|Year : 2017 | Volume
| Issue : 4 | Page : 580-584
Effect of Interleukin-28B polymorphism on Interleukin-28 expression and immunological recovery amongst HIV-1-infected individuals following antiretroviral therapy
BV Srinidhi1, G John Fletcher1, Jaiprasath Sachidanantham1, Priscilla Rupali2, Veena Vadhini Ramalingam1, JP Demosthenes1, OC Abraham2, Susanne A Pulimood3, Grace Rebekah4, Rajesh Kannangai1
1 Department of Clinical Virology, Christian Medical College, Vellore, Tamil Nadu, India
2 Department of Medicine and Infectious Diseases, Christian Medical College, Vellore, Tamil Nadu, India
3 Department of Dermatology, Christian Medical College, Vellore, Tamil Nadu, India
4 Department of Biostatistics, Christian Medical College, Vellore, Tamil Nadu, India
|Date of Web Publication||1-Feb-2018|
Dr. Rajesh Kannangai
Department of Clinical Virology, Christian Medical College, Vellore - 632 004, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Purpose: Type III interferon is well known to have diverse antiviral and immunomodulatory activities. Studies describing the association of interleukin (IL)-28 polymorphisms in treatment-experienced HIV participants are limited. This study was aimed to determine the association of IL-28B gene polymorphisms with immunological recovery in HIV patients on 6–9 months of antiretroviral therapy (ART). Methods: Eighty treatment-naive HIV patients were recruited, of which 48 patients were followed up after 6–9 months of ART. Whole blood samples were collected before and after 6–9 months of ART. CD4, CD8 and CD3 counts were enumerated flow cytometry. IL-28B polymorphisms (rs12979860 and rs8099917) were profiled by polymerase chain reaction (PCR)-restriction fragment length polymorphism. The IL-28 mRNA and plasma HIV-1 viral load were estimated using real-time PCR and plasma IL-28 level by ELISA. Results: The CD4, CD4/CD3%, IL-28 mRNA and reversal of CD4/CD8 ratio were significantly increased following 6–9 months of ART (P < 0.01). The rs12979860 CC genotype and rs12979860:rs8099917 (CC: TT) haplotype showed significant association with higher CD4+ T-cell count amongst treatment-naive HIV-infected individuals (P < 0.05). In addition, there was a significant association of rs12979860 CC genotype with increase in CD4/CD3% following 6–9 months of ART. IL-28 mRNA showed correlation with the HIV-1 viral load, and there was a significant increase in the IL-28 mRNA expression following 6–9 months of ART. Conclusion: Our preliminary findings suggest that IL-28 polymorphisms could influence both immunological recovery and therapeutic response in HIV infection. Hence, functional studies are warranted to understand the mechanistic basis of IL-28-mediated host genetic influence on HIV therapeutic response.
Keywords: CD4+ and CD8+ T-cell count, HIV, interleukin-28B mRNA, interleukin-28B polymorphism
|How to cite this article:|
Srinidhi B V, Fletcher G J, Sachidanantham J, Rupali P, Ramalingam VV, Demosthenes J P, Abraham O C, Pulimood SA, Rebekah G, Kannangai R. Effect of Interleukin-28B polymorphism on Interleukin-28 expression and immunological recovery amongst HIV-1-infected individuals following antiretroviral therapy. Indian J Med Microbiol 2017;35:580-4
|How to cite this URL:|
Srinidhi B V, Fletcher G J, Sachidanantham J, Rupali P, Ramalingam VV, Demosthenes J P, Abraham O C, Pulimood SA, Rebekah G, Kannangai R. Effect of Interleukin-28B polymorphism on Interleukin-28 expression and immunological recovery amongst HIV-1-infected individuals following antiretroviral therapy. Indian J Med Microbiol [serial online] 2017 [cited 2020 Jan 22];35:580-4. Available from: http://www.ijmm.org/text.asp?2017/35/4/580/224434
| ~ Introduction|| |
Interferons (IFNs) modulate innate and adaptive immune responses in viral infections. Human IFNs are classified as type I, II and III based on the type of receptors used for signalling. Type III IFNs (also known as IFN-λ) are composed of three structure-related cytokines, i.e. IFN-λ1 (interleukin [IL]-29), IFN-λ2 (IL-28A) and IFN-λ3 (IL-28B). IFN-λ signals through a receptor complex containing IL-10Rb and IL-28Ra., Peripheral blood mononuclear cells (PBMCs) and dendritic cells are considered to be the major producers of IFN-λ. IFN-λ is known to inhibit replication in a range of viruses, including hepatitis C virus (HCV) and hepatitis B virus, influenza virus, rotavirus, herpes simplex virus-1 and herpes simplex virus-2, cytomegalovirus and West Nile virus.,,,,
Four landmark studies published in 2009 described a clinical association of sustained virological response and spontaneous clearance of HCV in those with the CC genotype of the rs12979860 single-nucleotide polymorphism (SNP) on chromosome 19q13, 3 kb upstream of the IL-28B gene, which encodes IFN-λ3.,,,
Studies on the association of IL-28B polymorphism in HIV patients are contradicting and limited. Based on the anti-HCV properties of IFN-λ and the association of IL-28B polymorphism with better treatment outcome in HCV infection, in this study, we looked at the association of IL-28B polymorphism and immune recovery amongst HIV-1-infected individuals following 6–9 months of antiretroviral therapy (ART). It is also important to know the effect of these polymorphisms and the IL-28B cytokine level both in the expression and plasma level as these cytokines are found to have antiviral effect. In this reported study, an effort was also made in this direction.
| ~ Materials and Methods|| |
Subjects and study design
This longitudinal study was conducted at a tertiary care hospital in South India between August 2013 and September 2015 approved by the Institutional Review Board. Informed consent was obtained from all individual participants included in the study. Whole blood samples were collected in ethylenediaminetetraacetic acid tubes from eighty treatment-naive HIV-1-infected individuals and thirty HIV-negative controls. PBMCs were separated from whole blood using Ficoll-Paque™ PLUS (GE Healthcare, Uppsala, Sweden) and stored at −80°C in RNA later solution.
CD4+/CD8+ T-cell counts and CD3+ T-cell estimation were performed on blood samples collected by BD FACScount flow cytometer (Becton Dickinson, New Jersey, USA) as per the manufacturer's instruction as reported earlier.
Genomic DNA was extracted from PBMCs using QIAamp ® DNA Blood Mini Kit (Qiagen, Hilden, Germany) as per manufacturer's protocol. The extracted DNA was quantified spectrophotometrically using Take3, Gen5™, Biotek. IL-28B genotyping was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism. The digestion of the amplicons was carried out using restriction endonucleases BstUI and BsrDI (New England Biolabs, UK) for rs12979860 and rs8099917, respectively. The digested products were electrophoresed on a 3% agarose gel and the polymorphisms were identified [Figure 1].
|Figure 1: A representative gel picture of restriction fragment length polymorphism for detection of rs12979860 and rs8099917 genotypes|
Click here to view
IL-28 plasma level was estimated by a human IL-28B in-house quantitative ELISA (R and D Systems, Minneapolis, MN, USA) as per manufacturer's instructions. The average of concentration for each sample was calculated using standards in seven dilutions and zero standards; a four-parameter logistic curve was generated with concentrations on X-axis and delta optical density values on Y-axis using Gen5 software (Biotek, Winooski, USA) and concentration was expressed in pg/mL.
Interleukin-28 real-time polymerase chain reaction
Total cellular RNA was extracted using QIAamp ® RNeasy mini kit (Qiagen, Germany) according to the manufacturer's instructions. The following IL-28A/B consensus primers, outer forward (5'-AGTGCTGACCGTGACTGG-3'), outer reverse (5'-AGGTTAAGGTGACAGAGGC-3'), inner forward (5'-GCCACATAGCCCAGTTCAAG-3') and inner reverse (5'-GGGAGAGGATATGGTGCAGG-3'), were used for plasmid construction, and IL-28 mRNA expression detection was carried out by real-time PCR assay using the following primers, forward (5'-GCTGAAGGTTCTGGAGGCC-3') and reverse (5'-GGATATGGTGCAGGGTGTGA-3') in Rotor-Gene 6000 (Qiagen, Germany). For the quantitation, a standard curve was generated using the IL-28 plasmid cloned using Topo TA Clone/TOP10 (Invitrogen, USA) and was diluted serially 10-fold dilutions of Milli-Q water to obtain PCR standards.
HIV-1 viral load
The total RNA was extracted from plasma by Abbott m2000sp automated system (Abbott Diagnostics Gmbh, Wiesbaden, Germany). Following extraction, all the processed samples were then transferred to the Abbott m2000sp automated system and processed as per the manufacturer's instructions. The lower limit of detection of this assay is 40 copies/ml.
Continuous variables such as CD4+, CD8+, CD3+ T-cell counts, CD4/CD8 ratio, IL-28 mRNA, IL-28B plasma level and HIV-1 viral load were reported as mean ± standard deviation or median. Continuous variables of the follow-up after initiation of ART were analysed using paired t-test. The difference in the distribution of variables was checked using Kruskal–Wallis equality-of-populations rank test. Independent variables were analysed using Mann–Whitney U-test. Chi-square test was used to analyse the frequencies of IL-28B genotype/haplotype therapeutic response. P ≤ 0.05 was used as the criterion for statistical significance.
| ~ Results|| |
Amongst 80 HIV-infected individuals recruited from the four South Indian states, forty-eight HIV-1-infected individuals who fulfilled the inclusion criteria were started on ART and were followed up for 6–10 months. Of 48 HIV-infected individuals, 28 were male and 20 were female. The mean age of these 48 individuals was 43.27 years (range = 23–65) while that of the 30 controls was 41.93 years (range = 24–65).
Frequency distribution of interleukin-28B genotypes and haplotypes
Amongst the 48 HIV-infected individuals, the frequency distribution of rs1297960 genotype was CC 32 (67%), CT 14 (29%) and TT 2 (4%). Likewise, the frequency distribution of rs8099917 genotypes was TT 39 (81%), GT 8 (17%) and GG 1 (2%). The frequency of haplotypes in HIV-infected individuals was CC/TT 31 (64.5%), CT/GT 7 (14.5%), CT/TT 7 (14.5%), TT/GG 1 (2%) and TT/GT 1 (2%). One individual with homozygous TT genotype of rs12979860 SNP and homozygous GG genotype of rs8099917 SNP developed immune reconstitution inflammatory syndrome (IRIS). A representative gel with different patterns of bands showing genotypes is shown in [Figure 1].
Amongst the 30 controls, the frequency of genotypes at rs1297960 were CC 18 (60%), CT 10 (33%) and TT 2 (7%), and the frequency of genotypes at rs8099917 was TT 23 (77%), GT 7 (23%) and GG 0 (0%). The frequency of haplotypes in controls was CC/TT 15 (52%), CT/TT 7 (24%), CT/GT 3 (10%) and TT/GT 1 (4%).
There was no significant association of genotypes rs12979860 and rs8099917 with the median absolute CD4+, CD8+ T-cell count, IL-28B plasma level, IL-28 mRNA and HIV-1 viral load. However, the wild genotype CC at rs12979860 showed a significantly higher median CD4+ T-cell count when compared to other genotypes CT and TT before and after ART as shown in [Figure 2]a and [Figure 2]b (P = 0.03 and 0.04). Both wild C allele and CC genotype of rs12979860 were significantly associated with higher CD4/CD3% following 6–9 months of ART as shown in [Figure 2]c and [Figure 2]d (P = 0.01). IL-28 mRNA expression was significantly lower amongst HIV-infected individuals compared to the healthy controls and showed a significant correlation with the HIV-1 viral load. There was a significant increase in the IL-28 mRNA expression following 6–9 months of ART as shown in [Figure 3]. There was a reciprocal association of IL-28 mRNA expression with HIV viral replication in treatment-naive individuals (P ≤ 0.001).
|Figure 2: Box-and-whisker plot showing the association of absolute CD 4+ T-cell count and CD4% between CC and CC/TT genotypes before (a and c) and after (b and d) 6 to 9 months of antiretroviral therapy|
Click here to view
|Figure 3: Box-and-whisker plot showing the association of interleukin-28 expression before and after antiretroviral therapy|
Click here to view
| ~ Discussion|| |
The genetic influence of IL-28B polymorphism on HCV pathogenesis and outcome are strongly established in various populations.,,,, This study was undertaken because both HCV and HIV are RNA viruses involved in chronic pathology, and there is also paucity of studies on the influence of IL-28B polymorphism on HIV pathogenesis.
The frequency distribution of IL-28 genotypes rs12979860 and rs8099917 was comparable to several other studies published previously in the Indian population.,, The predominant rs12979860 wild CC genotype showed a significant association with higher CD4+ T-cell count compared to other genotypes (C/T and T/T) in treatment-naive and ART-experienced individuals. This association is consistent because Machmach et al. showed a similar association of rs12979860 CC genotype with spontaneous control of HIV. Hence, we speculate that the predominance of rs12979860 CC genotype may have certain functional relevance in HIV pathogenesis, and it can confer selective advantage in the Indian population. It is noteworthy that a patient with homozygous recessive genotype at both SNPs (rs12979860TT: rs8099917GG) developed IRIS with Pneumocystis carinii pneumonia. This clinical outcome requires validation to identify whether recessive homozygosity has any genetic preponderance to the development of IRIS in the Indian population. Ogola et al. reported that the non-synonymous variant rs1884444 T>G of IL-23R is associated with a decreased risk to schistosomiasis-associated IRIS.
In the present study, IL-28 mRNA expression was significantly higher in healthy controls compared to HIV-infected individuals. An earlier study showed that HIV-1-infected individuals with CD4+ T-cell numbers >800 cells/μL retained a similar level of plasma IFN-λ1 than the uninfected controls, and IL-λ1 was significantly elevated with the depletion of CD4+ T-cells in HIV-1-infected individuals. The IFN-λ1 production was dropped when CD4 numbers were lower than 200/μL; the reason may be due to defective cytokine secretion or the persistent depletion of blood myeloid cells, which are the main producers of IFN-λ. In this study, we could not find any significant (P ≥ 0.05) correlation of IL-28 mRNA expression and IL-28B plasma level with the CD4+, CD8+ absolute T-cell counts and CD4/CD8 ratio amongst treatment-naive HIV-infected individuals and following 6–9 months of ART. The lack of association could be because of mononuclear cells, and dendritic cells are considered to be the major producers of IFN-λ. Chi et al. reported that respiratory syncytial virus-infected monocyte-derived dendritic cells secrete IFN-λ that limits the in vitro proliferation of CD4+ T-cells. Misumi and Whitmire reported that IFN-λR limits T-cell responses and memory following transient infection but augments T-cell responses during persisting infection.
The transcriptional and translational influence of the IL-28B SNPs on IL-28B (IFN-λ3) production and activity is still inconclusive. We found no influence of IL-28B SNPs with IL-28 mRNA and plasma levels amongst treatment-naive and ART-experienced individuals. However, IL-28B mRNA expression showed significant (P ≤ 0.001) reciprocal correlation with HIV-1 viral load in treatment-naive individuals. Functionally, it is plausible because in vitro studies had shown antiviral effects of IFN-λ on HIV replication.,, Identifying the precise antiviral mechanisms of IFN-λ may contribute to emerging therapeutics in HIV infection.
| ~ Conclusion|| |
Our preliminary findings suggest that SNPs at rs12979860 and rs8099917 of IL-28B influence the CD4+ T-cells level prior and post-ART. IL-28 mRNA expression influences HIV viral replication kinetics before ART. The intricate genetic and functional link between Il-28B and immune recovery and therapeutic response requires further detailed studies to understand the precise mechanisms of IFN-λ-mediated immunopathogenesis in HIV infection.
Financial support and sponsorship
Acknowledge the financial support from Fluid Research Fund (IRB Ref Min No. 8237) and Special fund Department of Clinical Virology, Christian Medical College, Vellore.
Conflicts of interest
There are no conflicts of interest.
| ~ References|| |
Sheppard P, Kindsvogel W, Xu W, Henderson K, Schlutsmeyer S, Whitmore TE, et al.
IL-28, IL-29 and their class II cytokine receptor IL-28R. Nat Immunol 2003;4:63-8.
Hou W, Wang X, Ye L, Zhou L, Yang ZQ, Riedel E, et al.
Lambda interferon inhibits human immunodeficiency virus type 1 infection of macrophages. J Virol 2009;83:3834-42.
Doyle SE, Schreckhise H, Khuu-Duong K, Henderson K, Rosler R, Storey H, et al.
Interleukin-29 uses a type 1 interferon-like program to promote antiviral responses in human hepatocytes. Hepatology 2006;44:896-906.
Diegelmann J, Beigel F, Zitzmann K, Kaul A, Göke B, Auernhammer CJ, et al.
Comparative analysis of the lambda-interferons IL-28A and IL-29 regarding their transcriptome and their antiviral properties against hepatitis C virus. PLoS One 2010;5:e15200.
Wang J, Oberley-Deegan R, Wang S, Nikrad M, Funk CJ, Hartshorn KL, et al.
Differentiated human alveolar type II cells secrete antiviral IL-29 (IFN-lambda 1) in response to influenza A infection. J Immunol 2009;182:1296-304.
Jewell NA, Cline T, Mertz SE, Smirnov SV, Flaño E, Schindler C, et al.
Lambda interferon is the predominant interferon induced by influenza A virus infection in vivo
. J Virol 2010;84:11515-22.
Ank N, Iversen MB, Bartholdy C, Staeheli P, Hartmann R, Jensen UB, et al.
An important role for type III interferon (IFN-lambda/IL-28) in TLR-induced antiviral activity. J Immunol 2008;180:2474-85.
Suppiah V, Moldovan M, Ahlenstiel G, Berg T, Weltman M, Abate ML, et al.
IL28B is associated with response to chronic hepatitis C interferon-alpha and ribavirin therapy. Nat Genet 2009;41:1100-4.
Ge D, Fellay J, Thompson AJ, Simon JS, Shianna KV, Urban TJ, et al.
Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature 2009;461:399-401.
Tanaka Y, Nishida N, Sugiyama M, Kurosaki M, Matsuura K, Sakamoto N, et al.
Genome-wide association of IL28B with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis C. Nat Genet 2009;41:1105-9.
Thomas DL, Thio CL, Martin MP, Qi Y, Ge D, O'Huigin C, et al.
Genetic variation in IL28B and spontaneous clearance of hepatitis C virus. Nature 2009;461:798-801.
Machmach K, Abad-Molina C, Romero-Sánchez MC, Abad MA, Ferrando-Martínez S, Genebat M, et al.
IL28B single-nucleotide polymorphism rs12979860 is associated with spontaneous HIV control in white subjects. J Infect Dis 2013;207:651-5.
Ramalingam VV, Mani M, Sundaresan VC, Karunaiya RJ, Sachithanandham J, Kannangai R, et al.
Daily quality control in CD3+ and CD4+ T cell estimation by the FACSCount system at a tertiary care center in South India. Clin Vaccine Immunol 2012;19:1693-6.
Sharafi H, Pouryasin A, Alavian SM, Behnava B, Keshvari M, Mehrnoush L, et al.
Development and validation of a simple, rapid and inexpensive PCR-RFLP method for genotyping of common IL28B polymorphisms: A Useful pharmacogenetic tool for prediction of hepatitis C treatment response. Hepat Mon 2012;12:190-5.
Ding Q, Huang B, Lu J, Liu YJ, Zhong J. Hepatitis C virus NS3/4A protease blocks IL-28 production. Eur J Immunol 2012;42:2374-82.
David S, Sachithanandham J, Jerobin J, Parasuram S, Kannangai R. Comparison of HIV-1 RNA level estimated with plasma and DBS samples: A pilot study from India (South). Indian J Med Microbiol 2012;30:403-6.
] [Full text]
Honda M, Shirasaki T, Shimakami T, Sakai A, Horii R, Arai K, et al.
Hepatic interferon-stimulated genes are differentially regulated in the liver of chronic hepatitis C patients with different interleukin-28B genotypes. Hepatology 2014;59:828-38.
Sivaprasad S, Rao PN, Gupta R, Ashwini K, Reddy DN. The distribution of genotype and allelic frequency of IL28B gene polymorphism in Andhra Pradesh, India. J Clin Exp Hepatol 2012;2:112-5.
Firdaus R, Biswas A, Saha K, Mukherjee A, Chaudhuri S, Chandra A, et al.
Impact of host IL28B rs12979860, rs8099917 in interferon responsiveness and advanced liver disease in chronic genotype 3 hepatitis C patients. PLoS One 2014;9:e99126.
Gupta AC, Trehanpati N, Sukriti S, Hissar S, Midha V, Sood A, et al.
Interleukin-28b CC genotype predicts early treatment response and CT/TT genotypes predicts non-response in patients infected with HCV genotype 3. J Med Virol 2014;86:707-12.
Ogola GO, Ouma C, Jura WG, Muok EO, Colebunders R, Mwinzi PN, et al.
A non-synonymous polymorphism in IL-23R gene (rs1884444) is associated with reduced risk to schistosomiasis-associated immune reconstitution inflammatory syndrome in a Kenyan population. BMC Infect Dis 2014;14:316.
Tian RR, Guo HX, Wei JF, Yang CK, He SH, Wang JH, et al.
IFN-λ inhibits HIV-1 integration and post-transcriptional events in vitro
, but there is only limited in vivo
repression of viral production. Antiviral Res 2012;95:57-65.
Chi B, Dickensheets HL, Spann KM, Alston MA, Luongo C, Dumoutier L, et al.
Alpha and lambda interferon together mediate suppression of CD4 T cells induced by respiratory syncytial virus. J Virol 2006;80:5032-40.
Misumi I, Whitmire JK. IFN-λ exerts opposing effects on T cell responses depending on the chronicity of the virus infection. J Immunol 2014;192:3596-606.
Liu MQ, Zhou DJ, Wang X, Zhou W, Ye L, Li JL, et al.
IFN-λ3 inhibits HIV infection of macrophages through the JAK-STAT pathway. PLoS One 2012;7:e35902.
[Figure 1], [Figure 2], [Figure 3]