Fig 2: Tolerance induction by UOX-Ery enhances the pharmacokinetics and pharmacodynamics of UOX-based therapy for gout.(A and B) Pharmacokinetic and pharmacodynamic analysis in Wistar rats treated with rEry alone (n = 4), UOX-rEry alone (n = 2), CTX followed by three challenges with UOX-rEry (n = 4), or a single coadministration of UOX-rEry and CTX followed by three challenges with UOX-rEry (n = 6). (A) Pharmacokinetic profiles of UOX-rEry. (B) Serum uric acid levels. (C and D) Therapeutic evaluation in Uox−/− mice treated with mEry alone (n = 4), UOX-mEry alone (n = 4), a single coadministration of UOX-P and CTX followed by two challenges with UOX-mEry (n = 5), or a single coadministration of UOX-mEry and CTX followed by two challenges with UOX-mEry (n = 5). (C) Pharmacokinetic profiles of UOX-mEry. (D) Serum uric acid levels. (E) Pharmacokinetic profiles of free UOX-P in C57BL/6 mice pretreated with CTX or coadministered with UOX-mEry and CTX, followed by two challenges with UOX-P (n = 2 per group). Data are presented as means ± SEM. Half-life (T1/2) was calculated using a one-phase decay model [(A), (C), and (E)]. Statistical significance was assessed using two-way ANOVA with Tukey’s multiple comparisons test [(B) and (D)]. *P < 0.05; ***P < 0.001. d, days; NC, not calculable.

Fig 3: A single coadministration of UOX-Ery with CTX induces long-lasting and antigen-specific immune tolerance in rodents.(A) Tolerance induction to UOX-mEry in C57BL/6 mice pretreated with UOX-mEry alone, CTX alone, UOX-P coadministered with CTX, or UOX-mEry coadministered with CTX, followed by three challenges with UOX-mEry (n = 5 per group). (B) Tolerance induction to UOX-rEry in Wistar rats pretreated with UOX-rEry alone, CTX alone, or UOX-rEry coadministered with CTX, followed by 10 challenges with UOX-rEry (n = 4 per group). (C) Tolerance induction to UOX-P in C57BL/6 mice pretreated with CTX alone, UOX-P coadministered with CTX, or UOX-mEry coadministered with CTX, followed by four challenges with UOX-P (n = 3 per group). (D) Tolerance induction to OVA-P in C57BL/6 mice pretreated with OVA-mEry alone, CTX alone, or OVA-mEry coadministered with CTX, followed by four challenges with OVA-P (n = 3 per group). (E) Antigen specificity of antigen-Ery–induced tolerance. Mice were treated with CTX alone followed by two challenges with either UOX-P or OVA-P; OVA-mEry coadministered with CTX followed by two challenges with UOX-P or OVA-P; or UOX-mEry coadministered with CTX followed by two challenges with UOX-P (n = 3 per group). Anti-UOX and anti-OVA IgG levels were assessed on Day 35. Data are presented as means ± SEM. The dashed line indicates the lower limit of detection for antibody titers. Statistical significance was assessed using two-way ANOVA with Tukey’s multiple comparisons test [(A) to (D)] or unpaired t test (E). ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 4: Coadministration of UOX-Ery with CTX induces tolerogenic macrophages in the spleen.(A and B) Experimental design for assessing splenic uptake of UOX-mEry. C57BL/6 mice were injected with mEry (n = 2), FITC-labeled UOX-P (n = 3), or UOX-mEry (n = 3). Splenocytes were isolated 4 hours postinjection and analyzed by flow cytometry. (B) Left: Representative flow cytometry plots showing uptake of mEry, UOX-P, or UOX-mEry by F4/80+ macrophages. Right: Quantification of UOX-FITC+ cells across different immune cell types. (C to E) Imaging analysis of UOX-mEry phagocytosis by splenic macrophages. C57BL/6 mice were injected with Far Red–labeled UOX-mEry, and splenocytes were collected 4 hours later. F4/80+ macrophages were isolated by magnetic sorting. (D) Immunofluorescence images of isolated macrophages. The following markers were used: F4/80 (yellow), Far Red (red), and 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 10 μm. Representative data from two independent experiments. (E) Costaining of isolated macrophages with benzidine and Giemsa. Erythrocytes are stained brown, while macrophages are stained pink, purple, or light purple. (F) Enrichment of splenic macrophages, calculated as RO/E values relative to total splenocytes in each sample, as determined by scRNA-seq analysis (corresponding to samples in Fig. 4B). (G) Dot plot showing scaled expression of immunosuppressive genes and proinflammatory genes in macrophages across treatment groups. Data are presented as means ± SEM. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test (B). ns, not significant; ***P < 0.001.

Fig 5: Tolerance induced by UOX-Ery plus CTX is dependent on the spleen and expansion of splenic effector Treg cells.(A) Role of the spleen in tolerance induction. Mice with or without splenectomy were pretreated with CTX, UOX-P coadministered with CTX, or UOX-mEry coadministered with CTX, followed by two challenges with UOX-P (n ≥ 3 per group). Anti-UOX IgG levels were measured on Day 35. (B to G) Experimental design for immune profiling. C57BL/6 mice were treated with mEry alone (G1), UOX-mEry alone (G2), CTX followed by a single challenge with UOX-mEry (G3), or a single coadministration of UOX-mEry and CTX followed by one challenge with UOX-mEry (G4). Splenocytes were harvested on day 21 for scRNA-seq [(C) to (F)] or flow cytometry (G). (C) t-distributed stochastic neighbor embedding (t-SNE) plot of splenic CD4+ T cells from different treatment groups. (D) Dot plot showing scaled expression of signature genes across CD4+ T cell clusters. (E) Enrichment of CD4+ T cell subpopulations relative to the total CD4+ T cell population in each sample estimated by RO/E values. (F) Violin plots showing expression of suppressive function–associated genes in effector Treg cells across treatment groups. (G) Left: Representative flow cytometry plots of splenic Treg cells. Right: Quantification of Treg frequencies in different groups (n ≥ 2 per group). Data are presented as means ± SEM. Box and bar plots indicate the interquartile range (IQR); whiskers represent the minimum and maximum values within 1.5 × IQR. Statistical significance was determined by chi-square test (E) or one-way ANOVA with Tukey’s multiple comparisons test (G). ns, not significant; *P < 0.05; ***P < 0.001.