NLRP3, NLRC4 and NLRC5 Gene Polymorphisms Associate with Susceptibility of Pulmonary Aspergillosis in Non-Neutropenic Patients

Background: Non-neutropenic pulmonary aspergillosis is one of the most common and serious fungal infections. Previous studies have shown that single nucleotide polymorphisms (SNPs) of pattern recognition receptors genes are associated with susceptibility to aspergillosis. NOD-like receptors (NLRs) play an important role in the immunological response against fungal infection. In this study, we investigated the relationship between polymorphisms of three NLRs and susceptibility to pulmonary aspergillosis disease in non-neutropenic patients. Methods: We included 73 patients with proven pulmonary aspergillosis and 103 healthy controls. A total of sixteen SNPs in the NLRP3, NLRC4, and NLRC5 genes were detected by PCR-direct sequencing. Then, we evaluated the association between these polymorphisms and susceptibility to aspergillosis. Results: Fifteen SNPs were consistent with Hardy–Weinberg equilibrium except for NLRP3 rs7525979. A total of eight SNPs (NLRP3 rs3806265, NLRC4 rs212704 and NLRC5 rs1684579, rs12598522, rs3995817, rs3995818, rs34531240, rs28438857) were observed an association with susceptibility of pulmonary aspergillosis. The CC homozygote of NLRP3 rs3806265, TT homozygote of NLRC5 rs1684579 and T allele of NLRC5 rs12598522 were associated with a higher risk of aspergillosis while TT homozygote of NLRC4 rs212704 was associated with a lower risk of aspergillosis. Especially in the invasive pulmonary aspergillosis subgroup, the TT homozygote of NLRC5 rs1684579 and rs3995817, the CC homozygote of NLRC5 rs34531240 and rs28438857, GG homozygote of NLRC5 rs3995818, the C allele and CC homozygote of NLRP3 rs3806265 were associated with higher susceptibility. Conclusions: This study showed an association between polymorphisms of NLRP3, NLRC4, and NLRC5 and susceptibility to pulmonary aspergillosis for the first time. Further investigations in larger populations are needed, and functional studies are also required to investigate the function of these NLRs in aspergillosis, as well as other fungal infection diseases.


Introduction
Aspergillus is a saprotrophic fungus which spreads by asexual conidia. The airborne Aspergillus conidia can be inhaled into the respiratory tract and lungs of humans, causing different types of diseases, including invasive pulmonary aspergillosis (IPA), chronic pulmonary aspergillosis (CPA), and allergic bronchial pulmonary aspergillosis (ABPA) [1][2][3]. Since the late 1990s, aspergillosis has actually proved to be the most common invasive pulmonary fungal infection. Moreover, it has also become the most expensive fungal disease because of its prevalence and costly treatments [4].
Pattern recognition receptors (PRRs), including Toll-like receptors, RIG-I-like receptors, NOD-like receptors, and C-type lectin receptors, play an important role in host immunity against Aspergillus; thus, the genetic defects in PRRs may lead to susceptibility to aspergillosis [5][6][7]. Polymorphisms in Toll-like receptors such as TLR4, C-type lectin receptors such as dectin-1, and other pattern recognition receptors such as Pentraxin 3 (PTX3) have been found to be associated with susceptibility to aspergillosis [8][9][10].
The NOD-like receptors make up an important family of PRRs. Many of them can bind with apoptosis-associated speck-like protein containing a CARD(ASC) and caspase-1 to form inflammasome, such as NLRP3, NLRC4, NLRC5 inflammasome, and so on [11]. In an aspergillosis mouse model, several kinds of NLRs increased in the infected lungs, including NLRP3, NLRC4, and NLRC5, but their exact functions remain to be explored [12]. NLRP3 is the most well-characterized and most well-studied inflammasome sensor molecule. When the NLRP3 inflammasome is activated, Pro-Caspase-1 is cleaved, which leads to the release of proinflammatory cytokines like IL-1β and IL-18, as well as pyroptotic cell death [13,14]. Generally, NLRP3 is known to contribute to antifungal immunity and help control infection [5]. For instance, a GAG-deficient Aspergillus mutant, which failed to elicit protective NLRP3 inflammasome activation, exhibited enhanced virulence [15]. Likewise, the mice lacking AIM2 and NLRP3 were susceptible to Aspergillus infection [16]. Similarly, the NLRC4 inflammasome has been also found to protect mucosal barriers such as the lung, stomach, and intestine from invading pathogens [17]. NLRC5 is the largest one in the NLR family and works as the master transcriptional regulator of MHC class I and related genes [18,19]. Therefore, after infection, NLRC5 knockout mice showed increased bacterial load and impaired clearance of viruses due to strongly impaired MHCI-mediated CD8+ T cell activation [20][21][22]. However, the role of NLRC4 and NLRC5 in Aspergillus infection is still unknown. Since innate immunity and adaptive immune responses are all important parts for host defense against Aspergillus, we assume that they are probably involved in the host immune response in aspergillosis, as well as in other PRRs.
A few previous studies have shown the polymorphisms of NLRs genes affected the susceptibility of Aspergillus infection or colonization. Among transplant recipients after hematopoietic stem cell transplantation (HSCT), P268S (rs2066842) in NOD2 of the donors was associated with an increased risk of invasive aspergillosis [23]. For patients with cystic fibrosis, significant associations were found between A.fumigatus colonization and polymorphisms of NLRC4, including the haplotype ACTT (rs212704 rs455060 rs7562653 rs385076) and GG genotype of rs212704 [24]. In this study, we investigated 16 SNPs in NLRP3, NLRC4, and NLRC5 genes among the southeastern Han Chinese population and analyzed the relationships between these SNPs and susceptibility of pulmonary aspergillosis in non-neutropenic patients.

Study Population
This study included 73 pulmonary aspergillosis patients treated at Jinling Hospital from June 2016 to December 2019. The control group consisted of 103 healthy people undergoing physical examination. According to the updated IDSA guideline criteria and EORTC/MSGERC criteria, these aspergillosis patients were diagnosed as 30 IPA, 27 CPA (including chronic cavitary pulmonary aspergillosis, CCPA and aspergilloma), and 16 ABPA [25,26] (Table 1). Since the study subjects were non-neutropenia patients, we excluded patients who had previously undergone organ transplantation or chemotherapy.

Selection of SNPs and Genotyping
The single nucleotide polymorphisms of NLRP3 (rs3806265, rs7525979, rs35829419, rs10754558) and NLRC4 (rs12989936, rs212704, rs7562653, rs479333, rs385076) were selected from previous literature. Seven SNPs of NLRC5 (rs12598522, rs34531240, rs28438857, rs3995818, rs3995817, rs1684579, rs3751705) were selected based on information from the NCBI GenBank, dbSNP, and HapMap databases, with the minimum allele frequency set at 5% and r2 at 0.8. These SNPs were located within the coding region, 5 untranslated region (UTR), or 3 UTR that may possibly influence protein synthesis and gene transcription. Peripheral blood (1 mL) was collected in an EDTA tube from each subject. Genomic DNA was extracted from the whole blood using the QIAamp DNA Blood Mini Kit (Qiagen, Berlin, Germany) according to the manufacturer's instructions and then stored in a −80 • C freezer. Primers were designed using Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA, USA). PCR amplification was performed in Eppendorf PRO PCR System (Hamburg, Germany). All SNPs were genotyped by ABI Prism 377 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with technical support from the Shanghai Genesky Biotechnology Company (Shanghai, China). DNA sequences were read by Chromas 2.3 software (Technelysium Pty Ltd., Tewantin, Australia). Negative controls were included in each plate for accuracy.

Statistics
Each SNP was tested for Hardy-Weinberg equilibrium (HWE) in the healthy control group using the chi-square (χ 2 ) test. Statistical comparison was performed by independent Student's t-test or one-way analysis of variance. Differences among SNPs were evaluated using Pearson's χ 2 or Fisher's exact test. The strength of association between polymorphisms and the risk of aspergillosis was evaluated by odds ratio (OR) and 95% confidence interval (CI). HWE, linkage disequilibrium, and haplotype analysis were analyzed by HaploView [27,28]. Statistical analysis was performed by the SPSS 24.0 and the GraphPad Prism 7. All tests were considered significant with a p value of <0.05.

Characteristics of the Patient Population
We enrolled 73 patients with pulmonary aspergillosis and 103 healthy controls. Healthy controls were used to compare the frequency of mutations between the patient group and the general population, and there were no significant differences in age (58.48 ± 1.63 vs. 55.25 ± 1.038, p = 0.0819) or sex (male/female: 41/32 vs. 64/39, p = 0.4263) between the case and control groups. Table 1 summarizes the main characteristics of the study population. The case group included 20 (27.40%) patients with hypertension and 12 (16.44%) with diabetes, 11 (15.07%) with COPD, 5 (6.85%) with asthma, 23 (31.51%) with bronchiectasis, and 20 (27.40%) with tuberculosis. In addition, 21 (28.77%) in the case group had a history of smoking and 13 (17.81%) had a history of steroid treatment prior to onset.

Hardy-Weinberg Equilibrium Analysis and Minor Allele Frequencies of SNPs
We analyzed HWE and minor allele frequencies (MAF) of the 16 SNPs. All but NLRP3 rs7525979 were consistent with HWE in the control group (p > 0.05). No allele was detected at NLRP3 rs35829419 in either control group or case group. MAF of the other SNPs were >5%. So, we excluded these two SNPs in the following statistical analysis. Other information regarding these SNPs is shown in Table 2.

Association of NLRs Variants with Pulmonary Aspergillosis
First, we evaluated the association of 14 single nucleotide polymorphisms with pulmonary aspergillosis risk among all patients and healthy controls (Table 3). A total of 4 SNPs (NLRP3 rs3806265, NLRC4 rs212704, NLRC5 rs1684579, and rs12598522) were observed an association with aspergillosis risk. Genotype difference in NLRP3 rs3806265 between the case and control groups was statistically significant (p = 0.0451) and the CC homozygote of rs3806265 was associated with a higher risk of aspergillosis (p = 0.0130; OR = 2.567, 95% CI: 1.239 to 5.255). For the NLRC4 rs212704, the TT homozygote was associated with a lower risk of aspergillosis (p = 0.0447; OR = 0.4468, 95%CI: 0.2071 to 0.959) while for the NLRC5 rs1684579 it was opposite (p = 0.0261; OR = 2.066, 95%CI: 1.085 to 4.018). Furthermore, the T allele of NLRC5 rs12598522 was more frequent in aspergillosis patients than healthy controls (p = 0.0305; OR = 1.601, 95%CI: 1.048 to 2.47). The other SNPs did not show any association with aspergillosis risk (Supplementary Table S1).

Association of NLRs Variants with Different Kinds of Aspergillosis
Next, we divided the patients into non-ABPA subgroup and ABPA subgroup according to whether the pathological process was mainly an inflammatory or allergic response and then compared each of the two subgroups with healthy controls (Table 4). In the non-ABPA group, gene polymorphism of the rs3806265 in NLRP3 and rs12598522, rs1684579 in NLRC5 showed a significant association with susceptibility. Similar to the entire group analysis, genotype difference in NLRP3 rs3806265 was statistically significant (p = 0.0298) and the CC homozygote was associated with a higher risk (p = 0.0129; OR = 2.702, 95%CI: 1.172 to 5.924), as well as the T allele of NLRC5 rs12598522 (p = 0.0419; OR = 1.612, 95%CI: 1.012 to 2.529). For the NLRC5 rs1684579, there was also a significant genotype difference (p = 0.0125) and the TT homozygote (p = 0.0044; OR = 2.665, 95%CI: 1.371 to 5.292), as well as T allele (p = 0.0471; OR = 1.614, 95%CI: 0.9938 to 2.624) were significantly associated with a higher risk of Aspergillus infection. However, in the ABPA subgroup, there was no SNP associated with susceptibility (Supplementary Table S2).  Finally, we divided the non-ABPA group into IPA subgroup and CPA subgroup and compared them with control group (Table 5). It was worth noting that we found rs3806265 in NLRP3 and rs34531240, rs28438857, rs3995818, rs3995817, rs1684579 in NLRC5 were all associated with IPA risk. The CC homozygote (p = 0.0004; OR = 4.861, 95%CI: 2.007 to 11.9) and C allele (p = 0.0117; OR = 2.115, 95%CI: 1.196 to 3.741) of NLRP3 rs3806265, as well as TT homozygote of NLRC5 rs1684579 (p = 0.0036; OR = 3.385, 95%CI: 1.47 to 8.101) were associated with a higher risk of IPA and the genotype differences of them were significant between IPA patients and controls (p = 0.0017; p = 0092). The CC homozygote of rs34531240 and rs28438857, GG homozygote of rs3995818, TT homozygote of rs3995817 in NLRC5 were all more frequent in IPA patients than controls (p = 0.0420; OR = 2.386, 95%CI: 0.9749 to 5.567). However, in the ABPA subgroup, there was no SNP associated with susceptibility (Supplementary Table S3).

Discussion
The aim of this study was to investigate the relationship between NLRP3, NLRC4, and NLRC5 gene polymorphisms and susceptibility to pulmonary aspergillosis in non-neutropenic patients among the Chinese population. For the first time, we found rs3806265 in NLRP3, rs212704 in NLRC4 and rs12598522, rs34531240, rs28438857, rs3995818, rs3995817, rs1684579 in NLRC5 were associated with pulmonary aspergillosis in non-neutropenic patients.
NLR proteins are central mediators of microbial sensing with diverse functions, and they play an important role in the host antimicrobial immune responses including anti-Aspergillus response [38]. NLRs are usually composed of a tripartite structure, including an N-terminal effector domain, a central NACHT domain containing the nucleotide binding domain (NBD) for self-oligomerization and C-terminal leucine-rich repeats (LRRs) for recognizing PAMPs or DAMPs [39]. As for NLRs, previous studies have shown that rs2066842 in NOD2 of donors was associated with the risk of invasive aspergillosis after hematopoietic stem-cell transplantation, while rs212704 and ACTT (rs212704 rs455060 rs7562653 rs385076) in NLRC4 were associated with A. fumigatus colonization in cystic fibrosis patients [23,24]. In our study, we found the TT homozygote of rs212704 in NLRC4 and C allele of rs12598522 in NLRC5 was associated with a lower risk of aspergillosis. On the contrary, CC homozygote of rs3806265 in NLRP3 and TT homozygote of rs1684579 in NLRC5 was associated with a high risk of aspergillosis, especially of IPA. Besides, in the IPA subgroup, the CC homozygote of rs34531240 and rs28438857, GG homozygote of rs3995818, TT homozygote of rs3995817 in NLRC5 was more frequent than controls.
The polymorphism of rs3806265 in NLRP3 is associated with several kinds of diseases, such as myasthenia gravis (MG), psoriasis, recurrent aphthous stomatitis (RAS) and relapsing-remitting multiple sclerosis (RRMS) [40][41][42][43]. In the Iranian population, the C allele and CC homozygote of rs3806265 was more frequent in MG and RRMS patients than controls, whereas the T allele and TT homozygote were less frequent in RAS patients [40][41][42]. In the Chinese population, the T allele was associated with a higher risk of psoriasis and this locus might function as an enhancer in the immune-related system [43]. In our study, it was the opposite that C allele and CC homozygote were associated with a higher risk of IPA. This might be due to IPA occurring as a result of immune deficiency.
Previous research has found GG homozygote of rs212704 in NLRC4 was associated with A.fumigatus colonization in cystic fibrosis patients [24]. Our study showed that TT homozygote was associated with a decreased risk of aspergillosis, reflecting the role of NLRC4 in host defense against Aspergillus infection as well. Besides, C allele of rs212704 was associated with increased insulin and lower glucose levels while CC homozygote with a lower 2-h postprandial C-peptide level, suggesting that the polymorphism of NLRC4 may also relate to body metabolism [44,45].
The polymorphism of NLRC5 is also related to the susceptibility, severity, and prognosis of different kinds of diseases. In chronic periodontitis, the rs289723 in NLRC5 gene was associated with chronic slight and chronic localized periodontitis susceptibility and the AA genotype was correlated with increased risk of disease development [46]. It suggests that NLRC5 may play a promoting role in the insurgence of inflammation. So, it is reasonable that we found that the polymorphism of NLRC5 influenced the susceptibility to pulmonary aspergillosis, especially IPA. Some other studies reported that there was also an association between NLRC5 SNPs and the survival of colorectal and rectal cancer [47,48].
Our study demonstrated that the polymorphisms of NLRs (NLRP3, NLRC4, NLRC5) were associated with pulmonary aspergillosis risk, but there were still some limitations.
First, it was a single-center study in southeastern China. So, it was hard to get a large case group, and our results might be influenced by geographic, ethnic, and genetic factors. Second, it was a retrospective study so we could not measure the NLRs expression or the levels of downstream inflammatory factors at the onset of aspergillosis. Likewise, we could only select healthy people as the control group instead of patients with the same underlying diseases and health condition. Finally, functional evaluations are needed to unveil the function of different SNPs of NLRs in the progression of aspergillosis.

Conclusions
Our results identified the association of NLRP3, NLRC4, and NLRC5 genetic variation with the susceptibility of pulmonary aspergillosis for the first time. These NLRs are probably involved in host immune defense against Aspergillus infection. Their function and mechanism in aspergillosis have not yet been thoroughly studied. Our results can provide a reference for studies on the role of NLRs in aspergillosis and other fungal infections.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jcm11071870/s1, Table S1: Genotype distribution of NLRs SNPs in pulmonary aspergillosis cases and controls; TableS2: Genotype distribution of NLRs SNPs in non-ABPA group, ABPA group and controls; Table S3: Genotype distribution of NLRs SNPs in IPA group, CPA group and controls.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patient(s) to publish this paper.

Conflicts of Interest:
The authors declare no conflict of interest.