Lipopolysaccharide stimulation test on cultured PBMCs assists the discrimination of cryopyrin-associated periodic syndrome from systemic juvenile idiopathic arthritis

Systemic juvenile idiopathic arthritis (sJIA) and cryopyrin-associated periodic syndrome (CAPS) share many common manifestations. We aim to identify an applicable method to assist disease discrimination. Inflammatory cytokines were measured in the plasma of patients with CAPS, sJIA with persistent disease course and healthy controls. Supernatants collected from non-stimulated peripheral blood mononuclear cells (PBMCs) and those undergone inflammasome stimulation tests utilizing lipopolysaccharide (LPS) with and without adenosine triphosphate (ATP) were investigated. Inflammatory cytokines in patient plasma fail to differentiate sJIA from CAPS. PBMCs from sJIA secrets higher amount of IL-1β and IL-18 while CAPS PBMCs produces more caspase-1 without stimulation. IL-1β, IL-18, and caspase-1 were significantly elevated among CAPS PBMCs (all p < 0.05) upon LPS stimulation, but not when additional ATPs were provided. Levels of cytokines and PBMC responses to the stimulation assays were similar among all sJIA patients regardless of their history of macrophage activation syndrome. Unstimulated PBMC activities and the LPS inflammasome stimulation assay without exogenic ATPs can assist the differentiation of CAPS from sJIA with persistent disease course.

www.nature.com/scientificreports/ gen, Taiwan) according to the manufacturer's protocol. Sequencing reaction was performed with Big Dye Terminator v. 3.1 Ready Reaction Cycle Sequencing kit (Applied Biosystems, Warrington, UK). The electrophoretic profiles of NLRP3 sequences were analyzed on the ABI 3500 Genetic Analyzer. For those negative of NLRP3/CIAS1 missense mutations, whole exomes sequencing were performed to expand the coverage of other autoinflammatory disease related gene mutations. In detail, exome capture were performed using the Agilent SureSelect Human All Exon Kit V6 (Agilent Technologies) and massively parallel sequencing were carry out using the NovaSeq 6000 (Illumina, San Diego, CA). Raw image analyses and base calling were performed using Illumina's Pipeline with default parameters. Sequence data were aligned to the reference human genome (hs37d5) using the Burrows-Wheeler Aligner 21 , and duplicate reads were removed using Picard tools. We use the Genome Analysis ToolKit (GATK 4.1.2) Haplotype Caller for variant calling of SNVs and short (< 50 bp) indels 22 . Annovar were utilized to catalogue the detected variations 23 . Then, we filtered variations with a homopolymer length > 6 (and synonymous substitutions) or that were common (> 1%) in dbSNP150 (http:// www. ncbi. nlm. nih. gov/ proje cts/ SNP/), HapMap, the 1000 Genomes Project (http:// www. 1000g enomes. org), the Exome Aggregation Consortium database and the Genome Aggregation Database (GnomAD, https:// gnomad. broad insti tute. org). Integrated genome viewer was used to visualize the reads for manual checking.

Inflammasome stimulation test. After isolating human peripheral blood mononuclear cells via Ficoll-
Paque gradient centrifugation, cells were counted and seeded onto 96-well plates at a concentration of 3 × 10 6 cells/ml in culture medium (RPMI medium with 10% heat-inactivated autoserum, 100 U/ml penicillin and 2 mM L-glutamine). The cells were either stimulated with LPS 1 µg/ml (Sigma) for 1 or 4 h, or primed with LPS 1 µg/ml for 1 h then stimulated with ATP 2 mM (Sigma) for another 1 or 4 h after cell seeding. Supernatants collected at 1 h after PBMC seeding without stimulation were used as non-stimulation controls. The culture supernatants were collected for the analysis of IL-1β, IL-18 and caspase-1 secretion with and without stimulations. All tests were performed in duplicates.
Statistical analysis. Continuous data were summarized as means ± SDs and compared by unpaired t-test.
Nonparametric Mann-Whitney sum rank U test and Kruskal-Wallis (χ 2 ) test were used for the between-group comparison. The level of significance is determined to be at 0.05. Statistical analyses and graphic presentation were carried out using Prism 6.01 software (GraphPad Software, Inc., San Diego, California).

Ethics approval and consent to participate. This study was approved of by the Institutional Review
Board of the Chang Gung Memorial Hospital (IRB No.: 201802287A3).

Results
Clinical characteristics and inflammatory markers in cases with CAPS and sJIA. The average age of onset is 6.2 ± 5.7 years among cases with CAPS and 6.7 ± 4.8 for cases with persistent sJIA. Age at time of recruitment was 23.8 ± 18.0, 13.6 ± 7.8 and 18.3 ± 9.3 for CAPS, sJIA and healthy controls, respectively. The gender distribution, levels of acute phase reactants, laboratory profiles as well as the treatment regimens at time of sampling were summarized in Table 1. Three of the cases (25%) suffered from persistent sJIA experienced at least one MAS episodes after the diagnosis of sJIA.
Among the 11 patients with CAPS, three cases presented with a phenotype compatible with Muckle-Wells syndrome (MWS) while the other 8 experienced recurrent urticarial-like skin rashes especially upon cold or stress triggers. Five patients from 2 distinct family were found to harbor heterozygous c.1316 C > T (p.A439V) NLRP3 variants, which has been confirmed pathogenic according to the Infever database 17 . Additionally, two independent cases carrying heterozygous NLRP3 variants with undetermined clinical significance c.210 G > A (p.V70M) and c.1371 G > T (p.E457D) were also detached. The minor allele frequency for c.210 G > A and c.1371 G > T is 0.000741 and 0.00008 in the Trans-Omics for Precision Medicine program and 0.0077 and 0.0013 among the East Asians in the GnomAD database, respectively. Somatic mosaicism of NLRP3 were not identified using the sequencing technique applied. Cases diagnosed with persistent sJIA were negative for known genetic mutations associating autoinflammatory diseases. Clinical manifestations and relevant comorbidities of the patients diagnosed with CAPS were summarized in Supplementary Table 1.

Differences in the level of inflammatory cytokines between cases with CAPS and sJIA. CAPS
and sJIA were both systemic inflammatory diseases with overactivated innate immune responses. To investigate whether there are differences in the plasma profile of inflammatory cytokines, levels of IL-1β, IL-6, IL-18, TNF-α and SAA were examined from patients' plasma. As shown in Fig. 1, while an elevation in the level of IL-18 was observed in patients with CAPS when compared to the healthy controls (1366.1 ± 196.2 vs. 568.8 ± 171.9; p = 0.0008), the differences in levels of plasma IL-6 between CAPS and persistent sJIA were borderline significant (p = 0.051). Additionally, patients with sJIA have higher levels of inflammatory cytokines in all parameters

Differences in inflammasome stimulation test between cases with CAPS and sJIA. Consider-
ing the critical role of NLRP3 inflammasome in CAPS and its possible effect in sJIA 8,24,25 , we evaluated inflammatory cytokines production from patient PBMCs with and without NLRP3 stimuli using plain culture soup  Interestingly, caspase-1 production is increased in the CAPS PBMCs when compared to those isolated from sJIA and healthy controls before additional stimulants were applied (all p < 0.05). Moreover, the levels of IL-1β were also slightly higher in the sJIA PBMCs as compared to the healthy controls before stimulation (p = 0.03). Extracellular ATP is a potent activator for NLRP3 inflammasome 26 . When PBMCs were provided with additional ATPs followed by LPS priming, the levels of caspase 1 and IL-1β increased by 2 to 4 folds universally, as www.nature.com/scientificreports/ shown in Fig. 2B. However, as the production of inflammatory cytokines were significantly increased, the differences in the levels of IL-1β, IL-18 and caspase 1 between those with or without diseases may be limited. Indeed, no significant differences in the inflammatory mediators were detached in the culture soup of PMBCs isolated from patient diagnosed with CAPS, sJIA and healthy controls (all p > 0.05).

Differences in plasma cytokine levels and inflammasome responses in sJIA patients with and without a propensity of MAS.
To investigate whether the propensity of MAS correlated with inflammasome responses or levels of plasma cytokines in cases with sJIA, we compared the inflammatory mediators acquired from 3 sJIA who experienced at least one episodes of MAS during follow up to those without. As shown in Fig. 3A, no significant differences were observed in the levels of plasma IL-1β, IL-6, IL-18 or TNF-α between sJIA patients regardless of the history of MAS (all p > 0.05). Moreover, the production of IL-1β and IL-18 in PBMCs upon LPS stimuli were also similar (Fig. 3B).

Discussions
In the present study, we found that while inflammatory cytokines were generally elevated in persistent sJIA patients' plasma as compared to the healthy controls, differences between CAPS and sJIA patients were not statistically significance. Moreover, the production of IL-1β, IL-18, and caspase-1 in the inflammasome activation assay were significantly increased among CAPS patients in comparison with sJIA patients or healthy individuals 1 or 4 h following LPS stimulation. This reaction, however, is masked by the universal elevation of inflammatory cytokines when exogenic ATPs were added. Finally, our data suggested that the levels of plasma cytokines and PBMC responses to the LPS stimulation assays were similar among all sJIA patients regardless of their propensity of MAS. Nirmala et al. recently reviewed the similarities and differences of the protein, cellular, mRNA and DNA markers among patients suffering from sJIA and CAPS 16 . While inflammatory markers including IL-6, IL-18, S100A8/A9 and S100A12 were noted to elevate under both conditions 27-31 , limited reports have yet directly compared CAPS and sJIA for their expression of biomarkers. In a case series reported by Ohnishi et al., one patient with active JIA presented with higher levels of serum IL-6 and IL-18 in comparison with those with CAPS and healthy controls 3 . S100A8 and S100A9 have also been found to increase significantly among cases with sJIA, www.nature.com/scientificreports/ particularly during active status, when compared to patients with neonatal onset multisystem inflammatory disease (NOMID) and other inflammatory disorders 30 . As the activity status of sJIA largely influenced the level of serum cytokines 30,32 , sampling sJIA patients during chronic active phases, our data on inflammatory cytokines show no differences between cases suffered from CAPS and sJIA (Fig. 1). Thus, while our data echoed previous reports on a global increase of proinflammatory cytokines in cases with sJIA and CAPS 16 , no single cytokine along is capable of assisting the differentiation of CAPS from cases with persistent sJIA. Physiologically, NLRP3 cryopyrin, an intracellular sensor, forms inflammasome complex associating procaspase-1 and ASC proteins upon encountering its triggers 26 . The formation of inflammasome then catalyzes pro-IL-1β and pro-IL-18, produce upon the activation of nuclear factor kappa B (NF-κB) signaling, to their mature form and potently drives further inflammation 26,33 . According to our result, no spontaneous secretion of IL-1β and IL-18, but caspase 1 was noted in CAPS PBMCs before any stimulation was applied ( Fig. 2A,B). This may be explained by the necessity of NF-κB signaling for the transcription of pro-IL-1β and pro-IL-18, before they can be catalyzed by the over-active NLRP3 inflammasomes 34,35 . On the contrary, caspase-1 is a direct interaction partner for the NLRP3 inflammasome and is critical for its proteolytic activity in processing the precursors of various inflammatory cytokines 33 . Even without additional triggers, altered NLRP3 proteins oligomerized with pro-caspase-1 and displayed a higher basal caspase-1 activity as compared to healthy controls 36 . Nonetheless, while several reports also demonstrated the requirement of NF-κB signaling for the production of IL-1β and IL-18 in in vitro stimulation assays 3,37,38 , others reported high unstimulated cytokine production, including IL-6, IL-18, TNF, interferon (IFN)-γ and IL-12p70 in PBMCs isolated from patients suffered from CAPS 39,40 . These differences may be influenced by the disease status and severity at time of sampling. Furthermore, PBMCs from sJIA secreted higher amount of IL-1β and IL-18 before any additional stimulants were provided ( Fig. 2A,B). Although the exact mechanism is unknown, genes involved in innate immune reaction, such as IL-1, IL-18 and toll like receptor (TLR) signaling pathways have been found to upregulate in cases with sJIA 41 .
The most pronounced difference between CAPS and persistent sJIA in the present study is perhaps the reaction of PBMCs upon LPS stimulation ( Fig. 2A). While different mutations and its capacity to auto-activate the inflammasome can lead to large variability in the responses to stimulation 37 , IL-1β, IL-18, and caspase-1 were significantly elevated among CAPS PBMCs (all p < 0.05) upon LPS stimulation, but not when additional ATPs were provided. Experiments carry out in MWS murine models revealed a lower threshold and enhanced production of IL-1β and IL-18 42,43 . Likewise, enhanced IL-1β, IL-18 and caspase-1 release have been shown in CAPS PMBCs in comparison to control PBMCs 34,37 . Mutations in NLRP3 is believed to drive inflammation in cases with CAPS. Although polymorphisms in NLRP3 have also been reported to associate with sJIA 25 , it did not lead to an altered inflammatory response upon TLR stimulation. Additionally, while Reiber et al. observed a significant increase of mature IL-1β, IL-18, and caspase-1 in culture supernatants 4 h following LPS stimulation 37 , our result suggested that the differences may be detected at an earlier time point. The rapid and colossal secretion of inflammatory cytokines upon LPS stimulation is a reflection of the efficient processing of pro-cytokines in CAPS with constitutive active NLRP3 inflammasomes 1,2,4 . Moreover, priming the PBMCs with LPS followed by addition ATP in the samples showed global elevation in the inflammasome related products regardless of the underlining diseases (Fig. 2B). This was distinct from Reiber's report, perhaps because their PBMCs received dual stimulation with ATPs and LPS simultaneously at a dose 10 times higher 37 . ATP is a potent activator for NLRP3 inflammasome 26 . The exogenic ATPs provided in the present protocol somehow activated NLRP3 inflammasomes to a comparable degree as they were in CAPS and masked the differences when LPS stimulation was provided. Together, our data suggested that LPS stimulation test is better than LPS and ATP together in identifying CAPS from patients with persistent sJIA and healthy controls.
Marked increase of proinflammatory cytokines including IL-1, IL-6, IL-18, TNFα, IFNγ, ferritin and ST2 during MAS attacks portrayed a significant systemic inflammation in patients with various rheumatic diseases, particularly sJIA 28,[44][45][46][47] . Recently, Canna et al. reported that gain of function mutations in NLRC4 can display a MAS like clinical presentation 48 . In addition, repetitive TLR stimulation via administration of CpG has been shown to induce a MAS-like features in mice 49 . Even though infections and inflammasome dysregulation have been considered as possible triggers for MAS 44,45 , the discrimination of immune responses between sJIA from CAPS with LPS stimulation tests in not jeopardized by the tendency for MAS in the present study with limited cases. Although further study may be required to confirm this observation, the similarity in the level of inflammatory cytokines and PBMC responses to LPS suggested that the induction of MAS in patients with sJIA may less likely result from NLRP3 inflammasome dysregulation.
The present study is limited by its few case numbers and only CAPS patients with the clinical phenotype of familial cold autoinflammatory syndrome (FCAS) and MWS were studied. It would be worthwhile to also examine patients with NOMID phenotype since the disease activity and phenotype have also been implied with altered inflammasome responses 39 . Moreover, considering the association of aging and inflammasome activity 50 , the imperfectly age-matched healthy controls can also result in bias in the present study. Finally, the in vitro functional diagnostic tests were performed in CAPS and sJIA patients under medical treatment with a relatively long disease duration. Although in clinical settings, the consideration of rare diseases and genetic testing are usually reserved for those who suffered from persisted disease course or those who respond poorly to standard treatments, future validation of the results with treatment-naïve patients before being correctly diagnosed for CAPS or sJIA would definitely further strengthen the clinical application.

Conclusions
In summary, our data suggested that the rapid and excessive-production of IL-1β, IL-18 and caspase-1 in the LPS inflammasome stimulation assay on PBMC cultures can assist the differentiation of persistent sJIA and CAPS. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.