| Abstract|| |
Background: Dengue is an arboviral disease caused by four distinct serotypes of dengue virus. The pathogenesis of dengue is not very clearly understood. Various pro- and anti-inflammatory cytokines are involved in the immune pathogenesis of dengue. Interleukin (IL)-2/IL-2 receptor interaction is supposed to play a protective role, while IL-4 acts as pro-inflammatory whereas IL-10 acts as anti-inflammatory cytokines. So far, not much information is available regarding the established role of these cytokines with dengue infection and severity. Aims: our study aimed to show the association of IL-2, -4, and -10 with severity of dengue infection. Settings and Design: This was a cross-sectional study. Materials and Methods: The study was conducted in the year 2015; 150 blood samples from suspected dengue cases were confirmed for dengue and then with an equal number of healthy control samples were tested for cytokines levels (IL-2, -4, and -10) by ELISA. Severity of the dengue infection was determined on the basis of clinical manifestations based on the WHO criteria.Statistical Analysis: for statistical analysis, SPSS version 21 (IBM, New York, United States) was used. Results: Out of 150 samples, 56 samples came to be dengue positive. Thirty-eight (67.85%) cases were classified as nonsevere dengue and 18 (32.15%) were severe dengue. The serum levels of IL-4 and -10 were significantly raised in severe dengue cases as compared to nonsevere dengue cases. No significant association was observed between serum IL-2 levels and the severity of dengue. Conclusion: IL-4 and -10 levels can be used as marker of severe dengue and help in early preparedness to start the treatment in the line of severe dengue.
Keywords: Cytokines, IgM, NS1, pathogenesis, severe dengue
|How to cite this article:|
Abhishek KS, Chakravarti A, Baveja C P, Kumar N, Siddiqui O, Kumar S. Association of interleukin-2, -4 and -10 with dengue severity. Indian J Pathol Microbiol 2017;60:66-9
|How to cite this URL:|
Abhishek KS, Chakravarti A, Baveja C P, Kumar N, Siddiqui O, Kumar S. Association of interleukin-2, -4 and -10 with dengue severity. Indian J Pathol Microbiol [serial online] 2017 [cited 2017 Oct 17];60:66-9. Available from: http://www.ijpmonline.org/text.asp?2017/60/1/66/200027
| Introduction|| |
Dengue is an acute viral infection presenting with a wide array of clinical presentation, ranging from an asymptomatic case to potential fatal complications. There are four serotypes of dengue virus referred as DENV-1, -2, -3, and -4 belonging to family Flaviviridae with enveloped positive strand RNA. The fifth variant DENV-5 has been isolated in October 2013, which unlike the other four serotypes follows the sylvatic cycle. Genetic recombination, natural selection, and genetic bottlenecks could be the likely causes of emergence of new serotype. There is no indication of the presence of DENV-5 in India. Being an arthropod-borne virus, it is transmitted to humans by the bite of an infected female mosquito. The primary vector is the Aedes aegypti mosquito, but other species such as Aedes albopictus and less commonly Aedes polynesiensis can also transmit the virus. Dengue fever has reemerged as a major public health challenge worldwide, with 2.5 billion people at risk of infection, more than 100 million cases and 25,000 deaths being reported annually. Delhi is one of the dengue endemic states in India. It has so far witnessed several outbreaks during past years, viz., 1920, 1982, 1988, 1996, 2003, 2006, 2010, and 2013. Recently in 2015, an outbreak occurred in India during which a total of 99,913 dengue cases and 220 deaths were reported – more than twice the number of cases in previous year with 15,867 cases and sixty deaths in Delhi alone.
The pathogenesis of dengue is not yet very clear; a secondary infection with a different serotype has been suspected to be one of the risk factors., There are various hypotheses regarding the pathogenesis of dengue; antibody-dependent enhancement is the most accepted one., The immune pathogenesis of dengue involves antibody production, B-cell and T-cell response, and various pro-inflammatory and anti-inflammatory cytokines. Activation of T-cells, antibodies, and cytokines are influenced by various immunomodulators. Increase or decrease in the levels of these immunomodulators influences the outcome of viral infections. Tumor necrotic factor-α (TNF-α) and interleukin-4 (IL-4), -5, -6 act as pro-inflammatory cytokines while IL-10, -13, and interferon (IFN) act as anti-inflammatory cytokines. T-helper-1 (Th-1) cells secrete IFN-γ, IL-2, and TNF-β while Th-2 cells secrete IL-4, -5, -6, -10, and -13 and the levels of different cytokines vary with severity of dengue, suggesting an important role of these cytokines in the pathogenesis and severity of the disease.
IL-2 is produced by Th-1 during immune response. IL-2/IL-2 receptor interaction stimulates the growth, differentiation, and survival of antigen-specific CD4+ T-cells and CD8+ T-cells and thus plays a protective role in dengue infection. IL-4 is produced by Th-2. IL-4 has been called the “prototypic immunoregulatory cytokine.” IL-4 has important role in regulating antibody production, hematopoiesis and inflammation, and the development of effector T-cell responses. Arbovirus infectivity and pathogenicity have been correlated with Th-2-type immune reactions provoked by mosquito salivary compounds., IL-4 stimulated CD14+ dermal dendritic cells (dDCs) show increased viral load when infected with DENV. IL-10, which was originally named cytokine synthesis inhibitory factor, is a cytokine that is produced by Th-2 cells. IL-10 exhibits anti-inflammatory properties, including the inhibition of immune mediator secretion, antigen presentation, and phagocytosis. IL-10 is a cytokine with pleiotropic effects in immunoregulation and inflammation. IL-10 may play a role in DENV pathogenesis, reflecting an immunosuppressive function that causes IFN resistance, followed by impaired immune clearance and a persistent infectious effect for acute viral infection.
So far, not much information is available regarding the established role of these cytokines with dengue infection and severity. In the view of above, our study aimed to show the association of IL-2, -4, and -10 with severity of dengue infection to add up the information to already existing studies.
| Materials and Methods|| |
Five milliliters of venous blood was collected in the plain vial from the dengue suspected patients coming with complain of fever and other possible signs and symptoms of dengue to the emergency medicine outpatient department (OPD) for the routine diagnosis of dengue. Serum was separated from venous blood samples aseptically and aliquoted in Eppendorf vials and immediately transferred to –70°C until processed further.
The serum samples were tested for the following laboratory tests to confirm DENV infection:
- NS-1 dengue antigens: NS-1 dengue antigen were detected in serum in early phase (<5 days of fever) by NS-1 ELISA using Dengue NS1 Ag Microlisa Kits (J. Mitra and Co. Pvt. Ltd., New Delhi, India) as per the manufacturer's instructions
- Antidengue IgM antibodies: Antidengue IgM antibody was detected in serum in the late phase (≥5 days of fever) using NIV DEN IgM Capture ELISA Kits (National Institute of Virology, Pune, India) as per the manufacturer's instructions.
Samples positive for either NS1 antigen or IgM antibody and an equal number of samples from healthy controls were tested for cytokines levels (IL-2, -4, and -10) by commercially available ELISA kits as per the manufacturer's instructions.
Measurement of interleukin-2, -4, and -10 in serum
AviBion Human IL-2, -4, and -10 ELISA Kits (Orgenium Laboratories Business Unit, Finland) were used to measure serum IL-2, -4, and -10 levels as per the manufacturer's instructions.
Separate standard curves were generated by plotting the average absorbance of each standard on the vertical axis versus the corresponding IL-2, -4, and -10 standard concentrations on the horizontal axis.
The amount of IL-2, -4, and -10 in each sample was determined by extrapolating OD values against IL-2, -4, and -10 standard concentrations using the corresponding standard curves. Severity of the dengue infection was determined on the basis of clinical manifestations as per the WHO guidelines. New WHO guidelines 2009 categorizes dengue into two groups: Nonsevere dengue or mild disease with or without warning signs and severe dengue.
| Results|| |
This study included a total of 150 clinically suspected dengue patients who attended medical OPD or were hospitalized in the wards of Lok Nayak Hospital, New Delhi. The diagnosis of dengue infection was based on the presence of dengue NS-1 antigen or dengue IgM antibody by ELISA. Out of these 150 patients, 56 were positive either by dengue NS-1 antigen or dengue IgM antibody or both.
Out of 56 dengue positive cases, 35 were positive for dengue NS-1 antigen, 12 were positive for dengue IgM antibody, while 9 were positive by both [Table 1].
On the basis of clinical manifestations, 38 (67.85%) were classified as nonsevere dengue and 18 (32.15%) were severe dengue [Table 2].
None of the healthy volunteers showed detectable level of IL-2 in their serum. Among dengue cases, raised serum IL-2 level was noticed in only five cases. Out of these five dengue cases, only one case was of severe dengue. Hence, it was difficult to make out any kind of positive or negative significance of IL-2 serum level with dengue severity.
While the mean serum levels of IL-4 (2.54 ± 1.77 pg/ml) and IL-10 (57.15 ± 34.66 pg/ml) were significantly (P = 0.006, P < 0.0001, respectively) raised in severe dengue cases as compared to nonsevere dengue (IL-4: 1.18 ± 1.71 pg/ml and IL-10: 15.80 ± 15.53 pg/ml) and healthy controls (IL-4: 0.66 ± 0.66 pg/ml and IL-10: 3.28 ± 2.50 pg/ml) [Table 3].
| Discussion|| |
Dengue has emerged as a national threat considering its frequent outbreaks in the last few years in Delhi and other parts of the India.
The role of cytokines in the pathogenesis of dengue is an interesting subject to be studied. IL-2 produced by Th-1 cells induces plasma leakage in human when administered experimentally at dose of >105 U/kg., IL-2 is also known to induce lymphokine-activated killer cells  and thromboxane A2 and activate endothelial cells. Any of these may conceivably alter endothelial permeability to cause plasma leakage; IL-2 plays a central role in the regulation of the immune response, as it induces potent proliferation of T-cells and to a lesser extent of B-cells, stimulates synthesis of INF-γ and TNF-α, and may damage the integrity of endothelial cells. A study done by Kurane et al. clearly showed that the serum level of IL-2 is significantly raised in dengue fever and severe dengue during all the stages of illness. Unlike their study, in our study, no such significance was established; still not all but few cases at least showed elevated levels of IL-2 while it was undetectable in healthy controls. Small sample size might be the cause of such outcome.
IL-4 is produced by Th-2 cells. Th-2 type immune reaction is provoked by mosquito salivary compounds., A study done by Schaeffer et al. shown that the viral titer was significantly elevated when DCs were conditioned by IL-4. They also found a greatly increased viral load in IL-4 stimulated CD14+ dDCs as compared to untreated one. This could explain that the elevated IL-4 level somehow increases the affinity and favors viral replication. Similar results were obtained where pretreatment of human monocytes or macrophages with Th-2 cytokines (IL-4 or -13) enhanced their susceptibility to DENV infection. In our study, there is significant elevated serum IL-4 level in severe dengue cases as compare to dengue fever. Our study may be supported by the study done by Chaturvedi et al. in which, with regard to dengue disease, they showed shift from predominant Th-1 type response observed in cases of dengue fever to the Th-2 type response in severe dengue cases whereas Th-2 mediated IL-4 level was elevated in the serums of severe dengue cases in contrast to dengue fever cases. The exact mechanism to this shift and increased infectivity to DENV in response to IL-4 are yet to be established.
Our study also demonstrated the significant rise in serum IL-10 level in severe dengue infection as compared to healthy controls and uncomplicated dengue fever. Being an anti-inflammatory cytokine, IL-10 has been shown to inhibit TNF-α alpha production. Elevated IL-10 itself is unlikely to be the cause of plasma leakage as administration of IL-10 to healthy adults induces no clinical significant adverse reaction. Hence, we hypothesize that in severe dengue, elevated IL-10 plays a negative feedback role for pro-inflammatory cytokines. It has been shown to be associated with a worse outcome in many viral infections other than dengue, whereas serum IL-10 level was elevated including influenza and hepatitis B virus infections.,,, While in some viral infections as in Japanese encephalitis virus, elevated IL-10 levels were associated with favorable outcome. Shifting our interest back to DENV infection, on the basis of our results and others as mentioned above, we hypothesize that increasing serum levels of IL-10 can be a predictor for the developing severe dengue fever.
Compiling all these results, it can be said that the elevated levels of serum IL-4 and -10 in DENV-infected individuals can be an early predictor markers for the shifting of uncomplicated dengue to severe dengue even before the signs and symptoms of severe dengue appear. This would be helpful for the treating physicians for the early preparedness to start the treatment on the line of severe dengue to avoid complications arising out of dengue and also for the better dengue management ultimately benefiting the patients and humankind.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Gubler DJ. Dengue and dengue hemorrhagic fever. Clin Microbiol Rev 1998;11:480-96.
Mustafa MS, Rasotgi V, Jain S, Gupta V. Discovery of fifth serotype of dengue virus (DENV-5): A new public health dilemma in dengue control. Med J Armed Forces India 2015;71:67-70.
Senanayake S. Dengue fever and dengue haemorrhagic fever – A diagnostic challenge. Aust Fam Physician 2006;35:609-12.
Gupta E, Mohan S, Bajpai M, Choudhary A, Singh G. Circulation of Dengue virus-1 (DENV-1) serotype in Delhi, during 2010-11 after Dengue virus-3 (DENV-3) predominance: A single centre hospital-based study. J Vector Borne Dis 2012;49:82-5.
Halstead SB. Antibody, macrophages, dengue virus infection, shock, and hemorrhage: A pathogenetic cascade. Rev Infect Dis 1989;11 Suppl 4:S830-9.
Rico-Hesse R. Molecular evolution and distribution of dengue viruses type 1 and 2 in nature. Virology 1990;174:479-93.
Halstead SB. Observations related to pathogensis of dengue hemorrhagic fever. VI. Hypotheses and discussion. Yale J Biol Med 1970;42:350-62.
Halstead SB. Pathogenesis of dengue: Challenges to molecular biology. Science 1988;239:476-81.
Clyde K, Kyle JL, Harris E. Recent advances in deciphering viral and host determinants of dengue virus replication and pathogenesis. J Virol 2006;80:11418-31.
Beard JA, Bearden A, Striker R. Vitamin D and the anti-viral state. J Clin Virol 2011;50:194-200.
Pinto LM, Oliveira SA, Braga EL, Nogucira RM, Kubelka CF. Increased proinflammatory cytokines (TNF-α and IL-6) and anti-inflammatory compounds (sTNFRp55 and STNFRp75) in Brazilian patients during exanthematic dengue fever. Mem Inst Oswaldo Cruz 1996;94:387-94.
Hatch S, Endy TP, Thomas S, Mathew A, Potts J, Pazoles P, et al.
Intracellular cytokine production by dengue virus-specific T cells correlates with subclinical secondary infection. J Infect Dis 2011;203:1282-91.
Brown MA, Hural J. Functions of IL-4 and control of its expression. Crit Rev Immunol 1997;17:1-32.
Cox J, Mota J, Sukupolvi-Petty S, Diamond MS, Rico-Hesse R. Mosquito bite delivery of dengue virus enhances immunogenicity and pathogenesis in humanized mice. J Virol 2012;86:7637-49.
Styer LM, Lim PY, Louie KL, Albright RG, Kramer LD, Bernard KA. Mosquito saliva causes enhancement of West Nile virus infection in mice. J Virol 2011;85:1517-27.
Schaeffer E, Flacher V, Papageorgiou V, Decossas M, Fauny JD, Krämer M, et al.
Dermal CD14(+) dendritic cell and macrophage infection by dengue virus is stimulated by interleukin-4. J Invest Dermatol 2015;135:1743-51.
Fiorentino DF, Bond MW, Mosmann TR. Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. J Exp Med 1989;170:2081-95.
Jung M, Sabat R, Krätzschmar J, Seidel H, Wolk K, Schönbein C, et al.
Expression profiling of IL-10-regulated genes in human monocytes and peripheral blood mononuclear cells from psoriatic patients during IL-10 therapy. Eur J Immunol 2004;34:481-93.
Duell BL, Tan CK, Carey AJ, Wu F, Cripps AW, Ulett GC. Recent insights into microbial triggers of interleukin-10 production in the host and the impact on infectious disease pathogenesis. FEMS Immunol Med Microbiol 2012;64:295-313.
Rosenberg SA, Lotze MT, Muul LM, Leitman S, Chang AE, Ettinhausen SE, et al
. Observation on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. N Engl J Med 1985;313:1485-92.
Lotze MT, Matory YL, Ettinghausen SE, Rayner AA, Sharrow SO, Seipp CA, et al. In vivo
administration of purified human interleukin 2. II. Half life, immunologic effects, and expansion of peripheral lymphoid cells in vivo
with recombinant IL 2. J Immunol 1985;135:2865-75.
Peace DJ, Cheever MA. Toxicity and therapeutic efficacy of high-dose interleukin 2.In vivo
infusion of antibody to NK-1.1 attenuates toxicity without compromising efficacy against murine leukemia. J Exp Med 1989;169:161-73.
Ferro TJ, Johnson A, Everitt J, Malik AB. IL-2 induces pulmonary edema and vasoconstriction independent of circulating lymphocytes. J Immunol 1989;142:1916-21.
Cotran RS, Pober JS, Gimbrone MA Jr., Springer TA, Wiebke EA, Gaspari AA, et al.
Endothelial activation during interleukin 2 immunotherapy. A possible mechanism for the vascular leak syndrome. J Immunol 1988;140:1883-8.
Kurane I, Innis BL, Nimmannitya S, Nisalak A, Meager A, Janus J, et al.
Activation of T lymphocytes in dengue virus infections. High levels of soluble interleukin 2 receptor, soluble CD4, soluble CD8, interleukin 2, and interferon-gamma in sera of children with dengue. J Clin Invest 1991;88:1473-80.
Miller JL, de Wet BJ, Martinez-Pomares L, Radcliffe CM, Dwek RA, Rudd PM, et al.
The mannose receptor mediates dengue virus infection of macrophages. PLoS Pathog 2008;4:e17.
Chaturvedi UC, Agarwal R, Elbishbishi EA, Mustafa AS. Cytokine cascade in dengue hemorrhagic fever: Implications for pathogenesis. FEMS Immunol Med Microbiol 2000;28:183-8.
de Waal Malefyt R, Abrams J, Bennett B, Figdor CG, de Vries JE. Interleukin 10(IL-10) inhibits cytokine synthesis by human monocytes: An autoregulatory role of IL-10 produced by monocytes. J Exp Med 1991;174:1209-20.
Chernoff AE, Granowitz EV, Shapiro L, Vannier E, Lonnemann G, Angel JB, et al.
A randomized, controlled trial of IL-10 in humans. Inhibition of inflammatory cytokine production and immune responses. J Immunol 1995;154:5492-9.
Hasegawa S, Matsushige T, Inoue H, Shirabe K, Fukano R, Ichiyama T. Serum and cerebrospinal fluid cytokine profile of patients with 2009 pandemic H1N1 influenza virus-associated encephalopathy. Cytokine 2011;54:167-72.
Yu X, Zhang X, Zhao B, Wang J, Zhu Z, Teng Z, et al.
Intensive cytokine induction in pandemic H1N1 influenza virus infection accompanied by robust production of IL-10 and IL-6. PLoS One 2011;6:e28680.
Flynn JK, Dore GJ, Hellard M, Yeung B, Rawlinson WD, White PA, et al.
Early IL-10 predominant responses are associated with progression to chronic hepatitis C virus infection in injecting drug users. J Viral Hepat 2011;18:549-61.
Bai F, Town T, Qian F, Wang P, Kamanaka M, Connolly TM, et al.
IL-10 signaling blockade controls murine West Nile virus infection. PLoS Pathog 2009;5:E1000610.
Swarup V, Ghosh J, Duseja R, Ghosh S, Basu A. Japanese encephalitis virus infection decrease endogenous IL-10 production: Correlation with microglial activation and neuronal death. Neurosci Lett 2007;420:144-9.
Department of Microbiology, Maulana Azad Medical College, New Delhi - 110 002
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3]