The role of innate immunity in the initial detection of HIV-1 and in an efficient response against the virus has appeared increasingly important in recent years.1 Furthermore, the susceptibility to HIV-1 infection and pathogenesis has been associated with polymorphisms in genes involved in innate immune response.2–7
Pathogen recognizing receptors, including toll-like receptors and nod-like receptors (NLRs), expressed in phagocytes and dendritic cells are major triggers of innate immunity.
Three proteins belonging to NLRs, “NLR Family pyrin domain containing 1” (NALP1 NLRP1), “NLR Family pyrin domain containing 3” (NALP3 NLRP3), and “NLR family CARD domain containing 4” (IPAF NLRC4) interact with several adaptor proteins to form molecular complexes known as inflammasomes, leading to the activation of caspase-1 and the production of interleukin (IL)-1β.8 Inflammasome and IL-1β were previously reported as an innate mechanism, alternative to type-1 interferon, able to recognize nucleic acids and virus into the cytoplasm, and to induce a proinflammatory response.8 In particular, NLRP3 recognizes cytidine-phosphate-guanosine DNA motifs that are present in bacteria and viruses and it is able to sense ssRNA, dsRNA, and some virus (influenza virus, Sendai virus, adenovirus).8–10
To our knowledge, the involvement of NLRs or inflammasome in HIV recognition or in the anti-HIV response has not been yet demonstrated. However, we recently reported the association between a 3'UTR polymorphism in NALP3/NLRP3 gene (rs10754558) and the susceptibility to HIV-1 infection, suggesting a potential role of NLRP3 inflammasome in the pathogenesis of HIV.11
Here we evaluated the frequency distribution of 12 single nucleotide polymorphisms (SNPs) within 6 inflammasome genes (NLRP1, NLRP3, NLRC4, caspase recruitment domain family, member 8/CARD8, caspase 1/CASP1, interleukin 1 beta/IL1B) in patients chronically infected by HIV-1 and healthy individuals, all from Southern Brazil (Sao Paulo state), to investigate whether these SNPs could be associated with the susceptibility to HIV.
PATIENTS AND METHODS
Patients and Controls
One hundred fifty HIV-1–positive Brazilian adults (HIV+; 80 males/70 females; mean age 34 years ± SD: 12.4) were enrolled at the ambulatory of secondary immunodeficiencies (ADEE 3002) of “Hospital das Clinicas” (Faculty of Medicine, University of Sao Paulo/FMUSP, Sao Paulo, Brazil). One hundred fifty-eight healthy controls (HCs; 72 males/86 females; 29 years ± 7.8) from the same metropolitan area (Sao Paulo) were also recruited. Individuals were classified as European-derived or African-derived according to phenotypic characteristics and ethnicity data of parents/grandparents reported by the participants in an appropriate questionnaire. The issue concerning skin-color–based classification criteria adopted in Brazil is well documented and has been already assessed in previous studies.12,13 One hundred twenty-four HCs (78%) and 129 HIV+ (86%) were classified as European derived, whereas 34 HCs (22%) and 21 HIV+ (14%) as African derived.
Written informed consent was obtained according to the protocol of “Hospital das Clinicas” ethical committee (CAPPesq) (n 0791/09, 04/11/2009) (São Paulo, Brazil).
Genomic DNA was extracted from peripheral whole blood using the Qiagen genomic DNA purification kit (Qiagen, Milan, Italy) following standard laboratory protocols.
SNPs Selection and Genotyping
Twelve SNPs in 6 inflammasome genes (NALP1/NLRP1, NALP3/NLRP3, IPAF/NLRC4, CARD8, CASP1, IL1B) were selected from public databases Hapmap (www.hapmap.org) and GeneBrowser (www.genome.ucsc.edu) (Figure 1). Two SNPs in NLRP1 (rs12150220 and rs2670660) and in NLRP3 (rs10754558 and rs35829419) were chosen among others due to previously published association study.11 All polymorphisms in NLRC4, CARD8, CASP1, and IL1B genes, resulted from Hapmap and GeneBrowser databases, were included in the study (see Table, Supplemental Digital Content 1, http://links.lww.com/QAI/A230).
Genotyping was performed by commercially available TaqMan assays (Applied Biosystems/AB, LifeTechnologies, Carlsbad, CA). TaqMan reactions were set up based on the manufacturer's protocols and samples run on an ABI7300 real-time instrument (AB). Allelic discrimination was performed using the sequence detection system software (v.2.3) (AB).
Allelic and genotypic SNP frequencies were calculated using the Genotype Transposer software14 and then analyzed by Fisher exact test. The Haploview software15 was used to investigate the association and linkage disequilibrium pattern and for deriving the haplotypes. The open-source R package (www.r-project.org) was used for Fisher exact test and odds ratio (OR) calculation and for the analysis of covariance.
A formal Bonferroni correction for the number of the tests performed (considering alleles and genotypes comparisons) would require a significance threshold of P = 0.0021 (P0/N, P0 = 0.05, N = 24 test). When dominant/recessive model has been analyzed, according to Lewis,16 the significant threshold was P = 0.0019. The P values are presented in the text without correction.
Six inflammasome genes were considered for this association study: NLRP1, NLRP3, and NLRC4 as intracellular pathogen recognizing receptors, CARD8 as adaptor molecule, CASP1 and IL1B as effector molecules. A graphical representation of the selected SNPs within the inflammasome genes is reported in Figure 1.
Twelve inflammasome SNPs were genotyped in 150 HIV+ patients and 158 HCs. SNPs allelic and genotypic frequencies were in Hardy–Weinberg equilibrium in patients and controls and are reported in (Table 1).
Because this is the first study, to our knowledge, considering the selected polymorphisms in NLRC4, CARD8, CASP1, and IL1B genes in a Brazilian population, we remark that the allelic frequencies of the SNPs analyzed (Table 1) were between those reported in the HapMap database (http://www.hapmap.org/) for white and Afroamerican (see Table, Supplemental Digital Content 1, http://links.lww.com/QAI/A230), as expected due to the mixed ethnical origins of the Brazilian population.12,13
When considering SNPs in the inflammasome receptors genes, NLRP1 and NLRC4 polymorphisms was not associated with HIV-1 infection (Table 1).
NLRP3 rs35829419 polymorphism seems not to be associated with HIV infection, whereas the rs10754558 minor G allele was significantly less frequent in HIV+ than in HC (0.35 versus 0.48, P = 0.002) suggesting a protective role against the virus infection (OR = 0.61). rs10754558 genotype distribution varied in the 2 groups even if it was not significantly different after Bonferroni correction (P = 0.003) (Table 1). The G allele behaved according to a recessive model (P = 8exp-4; OR = 0.44; 95%CI = 0.26 to 0.72) rather than to a dominant one (P = 0.245).
The rs2043211 variation in the adaptor molecule CARD8 was not significantly associated to HIV infection (Table 1). The recently reported interaction between NLRP3 rs35829419 and CARD8 rs204321117 was also evaluated without any significant results (data not shown).
When looking at polymorphisms of inflammasome effector genes CASP1 and IL1B, a significant difference between patients and controls was observed only for IL1B rs1143634; the minor A allele was more frequent in HIV+ than in HC (0.41 versus 0.19; P = 1.56 exp-9) suggesting a predisposing effect on HIV susceptibility (OR = 3.0) (Table 1). The rs1143634 genotype distribution is also significantly different between the 2 groups (P = 1.14 exp-04) (Table 1) with the rs1143634 A allele behaved according to a recessive model (4.26 exp-07; OR = 3.31; 95% CI = 2.03 5.46) rather than to a dominant one (5.12 exp-05; OR = 4.9; 95% CI = 2.09 to 12.71).
The analysis of linkage disequilibrium for the SNPs in NLRP1, NLRP3, NLRC4, and IL-1B did not show any significant results (see Figure, Supplemental Digital Content 2, http://links.lww.com/QAI/A231).
The combined effect of the HIV-associated polymorphisms NLRP3 rs10754558 and IL1B rs1143634 has been evaluated: 4 allelic combinations resulted (C-G, G-G, C-A, G-A) and 3 were significantly differently distributed in our groups. In particular, the combination rs10754558 minor allele G and rs1143634 major allele G showed a protective effect against the viral infection (P = 3.82 exp-8;OR = 0.37), whereas the 2 combinations carrying the rs1143634 minor allele A showed a predisposing effect on HIV infection (P = 5.24 exp-6; OR=2.72 and P = 0.003; OR = 2.21) especially when the rs10754558 major C allele was present.
Considering the ethnical admixture characteristic of the Brazilian population, we evaluated the frequency distribution of the 12 SNPs in our case/control cohort stratified for European or African origin. All the polymorphisms presented a similar allele frequency in European and African HIV+/HC groups (see Data, Supplemental Digital Content 3, http://links.lww.com/QAI/A232). In subjects of European origin, the rs1143634 minor A allele resulted more frequent in HIV+ subjects than in HC (0.44 vs. 0.15; P = 3.3 exp-13; OR = 4.5; 95%CI = 2.89 to 7.12). NLRP3 rs10754558 appeared not to be statistically different distributed between HIV+ and HC (P = 0.03). In subjects of African origin, probably due to the low number of individuals, no significant association was observed. To exclude the influence of ethnicity on rs10754558 and rs1143634 frequency distribution, ethnic origin was put into covariate and analysis of covariance test (analysis of covariance) was carried out. After this correction, significant difference was observed again for rs1143634 in European-origin group (P = 1.5 exp-9).
Inflammasomes are known to be involved in recognizing several pathogens and in triggering the consequent innate immune response.8 Recently, we reported the association between NLRP3 and HIV-1 infection hypothesizing a role of NLRP3 inflammasome and IL-1β in HIV pathogenesis.11 To deeper investigate this association, 12 SNPs within 6 genes with a key function in inflammasome assembling or IL-1β secretion were selected (Fig. 1), and their distribution was evaluated in a novel cohort of south Brazilian HIV+ subjects.
Among the 12 SNPs, rs10754558 in NLRP3 and rs1143634 in IL1B were significantly associated to the HIV-1 infection.
The rs10754558 G allele resulted protective against the infection. This result was concordant with our previously published data reporting the association of rs10754558 with protection from HIV-1 infection in groups of patients characterized by different ethnic composition.11
Although the NLRP3 rs35829419 allele frequencies distribution in patients and controls was the same in the present and in the previous “Sao Paulo” cohort,11 the significant threshold has not reached in this study maybe due to the lower number of enrolled individuals (150/158 vs. 192/19211), the higher number of SNPs evaluated (12 versus 411), and the consequent complexity of statistical analysis.
Despite the quite limited size of the population studied, we believe that present findings support once again our previously reported hypothesis about the involvement of NLRP3 in the HIV-1/immune system interaction during the early steps of HIV infection.11
The augmented mRNA stability demonstrated for the rs10754558 G allele18 could augment the synthesis of NLRP3 increasing the number of anti-HIV available receptors and the rate of inflammasome assembling, resulting in a more effective early immune response against the virus. Whether NLRP3 acts directly as a HIV receptor or belongs to an intracellular antiviral pathway have not yet demonstrated, and functional studies are needed to clarify the NLRP3/HIV interaction.
The other 2 NLRs involved in the inflammasome assembling, NLRP1 and NLRC4, seemed not to be associated to HIV pathogenesis, reinforcing the hypothesis of a specific role of NLRP3 in HIV sensing.
The minor IL1B rs1143634 A allele was more frequent in HIV+ subjects suggesting a predisposing role of this allele to HIV infection. To our knowledge, it is the first time that this variation has been associated with HIV-1 infection. The rs1143634 polymorphism is a Tag SNP leading to a synonymous substitution (F105F) with a still unknown functional effect. This association emphasizes the importance of inflammasome and IL-1β secretion in HIV pathogenesis. High levels of IL-1β have been observed in patients from the early stages of HIV-1 infection,19 suggesting a role of NLRP3 inflammasome in driving this sustained inflammatory response.
Moreover NLRP3 inflammasome and IL-1β were reported to be prominent factors in the maturation and activation of dendritic cells8,20,21; playing a pivotal role not only in the innate immune response but also in the specific humoral and cellular immune response.
Whether a high activation of inflammasome is good or not for the immune activation against HIV-1 is an open question because a chronic inflammation is predictive of an adverse prognosis for the infected patients.19 In the first steps of infection, HIV enters the mucosal residential macrophages and dendritic cells inducing a inflammatory response that leads to early activation of the immune system against the virus, but also to the recruitment of a large number of immune cells targeted by the virus, enlarging its spreading.
Even if further investigations are needed to elucidate the role of NALP3 inflammasome in HIV-1 infection, our results demonstrate that genetic polymorphisms in NLRP3 and IL1B, 2 genes with a key role in the inflammasome biology, are associated to HIV-1 infection underlining once more the importance of innate immune genetic background in the susceptibility to the virus.
We thank the ambulatory of secondary immunodeficiencies (ADEE 3002) of Hospital das Clinicas (FMUSP, Sao Paulo, Brazil) for HIV+ patients' recruitment, Prof. Dr. Andrade Jr H. F. and the laboratory of Protozoology (Institute of Tropical Medicine, FMUSP) for the helpful technical support, and all patients for the collaboration.
1. Kawai T, Akira S. Innate immune recognition of viral infection. Nat Immunol. 2006;7:131–137.
2. Kaslow RA, Dorak T, Tang JJ. Inﬂuence of host genetic variation on susceptibility to HIV type 1 infection. J Infect Dis. 2005;191(suppl 1):S68–S77.
3. Gonzalez E, Bamshad M, Sato N, et al.. Race-speciﬁc HIV-1 disease-modifying effects associated with CCR5 haplotypes. Proc Natl Acad Sci U S A. 1999;96:12004–12009.
4. Gonzalez E, Kulkarni H, Bolivar H, et al.. The inﬂuence of CCL3L1 gene-containing segmental duplications on HIV-1/AIDS susceptibility. Science. 2005;307:1434–1440.
5. Dolan MJ, Kulkarni H, Camargo JF, et al.. CCL3L1 and CCR5 inﬂuence cell-mediated immunity and affect HIV-AIDS pathogenesis via viral entry-independent mechanisms. Nat Immunol. 2007;8:1324–1336.
6. Ji X, Gewurz H, Spear GT. Mannose binding lectin (MBL) and HIV. Mol Immunol. 2005;42:145–152.
7. Mangano A, Gonzalez E, Dhanda R, et al.. Concordance between the CC-chemokine receptor 5 genetic determinants that alter risks of transmission and disease progression in children exposed perinatally to human immunodeﬁciency virus. J Infect Dis. 2001;183:1574–1585.
8. Martinon F, Mayor A, Tschopp J. The inflammasomes: guardians of the body. Annu Rev Immunol. 2009;27:229–265.
9. Thomas PG, Dash P, Aldridge JR Jr, et al.. The intracellular sensor NLRP3 mediates key innate and healing responses to influenza A virus via the regulation of caspase-1. Immunity. 2009;30:566–575.
10. Hornung V, Ablasser A, Charrel-Dennis M, et al.. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature. 2009;458:514–518.
11. Pontillo A, Brandão LA, Guimarães RL, et al.. A 3'UTR SNP in NLRP3 gene is associated with susceptibility to HIV-1 infection. J Acquir Immune Defic Syndr. 2010;54:236–240.
12. Vargas AE, Marrero AR, Salzano FM, et al.. Frequency of CCR5delta32 in Brazilian populations. Braz J Med Biol Res. 2006;39:321–325.
13. Veit TD, Cordero EA, Mucenic T, et al.. Association of the HLA-G 14 bp polymorphism with systemic lupus erythematosus. Lupus. 2009;18:424–430.
14. Cox DG, Canzian F. Genotype transposer: automated genotype manipulation for linkage disequilibrium analysis. Bioinformatics. 2001;17:738–739.
15. Barrett JC, Fry B, Maller J, et al.. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics. 2005;21:263–265.
16. Lewis CM. Genetic association studies: design, analysis and interpretation. Brief Bioinform. 2002;3:146–153; Review.
17. Roberts RL, Topless RKG, Phipps-Green AJ, et al.. Evidence of interaction of CARD8 rs2043211 with NALP3 rs35829419 in Crohn's disease. Genes Immun. 2010;11:351–356.
18. Hitomi Y, Ebisawa M, Tomikawa M, et al.. Associations of functional NLRP3 polymorphisms with susceptibility to food-induced anaphylaxis and aspirin-induced asthma. J Allergy Clin Immunol. 2009;124:779–785.
19. Appay V, Sauce D. Immune activation and inflammation in HIV-1 infection: causes and consequences. J Pathol. 2008;214:231–241.
20. Kool M, P`etrilli V, De Smedt T, et al.. Alum adjuvant stimulates inflammatory dendritic cells through activation of the NALP3 inflammasome. J Immunol. 2008;205:869–882.
21. Eisenbarth SC, Colegio OR, O'Connor W, et al.. Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature. 2008;453:1122–1126.