Association of interleukin‐27 gene polymorphisms with susceptibility to HIV infection and disease progression

Abstract Interleukin‐27 (IL‐27) gene polymorphisms are linked to infectious disease susceptibility and IL‐27 plasma level is associated with HIV infection. Therefore, we aimed to investigate the association between IL‐27 polymorphisms and susceptibility to HIV infection and disease progression. A total of 300 patients with HIV infection (48 long‐term nonprogressors and 252 typical progressors) and 300 healthy controls were genotyped for three IL‐27 polymorphisms, rs17855750, rs181206, rs40837 which were performed by using multiple single nucleotide primer extension technique. Significant association was found between IL‐27 rs40837 polymorphisms with susceptibility to HIV infection (AG vs AA: adjusted OR = 1.60, 95% CI, 1.11‐2.30, P = 0.012; AG+GG vs AA: adjusted OR = 1.44, 95% CI, 1.02‐2.03, P = 0.038) and disease progression (LTNP: AG vs AA: adjusted OR = 2.33, 95% CI, 1.13‐4.80, P = 0.021; TP: AG vs AA: adjusted OR = 1.50, 95% CI, 1.04‐2.24, P = 0.030). Serum IL‐27 levels were significantly lower in cases compared to controls (P < 0.001). There were lower serum IL‐27 levels in TPs than in LTNPs (P < 0.001). We further found that LTNPs with rs40837 AG or GG genotype had lower serum IL‐27 levels than with AA genotype (P < 0.05). The CD4+T counts in cases were significantly lower than controls (P < 0.001). In contrast, individuals with rs40837 AG genotype had lower CD4+T counts than with AA genotype in cases (P < 0.05). In addition, CD4+T counts in TPs were significantly lower than LTNPs (P < 0.001). IL‐27 rs40837 polymorphism might influence the susceptibility to HIV infection and disease progression probably by regulating the level of serum IL‐27 or the quantity of CD4+T.


| INTRODUCTION
Acquired immunodeficiency syndrome (AIDS), caused by human immunodeficiency virus (HIV) infection and characterized by reduction in CD4 + T cells and subsequent immune function defects, is a worldwide infectious disease which seriously affect patients' physical and mental health because of its incurable property. The disease progression of HIV infection includes long-term nonprogression (LTNP) and typical progression (TP) in clinic. 1 On average, most long-term nonprogressors will progress to typical progression within 10 years without antiretroviral therapy, while a small number remain no progression maintaining CD4 + T counts above (over) 350 cells/μl for decades. 2,3 Previous studies suggested that genetic variations in genes that control immune responses may play an essential role in the HIV infection and disease progression. 4,5 Therefore, the study of the association between various gene polymorphisms and HIV infection and disease progression has become a hot topic at present.
Interleukin-27 (IL-27) composed of Epstein-Barr virus-induced gene (BBI3) and p28 protein, a member of IL-12 family, 6 is a heterodimeric immunoregulatory cytokine involved in early Th1 initiation 7 and can affect T-cell proliferation and cytotoxic activity. 8 The influences of IL-27 on HIV infection and replication have been discussed, an experiment using HIV infected mice as a model proved that IL-27 could increase dendritic cell functions, in addition, a more direct function of IL-27 is performed in an in vitro test, which is the inhibition of R5 and X4 HIV replication. 9 T lymphocyte subsets are the important detection in AIDS testing laboratory. As a significant immune cell in human immune system and the target of HIV, CD4 + T can directly reflect the immune function of human body, and it is the most definite indicator of immune system damage in patients with HIV infection. Cytotoxic T lymphocytes (CTL), mainly composed of CD8 + T cells, can actively kill infected cells and suppress viral replication. 10 Thus, we can get a better understanding of immunological status of body by CD4 + T and CD8 + T counts.
In recent years, IL-27 gene polymorphisms shown in quite a few studies were associated with infectious disease susceptibility. [11][12][13] Additionally, IL-27 plasma level becomes lower in HIVinfected patients, which was proved by Zheng et al. 14    Ltd., Shenzhen, China) whose linear range is 20-1 000 000 copies/mL.

| CD4 + T and CD8 + T count
CD4 + T and CD8 + T counts in fresh whole blood were obtained by flow cytometry on BD FACScantoII automatic flow cytometry (Becton Dickinson Immunocytometry System) within eight hours.
The reagent is BD Multitest 6-colour TBNK Reagent produced by Becton, Dickinson and Company, BD Biosciences.

| Statistical analysis
Conformity of genotype distribution to Hardy-Weinbery equilibruim (HWE) was tested by comparing the observed genotype frequencies and expected genotype frequencies by a goodness-of-fit χ 2 . Differences in allele frequencies in three SNPs between cases and control were determined using a chi-square test. Strength of the association between IL-27 SNPs polymorphism and risk of HIV infection and disease progression was expressed as odds ratio (OR) with 95% confidence interval (95% CI).OR, 95% CI and P were adjusted based on gender and age using unconditional logistic regression. Differences between continuous variables (IL-27 level, HIV viral road, CD4 + T and CD8 + T count) were compared using Mann-Whitney U test. Spearman correlation was used to analyse correlation. P < 0.05 was considered statistical significant. All statistical analyses were done by using SPSS 17.0 statistical software package (SPSS Inc., Chicago, USA).

| Clinical characteristics of the study subjects
The demographics and clinical parameters of HIV-infected patients and controls in this study are shown in Table 2. There were no significant difference between cases and controls in age (P = 0.345) and gender (P = 0.618). Both serum IL-27 levels and CD4 + T counts were significantly lower in patients of HIV infection than those in controls (P < 0.001, respectively). Group of HIV infection is divided into group LTNP and TP according to the progression of disease whose demographics and clinical parameters are shown in Table 3.

| Association of IL-27 polymorphism with HIV infection
The genotypes and allele frequencies of IL-27 rs17855750, rs181206, rs40837 polymorphisms between HIV-infected patients and controls are shown in Table 4. All genotype distributions in two groups were in Hardy-Weinbery equilibruim (HWE) (P > 0.05). In  were significantly lower than in controls (P < 0.001, Figure 1A). We found no significant associations of three polymorphisms with serum IL-27 level in cases (P > 0.05). When we compared LTNPs to TPs, difference can be observed, the median serum IL-27 level was 242.45 pg/mL (range 185.00-286.30 pg/mL) and 186.05 pg/mL (range 75.60-254.00 pg/mL), respectively (P < 0.001, Figure 1B). We further found that LTNPs with rs40837 AG genotype or GG genotype had lower serum IL-27 levels than with AA genotype (P < 0.05, respectively, Figure 1C), however, there were no difference in the serum IL-27 level of individuals with AG genotype and GG genotype (P > 0.05). We failed to find any associations of rs17855750 and rs181206 polymorphisms with serum IL-27 level in LTNPs (P > 0.05). There were no correlations between the three polymorphisms and TP (P > 0.05).

| Association of IL-27 polymorphisms and HIV viral road
Plasma HIV RNA levels in controls were below detection range. In TPs, there were much higher levels compared to LTNPs, the levels  Figure 2C). No significant difference was observed between IL-27 polymorphisms and CD4 + T count in LTNP and TP group (P > 0.05). We also analysed association of IL-27 polymorphisms and CD8 + T count, no association could be found between them in different group. We only found the difference in CD8 + T count between LTNP and TP group (P < 0.05, Figure 2D).

| DISCUSSION
Host genetic variation is an important determinant of HIV infection and disease progression. 18   CD4 + T is the target cell of HIV virus, when the human body is infected with HIV, the primary outcome is that CD4 + T counts continue to decline, thereby resulting in disease progresses. This was consistent to our finding that HIV viral road was negatively associated with CD4 + T counts and was confirmed by a previous study. 29  Up to now, only our study has shown that IL-27 gene polymorphism is associated with HIV infection and disease progression as far as we know. Our data, it is true, showed no direct association between IL-27 polymorphism and HIV viral road, but we suspect that IL-27 rs40837 polymorphism might influence the susceptibility to HIV infection and disease progression probably by regulating the level of serum IL-27 or the quantity of CD4 + T. However, the specific mechanism is not yet clear and more researches, especially for different countries, different races are warranted to verify our view as a result of the genetic heterogeneity in different ethnic groups.
Finally, it should be noted here that, although the results of our study could not completely elucidate the mechanism of HIV infection and disease progression, it can provide a useful direction for further research mechanism.

CONFLI CT OF INTEREST
The authors declare no competing financial interests.