Gout requires a unique approach to arthritis targets and biomarkers of the response to XOI-based ULT, due to variable phenotypes, and weaving of urate homeostasis, comorbidities, and inflammatory arthritis1–5,8. In contrast to the genetics of urate biology, genome-wide association studies have identified few genetic coding variants potentially involved in gouty arthritis30,31. Therefore, this biomarker study profiled the serum proteome of gout patient sera at 48wks sustained ULT to urate target, here using XOI, and with achievement of reduced flare burden and serum urate in two independent cohorts.
Specific serum proteomics findings at 48wks XOI-based treat to target ULT, in both cohorts studied, included decreased C8A and C8G chains, which play a major role in complement C5b-9 MAC assembly and activity that, along with C5a generation, contribute substantially to the inflammatory process in model gouty arthritis15,16,32. Paradoxically, we detected increase in serum of the NLRP3 inflammasome scaffolder and activation promoter VIM (vimentin)33, of interest because early increase in gout flares is seen in XOI-based ULT9, Increased serum sCD44 was noteworthy, since sCD44 inhibits macrophage phagocytosis of urate crystals and consequent NLRP3 inflammasome activation, by blocking crystal binding to transmembrane CD4434.
We also observed increase in serum of TGFB1, which promotes model gout flare resolution by suppressing macrophage activation by crystals35. Conversely, IGF-I, which cross-talks with and can synergize with TGF-beta, was decreased in serum at 48wks ULT36. We detected decrease in serum of the phagocyte-recruiting chemokine PPBP/CXCL737, and decreased lactoferrin, a neutrophil-released co-activator of the lubricin-degrading serine protease Cathepsin G 38. That finding was of note, since Cathepsin G is a major degrader of lubricin, which functions as a substantial constitutive suppressor of gouty inflammation and urate production by synovial resident macrophages39. We also observed an increase in monocyte/macrophage-expressed keratin-related proteins (KRT9,14,16), further validated by Cohort 1 gout patient PBMC proteomics. KRT16 is implicated in monocyte to macrophage differentiation, and MMP-1 and innate immune responses to tissue damage in epithelia40.
Last, STRING-db analyses of significantly altered proteins from both cohorts revealed that the tight serum protein interactome network altered by XOI-based ULT encompassed a core group of central mediators of gouty inflammation (including IL-1B, CXCL8, IL6, C5)4.
Robustness of our findings on effects of effective ULT on the serum protein interactome discovered here was buttressed by a group of parallel studies. First, in this context, previously published evidence in gout Cohort 1 that the ULT regimen altered the serum metabolome, and the serum lipidome in gout Cohorts 1 and 2, and effects of febuxostat on lipolysis in cultured adipocytes25. Moreover, the current study demonstrated that the serum metabolome was significantly altered for purine and pyrimidine metabolism in Cohort 2, associated with significant changes in multiple other pathways, most pronounced for linoleate metabolism at both 24wks and 48wks ULT. Second, analyses of the Cohort 1 proteome of gout patient PBMCs identified 42 high-confidence interacting proteins belonging largely to secretion, leukocyte, and neutrophil activation gene ontology pathways. The KRT findings for serum proteins were validated in the PBMC proteome. In addition, we found strong and high confidence (> 0.700) interactions between known gout mediators and EFS identified proteins, particularly in the proteins identified at 48wks of ULT, including MMP9. Whereas no significant difference in MMP9 abundance levels was identified between BL and 48wks of ULT, further study would be needed to validate significance of differences between PBMC proteome groups 1 and 2. The collective results of PBMC proteome analysis further teased apart the effects of XOI-based ULT in gout, and highlighted anti-inflammatory effects of XOI-based ULT on these leukocytes as a whole.
We employed in vitro studies that characterized effects of the selective XOI febuxostat on the proteome of cultured murine BMDMs stimulated by the major gouty inflammation driver IL-1b. Febuxostat suppressed multiple pro-inflammatory IL-1b-induced changes in the macrophage proteome. Analyses of gene ontology enrichment of proteins found in the macrophage protein interactome revealed that in vitro XOI treatment of activated BMDMs broadly reversed many pro-inflammatory responses. Notably, the most pronounced pathway changes were seen in classical and alternative pathway complement activation, which reinforced the impact of the findings for XOI-treatment effects on C8A and C8G in the gout patient serum proteome. Febuxostat also altered lymphocyte-mediated immunity, fibrinolysis, and cytolysis gene ontology pathways in cultured macrophages in response to IL-1b. Our findings in cultured macrophages and gout patient PBMCs were novel partly because previous studies have suggested that both hyperuricemia and urate crystals program elevated monocyte inflammatory responses in vitro and that hyperuricemia primes model gout inflammation in mice in vivo model gout41–43.
A pro-inflammatory serum proteome signature was recently characterized in asymptomatic hyperuricemia (AH) by targeted proteomics44. The approach used the Olink Target 96 Inflammation Panel™ 44, distinct from the unbiased mass spectrometry-based approach utilized in the current study. The methodology employed dual recognition by oligonucleotide-labelled antibody probe pairs and DNA-coupled quantitative PCR, designed to detect specific immunoregulatory proteins below mass spectrometry detection limits44. Upregulated serum immunoregulatory proteins in AH group included the mTOR effector 4E-BP1, IL-18R1, multiple growth factors, chemokines, and members of the IL-6 cytokine and TNF superfamily44. A Th17 cell signature, and increases in inflammation-dampening IL-10 and FGF21 also were identified44. Using the same targeted serum proteomics approach, a small sub-study of 13 subjects before and 3 months into successful XOI-based treat to target ULT revealed significant downregulation of LIF-R. CDCP1, IL-18, NT-3, IL10RB, CCL28, CCL11, and SLAMF144. A second, recent study of the serum proteome in gout flare, using the same targeted proteomics approach in two independent cohorts, identified four markers elevated during gout flare compared to the treat to target phase and in-between flare (intercritical) phase. These inflammation-mediating proteins were tumor necrosis factor superfamily 14 (TNFSF14), IL-6, colony-stimulating factor 1 and vascular endothelial growth factor A45.
The differentially detected proteins in both these referenced targeted proteomics studies 45were predominantly cytokines and growth factors below the detection limits of our unbiased mass spectrometry serum proteomics approach. Therefore, the design, approach, and sample size of the current study were unique and provided distinct information on the molecular signature of XOI effects on hyperuricemia in gout.
Hyperuricemia increases blood monocyte population expansion in vivo in humans42, however, monocytes, and other mononuclear leukocytes, are heterogeneous, and can be recruited into diseased or challenged tissues, and one limitation in this study is that monocytes are normally only a small fraction (ie, ≤ 10%) of PBMCs46. PBMCs remain a source of highly informative biomarkers for acute and chronic inflammatory diseases, but also are highly heterogeneous47, buttressing the limitation of this study that PBMCs only were obtained at the Cohort 1 site. This trial did not have a placebo, and the clinical trial did not include a uricosuric treatment arm, with the infrequently employed and frequently contraindicated USA-approved drug probenecid, to isolate effects due to serum urate-lowering without XOI. We did not exclude subjects with flare at onset of study enrollment and first and final blood sampling, or CRP higher than 2 mg/L, but it is noted that such CRP elevation was present at blood sampling in less than a handful of subjects. Also, we did not study gout patient controls from the same clinical trial that failed to achieve serum urate target. However, the proportion of such subjects overall in the VA STOP GOUT trial was low (ie, ~ 20%)19, and all those subjects were considered at least partially treated since they received XOI-based ULT.
In conclusion, a novel, functionally important network of physically interacting proteins in gouty inflammation emerged in association with response to sustained XOI-based ULT that effectively reduced gout flare burden. Potential clinical significance of the results, especially for data from the clinical trial, included that the treat to target ULT regimen is associated with early increase in flare activity before gout flares eventually decrease9. Moreover, the current study provides further support for the use of serum proteomics, including biomarker approaches highlighting the complement pathway and the inflammatory secretome, to help identify responsiveness of gouty inflammation to ULT pharmacotherapy, and for characterization and prognosis of different clinical phenotypes in gout39,44,48.