Summary
Global incidence of dengue has drastically increased in the last few years. Despite the global morbidity and mortality associated with dengue infection, mechanisms of immune control and viral pathogenesis are poorly explored. Pancytopenias, along with increased oxidative stress, are salient clinical findings in severe dengue patients. Previously, we demonstrated significant differences of circulating immune complexes (CICs) among severe and non‐severe dengue patients. Accordingly, here we sought to determine the contributory role of affinity‐purified antibody‐bound CICs in dengue severity. To characterize intracellular oxidative stress induced by antibody‐bound CICs, 5‐(and‐6)‐chloromethyl‐2′‐7′‐dichlorodihydrofluorescein diacetate (DCFDA) was measured by flow cytometry. At the same time, CICs sensitized healthy red blood cells (RBC) and patients’ RBC morphology was determined by scanning electron microscopy and flow cytometry analysis. Erythrophagocytosis and ferritin levels were further determined in severe and non‐severe dengue patients. Our results showed that the severe patients had high titres of immunoglobulin (Ig)M‐bound CICs (P < 0·0001) in their sera, increased intracellular oxidative stress (P < 0·0001), high ferritin levels (P < 0·0001), altered morphology of RBC and finally enhanced erythrophagocytosis. This study shows for the first time that RBC morphology is altered in severe dengue patients. Taken together, this study suggests that the enhanced IgM‐bound CICs could contribute to the increased oxidative stress and act directly on RBC destruction of severe dengue patients, and is an important pathophysiological determinant. Hence, IgM‐bound CICs can serve as an important laboratory parameter to monitor dengue infection progression.
Keywords: CICs, dengue, erythrophagocytosis, oxidative damage, RBC
Introduction
Dengue (DENV) is an arboviral infection affecting people worldwide, with an increasing mortality rate 1, 2. The pathophysiology behind severe manifestations of the viral infection is poorly understood, hindering early diagnosis of cases which leads to fatal complications for the patient. The virus belongs to the genus Flavivirus, family Flaviviridae, transmitted to human hosts by the Aedes mosquito. Being a mosquito‐borne infection, it has a huge disease burden in developing tropical countries suffering from unplanned urbanization, inadequate public health and poor sanitization infrastructure 3. Four different serotypes of DENV, namely, DENV‐1, DENV‐2, DENV‐3 and DENV‐4, are circulating in most of the affected countries, but new serotypes are also being characterized. Unfortunately, all these four serotypes are capable of producing the more severe and potentially fatal dengue haemorrhagic fever and dengue shock syndrome 4, 5, 6, 7. Until the present there are no accurate methods for early prediction of disease severity, as cases of dengue with warning signs (DWWS) later develops into severe dengue (SD) due to the lack of a proper biomarker, which can earmark the potentially fatal from non‐fatal cases 7. The exact role of high levels of viraemia in the development of SD is unknown, and often depends upon the host immunology. Therefore, host immune factors which can act as severity biomarkers become more promising in this field. In the acute phase of DENV infection, the high viral load triggers an activated immune response in the form of cytokines and other inflammatory mediators 8. Several studies have demonstrated that there is over‐production of cytokines and adhesion molecules from endothelial and other immune regulatory cells during DENV infection, and this situation accelerates the humoral immune responses 9 leading to the production of circulating immune complexes (CICs) within the host’s blood circulation. It has been reported that these CICs play a crucial role in the pathogenesis of several diseases, such as chronic hepatitis 10, measles 11 and malaria 12, as well as in dengue 13 and chronic viral infections 14, 15. CICs formed by antigen and antibodies are the main feature, resulting in disease severity 16. Studies from our laboratory revealed that high levels of CICs are present in patients with dengue haemorrhagic fever compared to dengue fever 17. However, their role in disease pathogenesis remains elusive. Recent studies have demonstrated that red blood cells (RBC) also play a crucial role in immunity and control the physiological homeostasis in disease severity 18. Previous studies have demonstrated that erythrocytes generate microparticles in patients with dengue infections of different degrees of disease severity 16. Elevated levels of RBC‐derived microparticles directly bind with NS1 antigen‐bound CIC for its clearance. Further, studies have demonstrated that immune modifications, inflammation and oxidative stress are closely related. Oxidative stress arises when there is imbalance between oxidants and anti‐oxidants within the cell. RBCs are predisposed to oxidative damage due to the high cellular concentration of oxygen and haemoglobin, a potentially significant promoter for the oxidative processes 19. Although RBCs have well‐equipped anti‐oxidant machinery such as reduced glutathione, thioredoxin, ascorbic acid and vitamin E, they are continuously exposed to high levels of endogenous reactive oxygen species (ROS) such as superoxide (O2 –) peroxide(H2O2), which impair their function 18. RBC also contains nicotinamide adenine dinucleotide (NADH) oxidases, which can generate endogenous ROS for immune modulation 19.These free radicals also play an important role in RBC scavenging. It has been demonstrated that in inflammatory disease RBC structure has been demonstrated to be dramatically affected in the presence of high levels of oxidative stress parameters 20.
In the present study we investigated, for the first time to our knowledge, the role of plasma IgM‐bound CICs in inducing oxidative stress in RBCs of patients with dengue infection, thereby contributing towards disease severity.
Material and methods
Ethics statement
The Institutional Human Ethical Committee approved the study (ethical number CREC‐STM/275, dated 18 April 2015) and necessary blood samples were taken from healthy control and patients. Written informed consent was taken from enrolled patients and a healthy control population.
Collection of blood samples
Patients with dengue‐like illness (n = 3066) were screened and 4·0 ml of venous blood were collected by venipuncture and stored in ethylenediamine tetraacetic acid (EDTA) anti‐coagulant tubes. Plasma were separated by centrifugation at 400 g and subjected to non‐structural protein 1 (NS1) human membrane attack complex enzyme‐linked immunosorbent assay (MAC‐ELISA), IgM/IgG ELISA (Pan Bio, East Brisbane, Australia) and reverse transcription–polymerase chain reaction (RT–PCR). The study was carried out at the Calcutta School of Tropical Medicine, India from January 2016 to December 2016. Of 3066 suspected patients, 151 confirmed NS1/IgM were selected for our study; 90 were categorized as dengue without warning signs (DWOWS) and nine patients as SD; another 52 were DWWS. Additionally, 30 healthy donor (HD) subjects were enrolled for this study. The ratio of IgM to IgG (1·78) indicated secondary infection of dengue. Whole blood (1 ml) was also collected for the RBC study and the plasma were collected for the entire study subjects and stored at –20°C until further use.
Case definition
Suspected dengue cases were defined as patients with reported or documented with high fever of ≥38°C of 2–7 days’ duration and two or more symptoms or signs, i.e. headache, rash, eye pain, myalgia, arthralgia, hypotension, haemorrhage, thrombocytopenia (platelet count <50 × 103/cmm), etc. All clinically suspected dengue cases were categorized into the following three groups (WHO 2009) – (1) DWOWS: nausea, vomiting, rash, aches and pains, tourniquet test positive and leucopenia; (2) DWWS: abdominal pain or persistent vomiting, clinical fluid accumulation, mucosal bleed, lethargy; restlessness, liver enlargement >2 cm, increase in haematocrit with a rapid decrease in platelet count; and (3) SD: severe plasma leakage, leading to shock, fluid accumulation with respiratory distress, severe bleeding as evaluated by the clinician, severe organ involvement liver, heart and other organs.
Quantification of biochemical parameters
To characterize the pathophysiological condition of the study subjects a detailed biochemical analysis of the blood samples was measured using a standard autoanalyser (ERBA model no. EM360). These included plasma glutamic oxaloacetic transaminase (SGOT), plasma glutamic pyruvic transaminase (SGPT), globulin and albumin. Additionally, white blood cell (WBC), RBC, blood haemoglobin (HB), haematocrit (HCT) and platelet (PLT) counts were measured using an automated cell counter (SYSMEX model no. KX100). The tests were carried out as per the manufacturer’s instructions. All the samples were tested in triplicate and the mean value taken for analysis.
Quantification of IgM‐bound CICs and DENV antigen
CICs were isolated from each category of dengue‐infected patients’ plasma by incubating with 8% polyethylene glycol (PEG) 6000 overnight at 4°C. The supernatants were removed and the precipitates were washed and resuspended in 3% PEG in phosphate‐buffered saline (PBS) (pH 7.2). This material was again centrifuged at 7900 g for 20 min at 4°C. The supernatants were then removed and the precipitates were dissolved in equal amounts of PBS and incubated at 37°C for 1 h. Further, acid dissociation of antigen and antibody present in CICs was performed, using glycine‐HCl buffer, as described by Gupta and Tan, with slight modification 21. The isolated CICs were dissolved in 200 µl glycine‐HCl, pH 1 and incubated at 47°C for 30 min to dissociate the antibody–antigen complex. Consequently, the acid‐treated dissociated CICs were neutralized with 2 M NaOH and were finally dialysed against PBS solution. To evaluate the concentration of IgM and DENV antigen in CICs, acid‐dissociated neutralized CICs were subjected to standard dengue IgM ELISA and dengue antigen ELISA kits (J. Mitra & Co. Pvt. Ltd, Delhi, India), respectively. Further, the glycosylation status of antigen‐bound CICs was determined by using a digoxygenin (DIG) glycan differentiation kit (Roche Pvt. Ltd, Mumbai, India). The tests were carried out as per the manufacturer’s instructions. All the samples were tested in triplicate and the mean value taken for analysis.
Affinity purification of CICs
To further characterize the role of antibody‐bound CICs, affinity purification of CICs antibody was performed using acid‐dissociated CICs as described by Gupta and Tan 21, with slight modification. Briefly, 4 ml of pooled dengue plasma samples (DWOWS, DWWS and SD separately) were precipitated with PEG and further treated with glycine‐HCl as described earlier. CICs were affinity‐purified using equilibrated Protein A Sepharose 4B column (Invitrogen, Carlsbad, CA, USA) at room temperature for 45 min. The immunoglobulin fraction bound to Protein A was further incubated for another 90 min followed by washing in PBS (0·1 M, pH 7.4). Subsequently, the immunoglobulin was eluted with citrate buffer (0·1 M, pH 3.0) and was finally dialysed against PBS. Protein concentrations were measured using the Lowry method 22. Additionally, purified IgM‐bound CICs were characterized by 7·5% sodium dodecyl sulphate‐ polyacrylamide gel electrophoresis (SDS‐PAGE).
Study of alteration of RBC morphology induced by affinity‐purified CIC IgM
Scanning electron microscopy
To study the morphological difference of RBC (if any) between dengue‐infected patients and healthy control with or without sensitization of antibody‐bound CICs. scanning electron microscopy was performed. Briefly, 500 μl of whole blood were added directly from the syringe to 1 ml of 2·5% glutaraldehyde. Fixation was allowed to proceed for at least 2 h before processing. The cells were washed with PBS, dehydrated with washes in 50% ethanol for 5 min, two washes in 70% ethanol for 30 min, two washes in 90% ethanol for 30 min and subsequently two washes in absolute ethanol and acetone. One drop of the cell suspension was applied to an acetone‐washed coverslip and allowed to dry. The coverslip was fixed to stub and gold‐coated using gold coater (Eiko Engineering Co., Ltd, Hitachinaka, Japan; model no. IB‐2). Electron microscopy was carried out using a scanning electron microscope (Hitachi S‐530) at 15 keV. Images were digitally captured. The RBC images were examined microscopically on the basis of their shapes.
Flow cytometry
To study the morphological variation of RBC (if any), flow cytometry was performed between dengue‐infected (DWWS/SD) patients and healthy controls with or without sensitization of antibody‐bound CICs. Briefly 5 × 106 RBC was incubated for 1 h (optimized) with 20 µg CICs at 37°C and then RBC were gated on the basis of forward‐ and side‐scatter properties with a flow cytometer (FACSCalibur; Becton Dickinson, San Jose, CA, USA). The data obtained were analysed using Cell Quest Pro software (BD Biosciences, San Jose, CA, USA).
Erythrophagocytosis assay
Erythrophagocytosis assay is a useful parameter to evaluate immune RBC destruction. To evaluate the rate of RBC destruction, an erythrophagocytosis assay was performed as described by Samanta et al. 23. Briefly, RBC (2·5 × 106) with or without sensitization with optimized 20 µg antibody‐bound CICs from patients with SD, DWWS and DWOWS for 45 min at 37°C were coated over macrophages adhered on coverslips and incubated at 37°C for 1 h. Non‐phagocytosed extra erythrocytes were removed by mild washing with PBS and erythrocyte lysate Tris‐NH4Cl for 5 min. The slides were stained with diaminobenzidine (DAB) solution and Giemsa stain. The percentage of macrophages that had ingested one or more erythrocytes under microscopy were calculated immediately, as described by Pradhan et al. 24.
Measurement of cellular oxidative stress parameters as induced by CIC IgM
Redox equilibrium is an important factor for normal cell function, and is involved in different functions such as activation, maturation and cell signalling, etc. Impaired balance of oxidants and anti‐oxidants leads to disease severity. Therefore, oxidants such as hydroxyl, peroxyl and other ROS were measured by 5‐(and‐6)‐chloromethyl‐2′‐7′‐dichlorodihydrofluorescein diacetate (DCFDA) to assess the degree of severity in dengue patients.
Estimation of intracellular reactive oxygen species by DCFDA
Measurement of intracellular ROS in RBCs with or without sensitization of IgM‐bound CIC (20 µg) was performed using DCFDA. This experiment was adapted from Mitra et al.; briefly, 5 × 105 RBC were incubated for 30 min with DCFDA at 37°C then acquiring the fluorescence with a flow cytometer (FACS verse; Becton Dickinson) and positive control with H2O2 25. The RBC was gated on the basis of forward‐ and side‐scatter properties. A total of 10 000 gated events were acquired. The data obtained were analysed using the BD FACSuiteTM software (BD Biosciences).
Statistical analysis
Results are presented as mean ± standard error of the mean (s.e.m.) by measuring three individual replicates. Statistical analysis was performed using the GraphPad Prism statistics software (GraphPad Software Inc., San Diego, CA, USA) and between‐group comparisons were performed by Mann–Whitney test. Differences with P‐values < 0·05 were considered to be statistically significant. Cut‐off values of IgM‐bound CICs were determined using the Youden index.
Results
Demography of the study population
In the present study, 3066 suspected dengue patients who attended the hospital out‐patient department (OPD) or were hospitalized during the onset of infections in the Carmicheal Hospital for Tropical Diseases, Kolkata, West Bengal, India were enrolled. Of 3066 patients, 151 (4·92%) were dengue IgM‐positive. Thirty healthy subjects served as controls and designated as HD. On the basis of clinical manifestation, 90 were classified as DWOWS, 52 were DWWS and nine were SD patients (Fig. 1). The classification was based on the medical records and clinical characteristics of selected patients followed by 2009 WHO guidelines. Fifty‐three patients (36%) required hospitalization during the time of enrolment and all were managed according to hospital guidelines. Among the dengue‐infected patients, approximately 54% (n = 81) were male and 46% (n = 70) were female; patients 48 were categorized as secondary and 103 were primary infection (based on the ratio of IgM to IgG).
Figure 1.
Population screening plan.
All the patients observed (DWOWS, DWWS and SD, respectively) had fever, together with myalgia (46, 56 and 88%), rash (51, 61 and 100%), headache (56, 81 and 88%), vomiting (9, 37and 66%) and abdominal pain (17, 25 and 100%) (Table 1).
Table 1.
Demography of study subjects at enrolment
| Study parameters | DWOWS | DWWS | SD | P‐value |
|---|---|---|---|---|
| Male (number, percentage) | 48 (59%) | 28 (35%) | 5 (6%) | 0.0005 |
| Female (number, percentage) | 42 (60%) | 24 (34%) | 4 (6%) | 0.0002 |
| Age (year, median) | 31.62 (27) | 32.33 (32) | 31.62 (27) | <0.0001 |
| Hospitalization (number, percentage) | 21 (24%) | 33 (66%) | 9 (100%) | 0.0027 |
| Positive dengue IgM test (number, percentage) | 90 (100%) | 52 (100%) | 9 (100%) | 0.0001 |
| Positive dengue NS1 test (number, percentage) | 89 (97%) | 47 (91%) | 9 (100%) | 0.224 |
| Predominant serotype (number, percentage) | DENV2 (18, 20%) | DENV2 (15, 30%) | DENV2 (11, 55%) | 0.0131 |
| Secondary dengue infection (number, percentage) | 22 (24%) | 23 (44%) | 3 (33%) | – |
| Day of fever on enrolment (mean, median) | 3.51 (3) | 3.56 (4) | 3.55 (3) | 0.002 |
| Rashes (number, percentage) | 46 (51%) | 32 (61%) | 9 (100%) | 0.0019 |
| Headache (number, percentage) | 51 (56%) | 42 (81%) | 8 (88%) | 0.5169 |
| Myalgia (number, percentage) | 42 (46%) | 29(56%) | 8 (88%) | 0.0026 |
| Abdominal pain (number, percentage) | 11 (12%) | 12 (23%) | 9 (100%) | <0.0001 |
| Vomiting (number, percentage) | 8 (9%) | 18 (35%) | 6 (66%) | 0.0099 |
| Loose motion (number, percentage) | 15 (17%) | 16 (31%) | 2 (22%) | 0.3154 |
| Major bleeding (number, percentage) | 0 | 0 | 9 (100%) | <0.0001 |
| Thrombocytopenia (number, percentage) | 0 | 0 | 9 (100%) | <0.0001 |
DWOWS = dengue without warning Signs; DWWS = dengue with warning signs; SD = severe dengue (SD was defined by the history of fever, plasma leakage, shock, fluid accumulation with respiratory distress and severe bleeding as evaluated by the clinician, severe organ involvement, central nervous system impaired consciousness, heart and other organs); NS1 = non‐structural protein; DENV2 = dengue serotype 2; χ2 test and analysis of variance (ANOVA) used for calculation of P‐value.
Blood profile
The results showed that low levels (mean ± s.e.m.) of SGOT (29·73 ± 0·75 IU/l) and SGPT (28·17 ± 0·79 IU/l) were detected in the sera of HD. In DWOWS and DWWS patients, the levels of SGOT (60·13 ± 3·847 IU/l and 91·01 ± 9·542 IU/l), SGPT (40·66 ± 3·53 IU/l and 73·85 ± 7·84 IU/l) were lower than in patients with severe dengue (378·7 ± 94·11 IU/l), (257·4 ± 53·10 IU/l), respectively. SD patients had an approximately 2·49‐fold decreased platelet count compared to DWOWS and DWWS. However, 7·68‐ and 1·27‐fold increased CRP and HCT were found in SD patients compared to DWWS. High levels of ferritin (3·62‐ and 2·21‐fold) were found in SD compared to DWOWS and DWWS, which was statistically significant (P < 0·0001) (Table 2).
Table 2.
Blood parameters of study subjects at enrolment
| Blood parameters | DWOWS (mean ± s.e.m.) | DWWS (mean ± s.e.m.) | SD (mean ± s.e.m.) | (P‐value) |
|---|---|---|---|---|
| Hb (gm/dl) | 15·27 ± 0·15 | 14·01 ± 0·24 | 9·164 ± 0·363 | <0·0001 |
| RBC (106 µl) | 4·812 ± 0·074 | 4·681 ± 0·099 | 3·91 ± 0·098 | <0·0001 |
| WBC (103 µl) | 4·97 ± 0·25 | 4·941 ± 0·256 | 7·77 ± 0·526 | <0·0001 |
| HCT (%) | 34·11 ± 0·684 | 34·10 ± 0·713 | 43·44 ± 1·502 | <0·0001 |
| SGOT (IU/l) | 60·13 ± 3·847 | 91·01 ± 9·542 | 378·7 ± 94·11 | <0·0001 |
| SGPT (IU/l) | 40·66 ± 3·53 | 73·85 ± 7·84 | 257·4 ± 53·10 | <0·0001 |
| CRP (pg/ml) | 10·45 ± 1·17 | 10·77 ± 2·065 | 82·77 ± 4·625 | <0·0001 |
| Platelets (103 µl) | 57·73 ± 3·38 | 57·54 ± 3·485 | 23·1 ± 2·372 | <0·0001 |
| ESR (mm/h) | 22·47 ± 0·49 | 22·07 ± 0·646 | 11·48 ± 0·328 | <0·0001 |
| Ferritin (ng/ml) | 443 ± 44·30 | 723 ± 71·15 | 1604 ± 91·18 | <0·0001 |
| Complement protein (C3) (pg/ml) | 53·72 ± 2·07 | 105 ± 6·5 | 223 ± 11·5 | <0·0001 |
DWOWS = dengue without warning signs; DWWS = dengue with warning signs; SD = severe dengue; Hb = haemoglobin; RBC = red blood cells; WBC = white blood cells; HCT = haematocrit; SGOT = serum glutamic oxaloacetic transaminase; SGPT = serum glutamate–pyruvate transaminase; CRP = C‐reactive protein; ESR = erythrocyte sedimentation rate; s.e.m. = standard error of the mean. One‐way analysis of variance (ANOVA) test used for calculation of P‐value.
Estimation of IgM‐bound CIC and DENV antigen from plasma by ELISA
Previously we have quantified the amount of CICs in different grades of dengue patients 17. Further, in this present study, we measured the amount of IgM‐bound CICs (Fig. 2). The mean ± s.e.m. of IgM CIC titres for DWWS and SD are 32·89 ± 1·587 and 69·74 ± 3·502, respectively. However, the levels of IgM CICs were very low in DWOWS patients (19·18 ± 0·879). Thus, 2·21‐ and 3·36‐fold increased IgM CICs titres were obtained in SD compared to DWWS and DWOWS, respectively. Analysis of variance (ANOVA) analysis among the groups revealed a P‐value of <0·0001, which was statistically significant (Fig. 2). Additionally, the cut‐off values for IgM‐bound CICs titres were determined using the Youden index. The calculated area under the ROC curve is 0·919, demonstrating that IgM‐bound CICs can serve as dengue severity biomarkers. Interestingly, 1·51‐fold higher dengue antigen‐bound glycosylated CICs were obtained in SD cases compared to DWWS (data not shown).
Figure 2.
(a) Level of immunoglobulin (IgM)‐bound circulating immune complex (CIC) titres in dengue without warning signs (DWOWS), dengue with warning signs (DWWS) and severe dengue (SD) fever. Study subjects are significantly different from each category. Line shows severity cut‐off value, analysis of variance (ANOVA) used for calculation of P‐value. (b) Receiver operating characteristic (ROC) analysis of IgM‐bound CIC titres of DWOWS, DWWS and SD.
Alteration of RBC morphology as induced by affinity‐purified IgM CICs
Scanning electron microscopic analysis
Scanning electron microscopic analysis of RBC revealed that the normal discoid shape was altered more in patients with SD compared to DWOWS, DWWS and HD. This study reveals for the first time, to our knowledge, that IgM CICs purified from SD upon 1 h incubation induce a greater number of ultrastructural morphological changes in healthy RBC compared to IgM CICs purified from DWOWS and DWWS patients. Conversely, IgM CICs purified from HD did not exhibit any remarkable changes when they bound with healthy RBC, which retained their normal discoid shape (Fig. 3).
Figure 3.
Morphological changes of red blood cells (RBC) after binding with immunoglobulin (Ig)M circulating immune complexes (CICs) by scanning electron microscope. (a) RBC from healthy donors (HD), (b) RBC from patients with dengue without warning signs (DWOWS), (c) RBC from patients with dengue with warning signs (DWWS), (d) RBC from patients with severe dengue (SD) fever, (e) healthy RBC after binding with healthy donor‐derived IgM CICs, (f) healthy RBC after binding with SD‐derived CICs, (g) healthy RBC after binding with DWOWS‐derived IgM CICs and (h) healthy RBC after binding with DWWS‐derived IgM CICs. (i–m) Representative histogram describing the morphological change in erythrocytes from different study subjects. RBC from patients are small and of various sizes and shapes, whereas RBCs from healthy donors are of normal and uniform sizes and shapes, as reflected from their MFI values. (i–l) Similar observations were found when healthy RBC were treated with IgM CICs of HD, DWWS and SD.
Flow cytometry analysis
Further demonstration of changes in RBC morphology upon IgM CICs binding was demonstrated using flow cytometry 23 by monitoring the forward light‐scatter of RBC before and after sensitization of IgM CICs with all these study cases. As forward‐scatter reveals cell size, representative histogram plots are represented as cell count versus forward‐scatter. Healthy erythrocytes are highest in number [325·11 mean fluorescence intensity (MFI)] and discoidal in size, whereas SD patients’ cells are fewer in number (94.80 MFI) as well as abnormal in size depicting abrupt morphological changes during the disease manifestation. In contrast, SD IgM CICs and DWWS IgM CICs sensitized healthy RBC exhibited 20·14 MFI and 24·08 MFI, respectively (Fig. 3). Thus, IgM CICs appear crucial in inducing RBC morphological change.
Eryhrophagocytosis
Deformed RBC produce the so‐called ‘eat me’ signals, which accumulate on its cell membrane, and these signals can trigger RBC clearance by macrophages 26, 27, 28. This study revealed that RBC of patients with SD show an increased rate of phagocytosis compared to DWWS patients, suggesting a deformed condition in these RBC. Interestingly, SD IgM CICs‐sensitized healthy RBC shows a greater number (54 ± 2) of erythrophagocytosis compared to DWWS IgM CICs‐sensitized healthy RBC (36 ± 2) (Table 3). Further, DWOWS IgM CICs‐sensitized RBC shows very low levels (32 ± 2) of erythrophagocytosis compared to DWWS IgM CICs‐sensitized RBC. However, HD IgM CICs‐sensitized healthy RBC did not exhibit any significant erythrophagocytosis (4 ± 1). Thus, a 13·5‐fold increased erythrophagocytosis was obtained with SD IgM CICs‐sensitized healthy RBC. Thus, IgM CICs appear vital in triggering erythrophagocytosis.
Table 3.
RBC DWOWS, RBC DWWS, RBC SD and RBC HD, with or without sensitization by IgM‐bound CICs and processed for erythrophagocytosis assay, as described in Material and methods
| RBC DWOWS | RBC DWWS | RBC SD | RBC HD | |
|---|---|---|---|---|
| Number of uptaking RBC by macrophage (mean ± s.e.m.) | 21 ± 2 | 28 ± 2 | 38 ± 2 | 3 ± 1 |
| Number of uptaking IgM CICs sensitized healthy RBC by macrophage (mean ± s.e.m.) | 32 ± 2 | 36 ± 2 | 54 ± 2 | 4 ± 1 |
Result represents the mean ± standard error of the mean (s.e.m.) of five separate determinations.
RBC = red blood cells; Ig = immunoglobulin; CIC = circulating immune complex; DWOWS = dengue without warning signs; DWWS = dengue with warning signs; SD = severe dengue.
Intracellular oxidative stress
Erythrocytic DCFDA is increased in severe patients
Intracellular ROS was determined by using the probe DCFDA. Negative control represents the absence of the probe and positive control represents probe, along with a natural source of free radical that is hydrogen peroxide. Cellular ROS level was determined by the number of fluorescent cells per sample in all the study subjects initially in the erythrocytes collected directly from the subjects and later treating healthy RBCs with IgM CICs purified from SD, DWOWS and DWWS patients. Statistically significant higher intracellular ROS was found in SD (primary infection: 266 ± 68 MFI of DCF; secondary infection: 419·60 ± 329·1 MFI of DCF), DWOWS (primary infection: 93·60 ± 15·16 MFI of DCF; secondary infection: 275·66 ± 72·72 MFI of DCF) and DWWS (primary infection: 99·93 ± 17·7 MFI of DCF; secondary infection: 109·93 ± 22·25 MFI of DCF) RBC (Fig. 4). Thus, 2·67‐ and 2·84‐fold increased intracellular ROS was obtained in SD patients compared to DWWS and DWOWS patients in primary dengue infection. However, a 2·50‐ and 1·52‐fold increased intracellular ROS was obtained in SD patients compared to DWWS and DWOWS in secondary infection. Interestingly, similar results were obtained when healthy RBCs were treated with IgM CICs purified from patients with DWOWS (primary infection: 141·1 ± 46·11 MFI of DCF; secondary infection: 964 ± 178 MFI of DCF ), SD (primary infection: 3192 ± 820·1 MFI of DCF; secondary infection: 3700 ± 915 MFI of DCF) and DWWS (primary infection: 866·1 ± 337·5 MFI of DCF; secondary infection: 1130·66 ± 294·83 MFI of DCF), respectively (Fig. 4). Two‐way ANOVA among the groups revealed a P‐value of < 0·0001, which was statistically significant. Thus, the capacity of IgM‐bound CIC purified from secondary dengue patients to trigger oxidative stress was greater compared to that purified from primary dengue.
Figure 4.
Comparative representative histogram depicting generation of reactive oxygen species (ROS) in red blood cells (RBC) after binding with immunoglobulin (Ig)M circulating immune complexes (CICs) purified from primary versus secondary in dengue without warning signs (DWOWS), dengue with warning signs (DWWS) and severe dengue (SD), respectively. Two‐way analysis of variance (ANOVA) was used to calculate level of significance.
Discussion
Dengue is an important viral disease, and the development from a non‐severe to a severe condition is unpredictable. Until the present, there is no specific laboratory parameter for early detection of dengue severity among patients. We have reported previously the enhanced presence of CICs in the circulation of SD patients 17. In this investigation, we demonstrate for the first time the status of IgM‐bound CICs, its utility as a DS biomarker and its role in RBC damage. To combat against the pathogen, our immune system deploys various interesting strategies; for example, the formation of immune complexes. CICs formed by antibody–antigen complexes and human proteins are the main feature indicating the severity of disease 15. With the objective of identifying severity biomarkers, we quantified IgM CICs in patients’ serum circulation that revealed 2·21‐ and 3·36‐fold increased IgM CICs titres in SD compared to DWWS and DWOWS patients. Subsequently, we also developed a dengue severity cut‐off using IgM CICs titres. At a cut‐off of 24·24, the biomarker sensitivity and specificity was 85%, and could successfully differentiate SD from milder forms of dengue (Fig. 2). The presence of such high titres of IgM CICs in the circulation of SD patients led us to further examine its pathophysiological role in dengue severity. CICs are reported to bind with complement receptor 1 (CR1) on primate RBC for clearance. Several investigators have proposed that RBC CR1 act as a buffer to adsorb immune complexes, keep them in the intravascular space and transport them to tissue macrophages of the mononuclear phagocyte system 29, 30, 31. Interestingly, scanning electron microscopic analysis in our study revealed definite morphological alterations of RBC when they were sensitized with IgM CICs, corroborating a key role of IgM CICs in SD (Fig. 2). Further, the role of IgM CICs in inducing morphological changes of RBC was confirmed through flow cytometric studies, which demonstrated a significant shift towards a lower forward‐scatter when RBC were sensitized with IgM CICs. The RBC SD cell size was typically more altered than RBC HD, due possibly to the IgM CICs interaction with RBC during the severe condition. Similar alterations in RBC membrane organization have been documented in acute childhood lymphoblastic leukaemia and leishmaniasis 32, 33. As IgM CICs primarily bind with CR1/DAF/glycophorin‐A on RBC, they might also influence ROS generation. Prolonged exposure to ROS can thus induce oxidative stress leading to damage of the RBC membrane 32. It is well known that RBC not only counteract oxidative stress, but also play an important role in maintaining immunological homeostasis in infectious diseases 33. More studies have demonstrated recently that higher oxidant exposure causes Hb and spectrin aggregation, leading to a change in RBC membrane structure 35. Mohanty et al. have demonstrated that increased levels of ROS affect the discoidal structure of RBC 34. Thus, the potency of IgM‐bound CICs to trigger ROS in RBC was also quantified by measuring the amount of intracellular free radicals generated. Interestingly, the study revealed 2·67‐ and 2·84‐fold increased ROS in RBC of SD compared to DWWS and DWOWS patients, which was statistically significant (P = 0·0004). Additionally, our studies demonstrated that the capacity of IgM‐bound CIC purified from secondary dengue patients to trigger ROS generation was greater compared to that purified from primary dengue (Fig. 4). de Back et al. demonstrated that macrophages are key players for the clearance of damaged RBC 35. Interestingly, this was reflected in erythrophagocytosis levels of both diseased and dengue IgM CICs‐sensitized RB HD. The erythrophagocytosis level was significantly higher in SD patients compared to DWWS and HD (Table 3).Thus, IgM CIC are one of the mainstays in inducing RBC damage in severe conditions. Increased RBC damage was also reflected by raised ferritin levels in sera of SD patients (P < 0·0001). Taken together, the present study demonstrates that high IgM CICs titres, along with high levels of ROS with increased RBC damage, are important clinical laboratory parameters for monitoring infection progression in dengue virus‐infected patients.
Disclosures
The authors declare that they have no conflicts of interest.
Author contributions
G. P. designed the study, performed the experiments, analysed the data and wrote the paper; B. S. helps in clinical classification and patient selection; S. M. designed the study, analysed the data and wrote the paper.
Acknowledgements
We are thankful to the Department of Instrumentation Science, Burdwan University for providing the facility of s.e.m. and their co‐operation in this experiment. We are thankful to WB DST (File no: 326(SANC)/ST/P/S and T/9G‐22/2015), Government of West Bengal, India for providing funding, which made it possible to carry out this work.
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