|Year : 2016 | Volume
| Issue : 1 | Page : 76-81
Expression of cytokine-mRNA in peripheral blood mononuclear cell of human immunodeficiency virus-1 subtype C infected individuals with opportunistic viral infections from India (South)
J Sachithanandham1, VV Ramalingam1, J Raja1, OC Abraham2, SA Pulimood3, R Kannangai1
1 Department of Clinical Virology, Christian Medical College, Vellore, Tamil Nadu, India
2 Department of Internal Medicine, Christian Medical College, Vellore, Tamil Nadu, India
3 Department of Dermatology, Christian Medical College, Vellore, Tamil Nadu, India
|Date of Submission||29-Mar-2014|
|Date of Acceptance||05-Aug-2015|
|Date of Web Publication||15-Jan-2016|
Department of Clinical Virology, Christian Medical College, Vellore, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Human immunodeficiency virus (HIV) disease progression is associated with a marked change in the level of plasma cytokines. The study reported here investigated the level of mRNA expression of different cytokines: Tumour necrosis factor-alpha (TNF-α), interferon (INF)-gamma, interleukin-10 (IL-10) and IL-21 in the peripheral blood mononuclear cell among the antiretroviral therapy naive subtype C HIV-1 infected individuals and normal healthy controls by real time polymerase chain reaction. The mRNA expressions of all the 4 cytokines in HIV-1 infected individuals were significantly higher compared to healthy controls (P value range 0.0004–0.01). The mean level of IL-10, INF-gamma and TNF-α were higher in HIV infected individuals with low CD4 counts (<300 cells/μl). The IL-10 expression showed a significant negative correlation with CD4 counts (r = −0.25, P = 0.04) while IL-21 showed a positive correlation with CD4 counts (r = 0.26, P = 0.03). There was a significant negative correlation between the cytomegalovirus (CMV) viral load and IL-21 expression. Cytokine levels by mRNA detection avoids the inherent problem of measuring plasma level and this study also provide information on the cytokine levels and CD4+ T cell level among HIV-1 subtype C infected individuals with opportunistic viral infections like CMV.
Keywords: Cytokines, human immunodeficiency virus-1, opportunistic viruses, real time polymerase chain reaction
|How to cite this article:|
Sachithanandham J, Ramalingam V V, Raja J, Abraham O C, Pulimood S A, Kannangai R. Expression of cytokine-mRNA in peripheral blood mononuclear cell of human immunodeficiency virus-1 subtype C infected individuals with opportunistic viral infections from India (South). Indian J Med Microbiol 2016;34:76-81
|How to cite this URL:|
Sachithanandham J, Ramalingam V V, Raja J, Abraham O C, Pulimood S A, Kannangai R. Expression of cytokine-mRNA in peripheral blood mononuclear cell of human immunodeficiency virus-1 subtype C infected individuals with opportunistic viral infections from India (South). Indian J Med Microbiol [serial online] 2016 [cited 2019 Nov 14];34:76-81. Available from: http://www.ijmm.org/text.asp?2016/34/1/76/174118
| ~ Introduction|| |
One of the factors that can affect the disease progression in human immunodeficiency virus-1 (HIV-1) infected individuals is the alteration in the cytokine level. To some extent, this cytokine profile depends on the virological, immunological features and the presence or absence of opportunistic viral infections in an infected individual. During the HIV-1 disease progression, the level of TH1 profile cytokines: Interleukin-2 (IL-2) and INF-gamma has been shown to be decreased and the level of TH2 immune cytokine profile: Tumour necrosis factor-alpha (TNF-α), IL-1, IL-4, IL-6, IL-8, and IL-10 shown to be increased. Among HIV infected individuals, the imbalance between TH1 and TH2 cytokine profiles is associated with enhanced severity of the disease.,, IL-21 is primarily secreted by CD4 + T cells and it is a multifunctional and a pleiotropic cytokine. It has a special relevance to HIV-1 infection. During HIV-1 disease progression, the level of the IL-21 is decreased due to the severe depletion of the CD4+ T cells, thus the decreased levels of IL-21 cytokine could act as a reliable biomarker for the progression towards AIDS. Most of the studies reported on the levels of cytokines till date was either the plasma level or the mRNA expression in stimulated peripheral blood mononuclear cell (PBMC). There are only a few studies that have looked at the level of mRNA expression of cytokines in un-stimulated PBMC. Hence, the aim of this study was to look at the real time level of mRNA expression of different cytokines such as TNF-α, INF-gamma, IL-10 and recently identified IL-21 in the un-stimulated PBMC among the HIV infected individuals and its association with CD4+ T cells. This study also looked at any association between the viral load levels of opportunistic infections with viruses such as cytomegalovirus (CMV) and Epstein–Barr virus (EBV) which reactivates during immunosuppression.
| ~ Materials and Methods|| |
The study was carried out at the Department of Clinical Virology of a Tertiary Care Hospital in South India with the approval from its Institutional Review Board. All the individuals were recruited into the study after obtaining informed consent. All the antiretroviral therapy (ART) naive HIV infected study subjects were categorized into two groups based on CD4 counts; group-1 <300 cells/µl and group-2 >350 cells/µl. 9 ml of whole blood was collected in a sterile K2 EDTA BD vacutainers (New Jersey, USA) from HIV infected and normal healthy individuals. The whole blood samples were stored in multiple aliquots of 400 µl at −70°C until testing. The PBMCs were processed using the Ficoll-Paque (GE Healthcare, Uppsala, Sweden) and stored at −70°C for a maximum period of 8 months until testing. The CD4+ T cell counts for all the study participants were performed using the standard procedures by BD FACS Count System with FACS Count CD4/CD4 SW, Version 1.0. The detection and quantitation (DNA) of opportunistic viruses was done as reported earlier.
mRNA extraction from peripheral blood mononuclear cells and DNase treatment
mRNA was extracted from PBMCs using RNA easy mini kit (Qiagen, Hilden, Germany). The extraction was carried out strictly as per the manufacturer's instructions. The eluted mRNA was immediately processed by DNase I treatment to remove possible contaminating DNA followed by cDNA synthesis.
cDNA synthesis was performed in a reaction volume of 15 µl in duplicate containing 20 picomoles of 2 µl random primers (InVitrogen, USA), 20 units of 0.5 µl RNase OUT inhibitor (InVitrogen, USA) 4 µl of ×5 first strand buffer (InVitrogen, USA), 2 µl of dithiothreitol, 0.8 µl of dNTPs, 0.2 µl of M-MLV reverse transcriptase and 5 µl of treated RNA. Reverse transcription was carried out in a thermal cycler (Veriti, Applied biosystem, USA) at 37°C for 60 min and the reaction was terminated at 95°C for 5 min.
Cytokine quantitative real time polymerase chain reaction
The cytokine quantification was carried out for all the four cytokines, TNF-α, INF-gamma, IL-10 and IL-21 by real time polymerase chain reaction (PCR) based on the TaqMan principle following the same methodology as reported earlier. The primers and probes for the quantification were chosen from the previously published literature., The endogenous retrovirus-3 (ERV-3) quantitation was done for all the study samples and the copy number calculated for the cytokines was based on the ERV-3 copies per 10,000 cells. The same protocol used for cytokine quantification was also followed for ERV-3 quantitation.
Human immunodeficiency virus sub typing
Among the 64 samples, randomly collected 35 (55%) samples were subjected to polPCR (1800 bp). The analysis and interpretation of HIV-1 sub typing determined based on the sequencing of PCR amplified products targeting polregion using the REGA HIV-1 subtyping tool - version 2.0 in HIV drug resistance stanford data base (http://www.hivdb.stanford.edu).
The two-tailed t-test probability and Welch test (for assuming the unequal variance of the independent sample) were used to analyse the difference in copies level between cytokines among two groups of HIV-1 infected individuals and controls. Spearman's coefficient of rank correlation test was used for comparison of cytokines, CD4 counts, EBV and CMV load. All the statistical analysis was done using the medical software version 126.96.36.199. (http://www.mescalc.be). A P < 0.05 was considered as significant.
| ~ Results|| |
Among the 32 HIV-1 infected individuals who were stratified based on the CD4 counts of >350 cells/µl, 16 were female (mean age ± standard deviation [SD] 34.5 ± 6.0 years) and 16 were male (mean age ± SD 36.8 ± 7.9 years). The 32 HIV-1 infected individuals who had a CD4 counts with <300 cells/µ l, 11 were female (mean age ± SD 41.1 ± 12.0 years) and 21 were male (mean age ± SD 41.6 ± 9.5 years). Among the 20 healthy controls, 9 were female (mean age ± SD 33.6 ± 3.5 years) and 11 were male (mean age ± SD 33.2 ± 2.4 years). All the 35 (100%) studied HIV strains were HIV-1 subtype C.
The multiplex real time PCR showed positive for viruses such as EBV, CMV, human herpesvirus-6 (HHV-6) and BK virus in whole blood samples while viruses such as herpes simplex virus-1 (HSV-1), HSV-2, varicella-zoster virus, HHV-8, adenovirus and JC virus were negative. The real time multiplex PCR percentage positivity of opportunistic viruses from the whole blood samples in the HIV infected and healthy individuals is shown in [Table 1].
|Table 1: The number, percentage positives and their mean viral loads detected by real time multiplex PCR from the whole blood samples in the HIV infected and healthy individuals|
Click here to view
The mRNA expression of all the 4 cytokines were significantly higher in both groups of HIV infected individuals compared to healthy controls (P = 0.0004–0.01). The INF-gamma and IL-10 levels in HIV-1 infected individuals with CD4 counts <300 cells/µl was significantly higher compared to group with CD4 counts >350 cells/µl (P value range from 0.05 to 0.005). The mRNA expression of various cytokines in un-stimulated PBMC among the controls and HIV-1 infected individuals are shown in [Table 2] and [Figure 1].
|Table 2: The mRNA level in PBMC of different cytokines (TNF-α, INF-γ, IL-10 and IL-21) among HIV-1 infected and healthy controls|
Click here to view
|Figure 1: The cytokine mRNA expression in a peripheral blood mononuclear cell among human immunodeficiency virus-1 infected individuals and normal healthy controls. (a) Tumor necrosis factor-alpha, (b) interferon-gamma, (c) interleukin-10, (d) interleukin-21|
Click here to view
The viral load levels of EBV and CMV in whole blood samples was also analysed to assess the association between the expressions of cytokines. Since the positive samples with HHV-6 and BK viruses were very few no analysis was carried out on these two viruses. All CMV positive individuals are having CD4 counts <300 cells/µl were identified as WHO clinical stage III and IV. The viral load levels of EBV and CMV was decided to have a cut-off of 1000 copies/ml in this study as in transplant patients who were immunosuppressed a copy number of 1000 copies/ml was considered as significant for CMV viral load. The expression of IL-21 showed a significantly higher level (P = 0.01) in individuals with CMV viral load <1000 compared to those with CMV load of >1000 copies/ml. The other cytokines fail to show a significant difference in the expression between the two viral load groups.
The overall correlation was estimated between each cytokine and other parameters such as CD4 counts, EBV and CMV viral load among all HIV infected individuals. IL-21 expression showed a significant positive correlation with CD4+ T cell counts (r = 0.26, P = 0.03) and interferon-gamma IFN- γ (r = 0.50, P = 0.0001) while significant negative correlation with CMV viral load (r = −0.28, P = 0.02). IL-10 expression showed a significant positive correlation with IFN-γ (r = 0.53, P = 0.0001) and TNF-α (r = 0.56, P < 0.0001) and a negative correlation with CD4 counts (−0.25 P = 0.04). EBV and CMV viral load showed significant negative correlation with CD4 counts (r = −0.34, P = 0.006 and r = –0.62, P ≤ 0.0001, respectively). This data are shown in [Table 3].
|Table 3: Correlation observed different cytokines (TNF-α, INF-γ, IL-10 and IL-21) with levels of CD4 T cell counts EBV and CMV viral loads among HIV-1 infected individuals|
Click here to view
| ~ Discussion|| |
The study reported here is on the expression of 4 cytokines in un-stimulated PBMC from HIV infected individuals and its association with CD4+ T cell counts and the concurrent opportunistic DNA viral infections with two major opportunistic DNA viruses such as EBV and CMV. There was a significant negative correlation observed for IL-10 cytokine expression with CD4+ T counts which may attributed to the shift toward the TH2/TH0 cytokine profile when the HIV infected individuals progress with the loss of CD4+ T cells. In the study reported here there was a significantly higher mRNA expression of IFN-γ in individuals with lower CD4+ T cell counts but the correlation with CD4+ T cell count was not significant. A previous study reported from our centre showed a significantly higher plasma IL-10 concentration among HIV infected individuals compared to normal healthy controls. However, there was no significant correlation of plasma IL-10 concentration with CD4+ T cell counts. That study also showed a significant negative correlation of plasma IFN-γ with CD4+ T cell absolute count and a positive correlation with plasma RNA levels (P = 0.017). A recent study from Mumbai had detected the mRNA levels of cytokines like TNF-α, INF-gamma, IL-10 and IL-4 in un-stimulated PBMC samples from HIV infected children by a semi-quantitative assay. They reported an increase in the IL-10, TNF-α expression with progression of disease while a decrease of INF-gamma mRNA expression with the HIV disease progression.
There are studies which have reported that the level of IL-21 decreased during the HIV disease progression.,, In one study, where in both longitudinal and cross-sectional data on the IL-21 production was analysed in HIV infected individuals from different clinical stages showed that only the Elite controllers (long-term non-progressors in whom viral load is undetectable) maintain normal production of the IL-21 cytokine and in individuals who were on highly active antiretroviral therapy (HAART) it was partially restored. The study also reported decreased serum levels of IL-21 in HIV infected individuals compared with that observed in HIV sero-negative healthy subjects. Our study also showed a significant decrease in the expression of IL-21 levels in HIV infected individuals with CD4 counts <300 cells/µl when compared to individuals with CD4 counts >350 cells/µl and also showed a positive correlation (r = 0.26, P = 0.03) with CD4 counts.
Most of the cytokine expression studies among HIV infected individuals were done using the stimulated PBMC because cytokines are detectable at the protein level only after the stimulation of these cells due to their short half-life. In contrast, measuring the cytokine mRNA expression in un-stimulated PBMC gives the exact information about that particular stage of infection and reflects on the in vivo cytokine production. Imami et al., from UK had utilized the un-stimulated PBMC for the assessment of cytokine expression of IFN-γ, IL-2, IL-4 and IL- 10 in HIV-1 infected individuals before and after the initiation of HAART. This study observed a continuous increase in IFN-γ and IL-2 mRNA expression as well as the significant reduction in HIV viral load and increase in CD4+ T cell counts following HAART. It shows that carrying out the mRNA expression on un-stimulated PBMC will give the real time status of the infection. In our study, we have estimated the cytokines by looking at the mRNA expression in un-stimulated PBMCs and found almost similar findings related to cytokine mRNA expression and disease stage. Few limitations of this study were HIV-1 viral load data was not available for all the study participants and no attempt was made to look at any opportunistic bacterial/parasitic/fungal infections in these individuals.
| ~ Conclusion|| |
The present study is one of the few to report changes in the cytokine levels in un-stimulated PBMCs as measured in mRNA avoiding the inherent problem of measuring plasma levels which are likely to be affected by moiety decay and antibodies to cytokines and soluble receptors which mop-up the free cytokines that interfere with in vitro assay. This study also showed the importance of IL-21 cytokine levels as a reliable biomarker for the progression of the disease and also adding new information on cytokine and viral opportunistic infections among ART naive HIV-1 subtype C infected individuals.
We gratefully acknowledge the funding received from Intramural (Christian Medical College, Vellore) support. The data presented in the manuscript form part of the Ph.D. thesis of Jaiprasath Sachithanandham. We would also sincerely thank Professor. Gopalan Sridharan for the help in designing the study and reading the manuscript and for his valuable comments.
Financial support and sponsorship
Intramural, Christian Medical College, Vellore.
Conflicts of interest
There are no conflicts of interest.
| ~ References|| |
Kedzierska K, Crowe SM. Cytokines and HIV-1: Interactions and clinical implications. Antivir Chem Chemother 2001;12:133-50.
Clerici M, Shearer GM. A TH1-->TH2 switch is a critical step in the etiology of HIV infection. Immunol Today 1993;14:107-11.
Kidd P. Th1/Th2 balance: The hypothesis, its limitations, and implications for health and disease. Altern Med Rev 2003;8:223-46.
Clerici M, Hakim FT, Venzon DJ, Blatt S, Hendrix CW, Wynn TA, et al.
Changes in interleukin-2 and interleukin-4 production in asymptomatic, human immunodeficiency virus-seropositive individuals. J Clin Invest 1993;91:759-65.
Iannello A, Tremblay C, Routy JP, Boulassel MR, Toma E, Ahmad A. Decreased levels of circulating IL-21 in HIV-infected AIDS patients: Correlation with CD4+T-cell counts. Viral Immunol 2008;21:385-8.
Marteau JB, Mohr S, Pfister M, Visvikis-Siest S. Collection and storage of human blood cells for mRNA expression profiling: A 15-month stability study. Clin Chem 2005;51:1250-2.
Pattanapanyasat K, Sukapirom K, Kowawisatsut L, Thepthai C. New BD FACSCount CD4 reagent system for simultaneous enumeration of percent and absolute CD4 T-lymphocytes in HIV-1-infected pediatric patients. Cytometry B Clin Cytom 2008;74 Suppl 1:S98-106.
Sachithanandham J, Kannangai R, Pulimood SA, Desai A, Abraham AM, Abraham OC, et al.
Significance of Epstein-Barr virus (HHV-4) and CMV (HHV-5) infection among subtype-C human immunodeficiency virus-infected individuals. Indian J Med Microbiol 2014;32:261-9.
Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C. An overview of real-time quantitative PCR: Applications to quantify cytokine gene expression. Methods 2001;25:386-401.
Jüngel A, Distler JH, Kurowska-Stolarska M, Seemayer CA, Seibl R, Forster A, et al.
Expression of interleukin-21 receptor, but not interleukin-21, in synovial fibroblasts and synovial macrophages of patients with rheumatoid arthritis. Arthritis Rheum 2004;50:1468-76.
Ramalingam S, Kannangai R, Vijayakumar TS, Mathai D, Abraham OC, Subramanian S, et al.
Subtype and cytokine profiles of HIV infected individuals from south India. Indian J Med Res 2005;121:226-34.
Kothari ST, Deshmukh RA. Estimation of mRNA levels of interleukin (IL)-10, tumor necrosis factor (TNF)-a, IL-4 and interferon (IFN)-γ in HIV infected children in Mumbai. Indian J Clin Biochem 2006;21:15-26.
Iannello A, Boulassel MR, Samarani S, Debbeche O, Tremblay C, Toma E, et al.
Dynamics and consequences of IL-21 production in HIV-infected individuals: A longitudinal and cross-sectional study. J Immunol 2010;184:114-26.
Pallikkuth S, Parmigiani A, Pahwa S. The role of interleukin-21 in HIV infection. Cytokine Growth Factor Rev 2012;23:173-80.
Altfeld M, Addo MM, Kreuzer KA, Rockstroh JK, Dumoulin FL, Schliefer K, et al.
T (H) 1 to T (H) 2 shift of cytokines in peripheral blood of HIV-infected patients is detectable by reverse transcriptase polymerase chain reaction but not by enzyme-linked immunosorbent assay under nonstimulated conditions. J Acquir Immune Defic Syndr 2000;23:287-94.
Imami N, Antonopoulos C, Hardy GA, Gazzard B, Gotch FM. Assessment of type 1 and type 2 cytokines in HIV type 1-infected individuals: Impact of highly active antiretroviral therapy. AIDS Res Hum Retroviruses 1999;15:1499-508.
Aziz N, Nishanian P, Mitsuyasu R, Detels R, Fahey JL. Variables that affect assays for plasma cytokines and soluble activation markers. Clin Diagn Lab Immunol 1999;6:89-95.
[Table 1], [Table 2], [Table 3]