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Basic and Translational Science

A 3′UTR SNP in NLRP3 Gene is Associated With Susceptibility to HIV-1 Infection

Pontillo, Alessandra PhD*; Brandão, Lucas A BSc; Guimarães, Rafael L BSc; Segat, Ludovica BSc*†; Athanasakis, Emmanouil BSc*; Crovella, Sergio PhD*‡

Author Information
JAIDS Journal of Acquired Immune Deficiency Syndromes: July 1, 2010 - Volume 54 - Issue 3 - p 236-240
doi: 10.1097/QAI.0b013e3181dd17d4

Abstract

INTRODUCTION

Many evidences showed that the interpersonal variations in the susceptibility to HIV-1 infection and in the AIDS disease progression, both multifactorial, are partially due to polymorphisms in genes involved in innate and acquired immunity.1-6

In the last decade, several studies underlined the out-coming importance of the host innate immunity in the initial detection of HIV-1 and in mounting an efficient response against the virus.7 Innate immune recognition of pathogens works through a big family of Pathogen Recognizing Receptors presented on the plasma membrane (Toll-like receptors) and into the cytosol (NOD-like receptors; retinoic acid-inducible gene I-like).

Belonging to NLRs, cytoplasmatic NALP receptors interact with several adaptor proteins to form molecular complexes, known as inflammasomes, leading to the activation of caspase-1 and to the production of inflammatory cytokines, principally IL-1beta. Until now, 3 inflammasomes were characterized: NALP1-, NALP3-, and IPAF-inflammasomes. Each inflammasome could recognize specific pathogen associated molecular patterns and/or danger associated molecular patterns, amplifying the stress signal level recognized by innate immune system.8

Inflammasomes and IL-1beta seem to be an innate mechanism alternative to type-1 interferon able to recognize nucleic acids and virus into the cytoplasm and to induce a pro-inflammatory response.8 NALP3 recognizes cytidine-phosphate-guanosine (CpG) DNA motifs that are present in bacteria and viruses and is able to sense ssRNA, dsRNA, and some virus (influenza virus, Sendai virus, adenovirus).8-11 Moreover, Mixoma virus encodes for a protein that inhibit the activation of inflammasome and caspase-1 and the cell death after virus infection.8 Starting from the recent discovered interaction between NALP3 and viruses, we hypothesized that NALP proteins could be similarly activated by HIV-1 leading an inflammatory response able to drive the first step of infection.

In our study, we analyzed the distribution of 4 single-nucleotide polymorphisms (SNPs) frequencies in NLRP1 (2 SNPs) and NLRP3 (2 SNPs) genes-the most known inflammasome's key molecules-in 2 groups of Brazilian HIV-1-positive (HIV+) subjects, children born to HIV-1-infected mothers and adults patients, and in a group of Italian HIV+ adults with the aim of disclosing an eventual association between NLRPs SNPs and susceptibility for HIV infection. NLRP1 gene SNPs rs12150220 and rs2670660 were chosen since they have been previously associated with immune disorders.12,13 For NLRP3, we decided to analyze a missense polymorphism already associated with inflammatory deregulation, the Q705K (rs35829419),14 and the SNP rs10754558 at the 3′ untranslated region of NLRP3 gene (3′UTR), considering the recently reported effect on mRNA stability.15

PATIENTS AND METHODS

Patients and Controls

One hundred thirty-five HIV-1-positive Brazilian children (68 males, 67 females; mean age 6.9 years ± SD 5.1), infected during delivery by HIV-1-positive mothers, and one hundred thirty-five healthy children born to seronegative mothers (63 males, 72 females; mean age 7.5 years ± SD 5.4), matched for age, were enrolled from metropolitan area of Recife (Pernambuco, North-East Brazil).

One hundred ninety-two HIV-1-positive Brazilian adults (102 males, 90 females; mean age 32 years ± SD 10.3) and one hundred ninety-two healthy controls (HCs) (87 males, 105 females; mean age 27 ± SD 4.6) were enrolled from metropolitan area of Sao Paulo (Sao Paulo, South-East Brazil).

Ethnicity of Brazilian cases and controls from Recife was an admixture of white (around 40%), Afro-American (around 40%), and Amerindian (around 20%) as described by Alves-Silva et al,16 whereas cases and controls from Sao Paulo were mostly of white origin. Ethnicity was determined by a questionnaire filled by all the participants to the study.

The ethical committee of the Instituto de Medicina Integrada Prof. Fernando Figueira approved the study on Brazilian subjects (CONEP 3127, n 25000.127120/2001-73).

Finally, one hundred ninety-two HIV-1-positive Italian HIV+ adults (92 males, 100 females; mean age 46.7 ± SD 9.9) and one hundred ninety-two HCss (94 males, 98 females; mean age 29 ± SD 12.4) from North Eastern Italy were enrolled at the Virological Service of the IRCCS Burlo Garofolo of Trieste (Italy). All subjects came from the same geographical area and were white. The ethical committee of the IRCCS Burlo Garofolo approved the study performed on Italian patients (CIB 26/08 31/12/2008).

DNA Extraction

Genomic DNA was extracted from peripheral whole blood using the Wizard genomic DNA purification kit (Promega, Madison, MA) using standard laboratory protocols.

SNPs Selection and Genotyping

We analyzed 2 SNP in the NLRP1 gene (rs12150220 and rs2670660) and 2 in NLRP3 (rs35829419 and rs10754558).

Genotyping was performed by commercially available Taqman assays (C_1600653_10, C_1600689_10, C_25648615_10, C_26052028_10; Applied Biosystems, Foster City, CA) (Nucleotide variations and context sequences are included in an online Table, Supplemental Digital Content 1,https://links.lww.com/QAI/A47). TaqMan reactions were set up based on the manufacturer's protocol, and the samples were run on an ABI7900HT II Fast Real-Time instrument (Applied Biosystems). Allelic discrimination was performed using the SDS software (v.2.3) (Applied Biosystems).

Statistical Analysis

Test for Hardy-Weinberg equilibrium and linkage disequilibrium test were performed using the Haploview program.17 The data were analyzed with Fisher exact test. A formal Bonferroni correction for the number of SNPs performed for the primary hypothesis would require a significance threshold of P = 0.0125 (P0/N1, P0 = 0.05, N1 = 4 SNPs). The P values were presented in the text without correction.

RESULTS

We analyzed the rs12150220, rs2670660 SNPs in NLRP1 gene, and rs35829419 and rs10754558 in NLRP3 gene in 135 HIV-1-positive children coming from the metropolitan area of Recife (Pernambuco, North-East Brazil), in 192 HIV+ adults coming from metropolitan area of Sao Paulo (Sao Paulo, South-East Brazil), in 192 Italian HIV+ adults (Trieste, North-East Italy) and in HCs, matched for geographical provenience with the 3 groups analyzed.

Allelic and genotypic frequencies of the SNPs were in Hardy-Weinberg equilibrium in patients and controls.

For NLRP1 rs12150220 and rs2670660 polymorphisms, we did not evidence any significant association with HIV infection in Brazilian children and in Italian adults, being SNPs' allelic and genotypes frequencies quite similar in HIV+ groups and controls (Table 1). In Sao Paulo HIV+ subjects, although rs12150220 SNP allelic frequencies were quite similar in HIV+ and HC, genotypes distribution was significantly different (P = 0.003) (Table 1). In particular, the frequency of A/T heterozygous was reduced in HIV+ subjects when compared with HCs [58 of 192, 0.30, versus 90 of 192, 0.47; P = 0.001, odds ratio (OR) = 2.03, 95% confidence interval (CI): 1.31 to 3.17]. A similar finding was observed for the rs2670660 SNP, also in this case, genotypes frequencies were differently distributed between HIV+ and HCs (P = 0.009) (Table 1). The frequency of A/G heterozygous was reduced in HIV+ (83 of 192, 0.43, versus 106 of 192, 0.55; P = 0.025, OR: 0.62, 95% CI: 0.40 to 0.94); moreover, the effect of the rs2670660 G allele was consistent with a recessive genetic model (G/G versus A/A+A/G genotypes P = 0.005; OR = 0.47, 95% CI: 0.27 to 0.81), rather than with a dominant one (A/A versus G/G+A/G genotypes P = 0.025; OR = 0.62, 95% CI: 0.40 to 0.94).

T1-2
TABLE 1:
Allele and Genotype Frequencies for NLRP1 SNPs Rs12150220 (A/T) and Rs2670660 (A/G) in HIV and HC from Recife (North-East Brazil), Sao Paulo (South-East Brazil), and Trieste (North-East Italy)

The 2 NLRP1 SNPs were in Linkage disequilibrium (LD, r2 = 0.47; r2 = 0.61; r2 = 0.81, respectively, in Recife, Sao Paulo, and Trieste groups), confirming previously reported data,12,13 and combines to form 4 main haplotypes (see Table, Supplemental Digital Content 2, https://links.lww.com/QAI/A48). No differences in haplotypes frequency were observed between HIV-1+ Recife children and HCs, whereas a different distribution was found between Sao Paulo HIV+ and HC (P = 1.3exp-4) (Table 2): A-A (rs12150220-rs2670660) and A-G haplotypes were significantly differently distributed (160 of 384, 0.42, versus 196 of 384, 0.51; P = 0.011; OR = 0.69, 95% CI: 0.51 to 0.92; 74 of 384, 0.19, versus 36 of 384, 0.10; P = 1exp-4; OR = 2.31, 95% CI: 1.48 to 3.64, respectively) (Table 2). Similarly, we observed a different distribution of rs12150220-rs2670660 haplotypes between Italian HIV+ and HC (P = 0.005). In particular, the less frequent haplotype T-A was significantly reduced in HIV+ when compared with HC (7 of 384, 0.02, versus 24 of 384, 0.07; P = 8exp-4; OR = 0.26, 95% CI: 0.09 to 0.62) (Table 2).

T2-2
TABLE 2:
NLRP1 SNPs Rs12150220 (A/T) and Rs2670660 (A/G) Haplotype Frequencies in HIV and HC from Recife (North-East Brazil), Sao Paulo (South-East Brazil), and Trieste (North-East Italy)

NLRP3 rs35829419 allele and genotype frequencies distributions were similar between Recife HIV-1 positive children and controls (Table 3), whereas they were different in Sao Paulo (alleles P = 0.001, genotypes P = 0.001) and in Italian groups (alleles P = 0.082, genotypes P = 0.047) (Table 3). Both in Sao Paulo and in Italian groups, a diminished frequency of C/A heterozygous was evidenced in HIV+ subjects when compared with HCs (3 of 192, 0.01, versus 16 of 192, 0.08, P = 0.004; 11 of 192, 0.05, versus 23 of 192, 0.12, P = 0.046).

An association with HIV-1 infection has been evidenced for the NLRP3 c.*230 G>C polymorphism (rs10754558) in all the analyzed populations. The minor allele G was less frequent among patients than among healthy subjects (Recife: 91 of 270, 0.34 versus 124 of 270, 0.46, P = 0.005; Sao Paulo: 150 of 384, 0.39, versus 180 of 384, 0.47, P = 0.034; Trieste: 146 of 384, 0.38, versus 184 of 384, 0.48, P = 0.007) (Table 3). When considering genotypes the difference remained and it was statistically significant (Recife: P = 0.001; Sao Paulo: P = 0.012; Trieste: P = 0.013), being the GG genotype less frequent in HIV+ than in HC (Recife: 12 of 135, 0.09, versus 34 of 135, 0.25; Sao Paulo: 31 of 192, 0.16, versus 42 of 192, 0.22; 34 of 192, 0.17, versus 39 of 192, 0.28) (Table 3). Then we compared the rs10754558 SNP genotypes distribution between HIV+ subjects and controls using a dominant and a recessive model. In Recife paediatric population, we observed that there was no association between the G allele and HIV-1 infection in the dominant model (G/G+G/C versus C/C genotypes, P = 0.129), whereas the effect of the G allele was consistent with a recessive genetic model (G/G versus G/C+C/C genotypes P = 5.6 exp-4). Therefore, the G allele exerts a protective effect against HIV infection only when the polymorphism is present in homozygosis (OR = 0.29, 95% CI: 0.13 to 0.61).

T3-2
TABLE 3:
NLRP3 SNPs Rs35829419 (C/A) and Rs10754558 (C/G) Allele and Genotype Frequencies and Counts in HIV and HCs from Recife (North-East Brazil), Sao Paulo (South-East Brazil), and Trieste (North-East Italy)

On the other hand, in Sao Paulo and Trieste groups, the effect of the G allele was consistent with a dominant model (G/G+G/C versus C/C genotypes P = 0.02 and P = 8 exp-4, respectively) rather than with a recessive one (G/G versus G/C+C/C genotypes P = 0.193 and P = 0.360, respectively). In these populations, the rs10754558 G allele seemed to exert a similar protective effect against the virus but when present either in homozygosis or heterozygosis (Sao Paulo: OR = 0.60, 95% CI: 0.39 to 0.93; Trieste: OR = 0.47, 95% CI: 0.29 to 0.74).

No linkage disequilibrium was found for the 2 NLRP3 SNPs (r2 = 0.0; r2 = 0.01; r2 = 0.05, respectively, in Recife, Sao Paulo and Trieste populations).

DISCUSSION

NLRP1 and NLRP3 are known to be involved in recognizing several PAMPs/DAMPs and in the consequent innate immune response8 and were previously associated to immunologic disorders.12-15

Among the 4 SNPs analyzed in NLRP1 and NLRP3, the 3′UTR polymorphism rs10754558 in NLRP3 was significantly associated to the HIV-1 infection in the 3 groups (Brazilian children from Recife, Brazilian adults from Sao Paulo, and North Eatern Italians) analyzed. The minor G allele (and the homozygote GG genotypes even more) seems to be protective against the infection. It was recently demonstrated that the G allele of rs10754558 polymorphism enhanced NLRP3 mRNA stability.15 The association between NLRP3 SNPs and susceptibility to HIV-1 infection was once more underlined by the differently distribution of rs35829419 C/A SNP among HIV+ patients and HCs in the adults from Sao Paolo and Trieste. Differences in allelic and genotypic frequencies within populations reflect their different ethnicity, but the association was retained.

We can hypothesize that if NALP3 works as an intracellular receptor/sensor for the HIV-1 virus, the level of protein could be a discriminant factor in inducing a correct response.

In our opinion, NALP3 could be activated by HIV-1 due to its previously reported ability in recognizing microbial RNA and DNA containing CpG motifs (CpG-DNA).8 In fact, in the first step of macrophages or dendritic cells infection, HIV-1 is present as RNA, and moreover, during reverse transcription in the cytoplasm, HIV-1 produces CpG-DNA.18

The activation of NALP3-inflammasome results in an inflammatory response principally driven by IL-1beta. This mechanism seems to match with the hypothesis on the role played by chronic immune activation and inflammation in HIV-1 pathogenesis. High levels of IL-1beta are observed in patients from the early stages of HIV-1 infection.19

Despite its role in the secretion of IL-1beta, activating CD4+ T cells, NALP3-inflammasome induces the MHC-II exposition on the macrophage/antigen presenting cells surface for a rapid nonself antigen presentation.20 So the activation of NALP3 not only drives the local inflammation but also an acquired immune response. Moreover, NALP3 plays a key role in the maturation and activation of dendritic cells,8,21,22 and this seems to be crucial for an appropriate specific humoral and cellular immune response.

A chronic inflammation and an immune activation against HIV-1 are predictive of an adverse prognosis for the infected patients.19

Polymorphisms in NLRP3 could have a role in a not yet known pathologic defect on inflammasome activation and contribute to a HIV susceptibility genetic background. However, this is only a preliminary association study and presents some limitations. The major limitation of this study is probably the lack of mothers' biological samples. In fact, it's plausible that the mother's genetic background can influence vertical transmission: genotype of the mothers, that is partly reflected on the child genotype that we studied, would affect susceptibility. It is also true that within all the described NLRP3 SNPs, we only considered NLRP3 SNPs that have been already correlated with a known functional effect or associated with pathologies.

Even if further investigations are needed to elucidate the role of NLRP3 in HIV-1 infection, our results demonstrate that the NLRP3 rs10754558 SNP is associated to HIV-1 infection, suggesting that the NALP3-inflammasome could be possibly involved in the augmented susceptibility to HIV-1 infection.

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Keywords:

genetics; HIV-1; inflammasome; IL-1beta; innate immunity; NALP3/NLRP3/CIAS1; NALP1/NLRP1; polymorphisms

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