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.
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genetics; HIV-1; inflammasome; IL-1β; innate immunity; NALP3/NLRP3; polymorphisms
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