 respectively. The C3 levels were measured by ELISA. Values shown are means and standard deviations. The p-values of two-tailed t-tests on the log-transformed data are indicated above each analysis.

Figure 2. Levels of complement factors C3a and C5a; plotted values are nonlog for ease of comprehension.

(A) Plasma levels of C3a and (B) C5a in dengue-infected individuals during the acute phase and in healthy subjects. C3a and C5a levels were measured by cytometric bead assay. Values shown are means and standard deviations. The p-values of two-tailed t-tests on the log-tranformed data are indicated above each analysis.

Figure 3. Levels of C4a; plotted values are nonlog for ease of comprehension.

Plasma levels of C4a in dengue-infected individuals during the acute phase and in healthy subjects. C4a levels were measured by cytometric bead assay. Values shown are means and standard deviations. The p-values of two-tailed t-tests on the log-transformed data are indicated above each analysis.

Figure 4. Levels of factors of the CP and LP; plotted values are nonlog for ease of comprehension.

(A) Plasma levels of C1q, (B) MBL oligomer, (C) C-reactive protein, and (D) immunocomplex-CIC-C1q in dengue-infected individuals at different stages of infection (acute and convalescent) and in healthy subjects. All complement proteins were measured by ELISA. Values shown are means and standard deviations. The p-values of two-tailed t-tests on the log-transformed data are indicated above each analysis.

Figure 5. Levels of AP factors; plotted values are nonlog for ease of comprehension.

(A) Plasma levels of factor D and (B) factor H in dengue-infected individuals and healthy subjects. (C) Paired comparisons of the factor D levels during the acute and convalescent phases in DHF patients. The mean levels of factor D on DF patients was 555+246 and 704+278 during acute and convalescence phases respectively (D) Paired comparisons of factor H levels during the acute and convalescent phases in DF patients. The mean levels of factor H on DHF patients was 138+49 and 210+83 during acute and convalescence phases respectively All complement proteins were measured by ELISA. Values shown are means and standard deviations. The p-values of two-tailed t-tests on the log-transformed data are indicated above each analysis

Figure 6. Mean log-ratios of concentration of factor H over the concentration of factor D for each diagnostic category, subtracted from the mean log-ratio in the healthy (ND) category.

A value of zero indicates that the levels of both factors were present in the same equilibrium as in healthy control subjects. Negative values indicate reduced AP regulation and favorable formation of (C3aBb) C3 convertase, whereas positive values indicate the opposite. The standard deviation bars give the interval (mean + err, mean –err), where err is the usual standard error of difference between sample means, except in the baseline (ND) category, where err is the standard error of the sample mean for that category.

Figure 7. Correlation between levels of factor H and C3.

Analysis of correlation was carried out between log-transformed levels of factor H and C3 dengue-infected patients during the acute phase.