IL-1 β gene (+3954 C/T, exon 5, rs1143634) and NOS2 (exon 22) polymorphisms associate with early aseptic loosening of arthroplasties

Aseptic prosthetic loosening (APL) and prosthetic joint infections (PJI) are frequent complications of hip and knee implants. Polymorphisms of cytokines and nitric oxide (NO), key inflammatory molecules in APL and PJI pathogenesis, could explain individual susceptibility to these complications. Three cytokines (IL-1-a, IL-1-β, TNF-α) and two nitric oxide synthase (NOS2, NOS3) genes polymorphisms were genotyped in 77 APL and 117 PJI patients and 145 controls with aseptic hip or knee implants that were implanted for > 16 years. Plasma cytokines and nitrate-nitrite (NOx) levels also were measured. The TT genotype and T allele of (+3954 C/T, exon 5, rs1143634) IL-1β polymorphism were more frequent in APL patients compared to controls (P = 0.03 and P = 0.02, respectively). No genotypic associations in PJI patients were observed. Plasma IL-6, TNF-α and NOx were significantly different between APL and controls (P < 0.0001). Plasma IL-1β and IL-6 were significantly higher in APL T allele carriers vs. non-carriers (P < 0.03). Knee implant (HR 2.488, 95% CI 1.307–4.739, P = 0.005), male gender (HR 2.252, 95% CI 1.121–4.525, P = 0.023), carriages of the TT genotype of the (+3954 C/T) IL-1β polymorphism (HR 3.704, 95% CI 1.274–10.753, P = 0.016) and AA genotype of the (exon 22) NOS2 polymorphism (HR 3.509, 95% CI 1.266–9.709, P = 0.016) were independently associated with a shorter implant survival by Cox regression. No genotypic associations in PJI patients were observed. Genotyping of IL-1β (+3954 C/T, exon 5, rs1143634) and NOS2 (exon 22) polymorphisms could be useful as predictors of early hip or knee APL.

Some NOS3 polymorphisms have been linked to differences in NO blood levels or in protein expression in response to several stimuli, including the 27-bp repeat in intron 4, the (-786 T/C,) in the promoter region (rs2070744), and the missense (E298D) in exon 7 (rs1799983). These NOS3 polymorphisms are associated with bone diseases 27,28 . Several polymorphisms of NOS2, such as the highly polymorphic CCTTT micro-satellite at the promoter region, the G/A substitution at position 37498, and G/A in exon 22 (iNOS 22), are associated with rheumatoid arthritis 29,30 . However, the association between NOS3 or NOS2 polymorphisms and APL has not been studied so far.
The aim of this study was to analyze a potential association between hip and knee PJI and APL and some polymorphisms of cytokines and NOS that were previously associated with inflammatory bone diseases, ([-889

C/T] IL-1α, [+ 3954 C/T, exon 5] IL-1β, [-308 G/A] TNF-α]), ([-786 T/C] NOS3 and [exon 22, NOS2]).
We compared a group of APL and PJI patients with hip or knee implants to control patients who had neither complication after ≥ 16 years. A secondary aim was to study the expression of cytokines and NOS in the different genotypes by measuring IL-1β, IL-6, TNF-α and nitrate-nitrite (NOx) plasma levels in APL and PJI patients and controls.

Material and methods
Patients. We recruited patients who were admitted with APL of hip or knee for a second arthroplasty or with PJI for antimicrobial treatment at the Hospital Universitario Central de Asturias (HUCA) between January 2003 and April 2021. Surgical confirmation of the APL and a negative bacteriological culture of surgical samples were required for inclusion as APL. PJI patients were included if they fulfilled the 2013 Infectious Diseases Society of America PJI diagnostic criteria 34 . Cultures of surgical and sinus tract samples from PJI patients were collected.
The main indication for the primary arthroplasty was degenerative osteoarthritis in all the APL and PJI patients, and controls. Other diseases which could affect the bone metabolism, such as hyperparathyroidism, chronic kidney insufficiency, rheumatoid arthritis and other inflammatory arthropathies, steroids use, autoimmune diseases, and avascular necrosis of the femoral head, were excluded from the study.
Controls were individuals admitted to the HUCA to undergo a primary hip or knee arthroplasty since January 2003 and were followed ≥ 16 years until April 2022 or implant-unrelated death without developing APL or PJI.
Patients and controls were members of a homogeneous population; all were Europeans and residents of the same region (Asturias, Northern Spain). Each participant gave written informed consent for the study, which was approved by the Ethics Committee of the HUCA with the number 2020.113. Genotypic analysis. Genomic DNA was extracted from peripheral leukocytes following a salting-out method 35 18,27 . The PCR results were confirmed by sequencing representative samples for each genotype of each polymorphism. PCR products were electrophoresed on a 2% low-melting agarose gel and the fragments were excised from the gel, purified with spin columns (DNA gel extraction Kit, Millipore, Billerica, MA, USA) and sequenced on an ABI Prism 310 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Plasma nitrate and nitrite levels. NO is a short-lived free radical gas that rapidly reacts with oxygen to generate the stable metabolites nitrate and nitrite. Thus, NO levels were assessed indirectly by measuring the accumulation of nitrates and nitrites. Plasma nitrate and nitrite (NO x ) determinations were performed using the Griess reaction 27,36 . Results were expressed as μM of NO x /sample.

Statistical analysis.
Non-parametric tests were used to assess the comparison of continuous variables because of their non-Gaussian distribution. Values are reported as median and interquartile range (IQR) or percentage as appropriate. Mann-Whitney U and Kruskal-Wallis tests were used to compare two or more groups, respectively, and the-chi-square test to compare proportions. Multivariate Cox proportional hazards models were constructed to assess the relationship of prosthesis loosening and infection with the diverse genotypes and the factors independently associated with such clinical outcomes. All the reported p values are two-sided. A P There were no differences in IL-1β levels among the groups, but controls had substantially higher levels of IL-6 than the APL and PJI groups, whereas APL patients had higher TNF-α serum levels than the other two groups. Plasma NOx levels were significantly higher in the controls compared to APL patients (Table 3). Table 1. Oligonucleotide primer sequences, PCR conditions and restriction enzymes used for genotyping and sequencing of the different polymorphisms studied. The underlined bases in the primers differ from the original sequences and served to introduce a restriction site or to disrupt a natural restriction site within the primer sequence.  www.nature.com/scientificreports/ All genotypes and alleles analyzed were in Hardy Weinberg equilibrium except for the IL-1α (-889 C/T) polymorphism in the controls. Table 4 describes the relationships among the diverse polymorphisms analyzed and the three groups of patients. Overall, there were no statistically significant associations when the three groups were considered altogether. However, when compared individually, the genotypes and alleles of the IL-1β (+3954 C/T, exon 5, rs1143634) polymorphisms were differently distributed (P = 0.03 and P = 0.02, respectively) between APL patients and controls, mainly due to a higher representation of the variant T allele and TT genotype in the APL group (these individual comparisons data are not shown in Table 4).
Plasma IL-1β and IL-6 were significantly higher in APL IL-1β (+3954C/T, exon 5) T allele carriers compared to non-carriers (IL- Multivariate Cox proportional hazard models were elaborated for each polymorphism to evaluate the associations between the diverse genotypes with prosthesis loosening and infection over time. No polymorphisms genotypes were significantly associated with PJI, but genotypes of the IL-1β (+3954 C/T, exon 5) and NOS2 (exon 22) SNPs showed different behavior in the APL group. Table 5 shows the results of these two multivariate regressions. IL-1β (+ 3954 C/T, exon 5) SNP independently associated with knee prosthesis while the NOS2 (exon 22) SNP also associated with knee prosthesis and male gender. Figure 1 depicts the Cox regression curves survival of the joint replacement according to the IL-1β (+3954 C/T) and NOS2 (exon 22) genotypes. Patients carrying the respective variant homozygous genotypes experienced earlier prosthesis loosening than those carrying the heterozygous and homozygous wild genotypes, an effect that was mainly noted during the first 5 years after surgery.

Discussion
Total hip and knee replacements are some of the most successful and cost-effective surgeries performed. Although the success rate at 10 years exceeds 90% in hip 37,38 and knee arthroplasties [39][40][41][42][43] , prosthesis, failure remains a problem necessitating revision surgery, which is costly both in monetary terms and morbidity 42,43 . It is difficult to predict who will require an early replacement. www.nature.com/scientificreports/ We found a significant association between a polymorphism within exon 5 of IL-1β (+3954 C/T, rs1143634) and early APL. The TT genotype of this IL-1β polymorphism showed a 3.7-fold higher probability of requiring a joint replacement because of loosening within the first 5 years after surgery, compared to controls with implants lasting ≥ 16 years. This association between IL-1β polymorphism and early loosening association was especially strong with knee APL (Table 5).
IL-1 and TNF-α genes are transcriptionally activated in patients and in experimental models of APL [44][45][46][47] . IL-1 and TNF-α activate bone-resorbing giant cells that can increase bone loss around implants, which is characteristic of APL 48 . Osteoporotic fractures due to a reduction in the bone mineral density are associated with an 86-base pair repeat polymorphism in the IL-1RN gene 49 . There is a linkage disequilibrium between IL-1α, IL-1β and IL-1RN genes, all of which are encoded very close to each other on the long arm of chromosome 2 50 . Thus, it is difficult to be sure whether these associations are specific for a particular gene in the IL-1 family, or even depend on other unknown gene from the same chromosome. Some haplotypes of the IL1R1-IL1A-IL1B-IL1RN gene cluster associated with enhancement to (IL1A-IL1B-IL1RN haplotype) or protection against knee osteoarthritis (IL1B-IL1RN haplotype) 20 .
Associations between polymorphisms in the IL-1 gene and aseptic loosening have been studied in maxillofacial surgery. An association between carriage of the IL-1β (+3954 C/T, exon 5) T allele and other IL-1 polymorphisms and unsuccessful retaining overdentures and periodontitis in smokers and non-smokers was reported 21,[51][52][53][54][55][56] . An association between a (-889) polymorphism of the promoter region of IL-1α and susceptibility to juvenile rheumatoid arthritis and to osteomyelitis was also reported 11,18 .
The IL-1 gene variations were previously examined for associations with various chronic inflammatory diseases and with altered levels of inflammatory mediators 56,57 . We observed that APL patients with the TT genotype of the IL-1β polymorphism had the highest plasma levels of IL-1β and IL-6 compared to carriers of other genotypes of the APL group. Although immunohistochemistry studies of the removed synovial membranes or measure of the cytokine levels in synovial fluid were not performed in our study, based on the work of others 58 we would expect that patients with the TT genotype of the IL-1β polymorphism might have proinflammatory cytokines (IL-1β and IL-6) overexpressed in synovial fluids around the implants, facilitating the aseptic loosening of the prosthesis.
We also found an association of the NOS2 (exon 22) polymorphisms with early APL. Carriers of the AA genotype and A allele of this NOS2 polymorphism had a 3.5 fold higher probability of early loss of their implants, mainly during the first 5 years after surgery. The association of NOS2 (exon 22 polymorphism with APL was especially strong with knee prosthesis loosening and with male gender. Although plasma NOx levels were significantly decreased in APL patients compared to controls, we could find no differences in plasma NOx levels among carriers of the different genotypes of the NOS2 and NOS3 polymorphisms. However we have measured plasma NOx only at one point in time, rather than a 24 h urine nitrate assay that would give a better picture of how much NOx is produced. A relationship of the NOS3 (27-bp, intron 4) polymorphism and osteomyelitis 27 and of the NOS3(Glu298Asp) polymorphism and hip osteoporotic fractures 28 was previously reported emphasizing the key role of NO in bone metabolism.Thus, the mechanism underneath the association of this NOS2 (exon 22) polymorphism and APL remain elusive.
Regarding PJI, no association of cytokines or NOS polymorphism were observed in our study. An association of the other IL-1 β genetic variant, the IL-1β (-511C/T, rs 16944) polymorphism, not genotyped here, and PJI was reported 32,33 . However the TNF-α (-308 G/A rs1800629) polymorphism negative association with PJI we observed fully agree with others 59 .
The main limitations of this study is the relatively small number of APL patients included and the limited number of cytokines and NOS2 and NOS3 polymorphisms genotyped due to budget limitations. The strong points of the work are the novelty of genotyping NOS2 and NOS3 polymorphisms, the inclusion of a good number of PJI patients and the use of patients with orthopaedic prosthesis lasting > 16 years as controls.
To our knowledge this the first study describing an association of IL-1β gene (+3954 C/T, exon 5, rs1143634) and NOS2 (exon 22) polymorphisms and early APL. However, further research in larger European and non-European populations is needed to clarify these genetic associations with early APL.

Data availability
All data generated or analysed during this study are included in this published article.