Lysosomal β-glucuronidase regulates Lyme and rheumatoid arthritis severity

Abstract

Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most prevalent arthropod-borne illness in the United States and remains a clinical and social challenge. The spectrum of disease severity among infected patients suggests that host genetics contribute to pathogenic outcomes, particularly in patients who develop arthritis. Using a forward genetics approach, we identified the lysosomal enzyme β-glucuronidase (GUSB), a member of a large family of coregulated lysosomal enzymes, as a key regulator of Lyme-associated arthritis severity. Severely arthritic C3H mice possessed a naturally occurring hypomorphic allele, Gusbh. C57BL/6 mice congenic for the C3H Gusb allele were prone to increased Lyme-associated arthritis severity. Radiation chimera experiments revealed that resident joint cells drive arthritis susceptibility. C3H mice expressing WT Gusb as a transgene were protected from severe Lyme arthritis. Importantly, the Gusbh allele also exacerbated disease in a serum transfer model of rheumatoid arthritis. A known GUSB function is the prevention of lysosomal accumulation of glycosaminoglycans (GAGs). Development of Lyme and rheumatoid arthritis in Gusbh-expressing mice was associated with heightened accumulation of GAGs in joint tissue. We propose that GUSB modulates arthritis pathogenesis by preventing accumulation of proinflammatory GAGs within inflamed joint tissue, a trait that may be shared by other lysosomal exoglycosidases.

Figure 1. Positional mapping and characterization of the Gusb^h allele.

(A) Advanced congenic lines identify regulatory subintervals within Bbaa2. Each row represents the genetic makeup of 1 congenic mouse line across the Bbaa2 interval (120.3 to 141.2 Mb) on mouse chromosome 5. White and black portions of each row represent areas inherited from the B6 or C3H background, respectively. Ankle swelling measurements taken 4 weeks after B. burgdorferi infection (n = 12 to 35 mice per group; overall P < 0.0001). Significance of cosegregation (right) between ankle swelling and blinded scores of joint histopathology and PMN infiltration, assessed by 1-tailed Mann-Whitney test. (B) Inheritance of the Gusb polymorphism among strains included in the Sanger SNP resequencing database. (C) C3H mice and congenics carrying the Gusb^h allele exhibited enzymatic hypomorphism in serum and bone marrow–derived macrophage cell extracts and supernatants (n = 4). (D) CBA/Ca expressed near normal serum GUSB activity, while CBA/J shared the C3H GUSB hypomorphism. (E) CBA/J developed severe Lyme arthritis, while CBA/Ca were resistant (n = 5 [B6 and C3H] and 10 [CBA substrains] mice in each group; overall P < 0.0001). Significance assessed by 1-way ANOVA followed by Dunnet’s multiple comparison test versus B6 (A and E) or Bonferroni’s post test (C and D). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 2. Loss of GUSB function exacerbates Lyme arthritis severity in a genetically recessive manner.

(A) Gusb^Null mice do not exhibit a defect in host defense. The observed difference between B6 and C3H genetic backgrounds in heart bacterial burden has been previously described (59). (B) Serum GUSB activity of infected B6 and C3H controls, Gusb^Null homozygotes, and Gusb^Null heterozygous littermates (n = 5 to 6 per group). (C) Arthritis severity measurements of Gusb^Null homozygotes, heterozygous littermates, and WT B6 and C3H controls (n = 5 to 6 per group; overall P < 0.0001). (D and E) Arthritis severity measurements of B6.C3H-Bbaa2 and B6.C3H-Gusb^h heterozygotes were statistically indistinguishable from B6 control animals (n = 5 per group; overall P < 0.001). Significance assessed by 1-way ANOVA followed by Bonferroni’s multiple comparison test versus B6. *P < 0.05; ***P < 0.001; ****P < 0.0001.

Figure 3. GUSB hypomorphism influences arthritis severity through a cell-intrinsic mechanism.

Radiation chimeras were generated between B6 and B6.C3H-Bbaa2 in all pairwise combinations. We achieved high level (>90%) engraftment of B cells and myeloid lineages (Supplemental Figure 6). (A) Chimera serum GUSB activity levels were determined by the donor cell source. (B and C) The C3H Bbaa2 locus contributed to more severe Lyme arthritis, primarily through the activity of radiation resistant joint resident cells. Notably, the B6→Bbaa2 group developed severe Lyme arthritis despite high serum GUSB levels, and the Bbaa2→B6 group was resistant despite low serum GUSB levels (n = 16 to 20 rear ankle joints, 8 to 10 mice per group; overall P < 0.0001). Significance of ankle swelling assessed by 1-way ANOVA followed by Dunnet’s multiple comparison test versus the B6→B6 transplant control. Significance of overall lesion scores assessed by Mann-Whitney test versus the B6→B6 transplant control, with Bonferroni correction. *P < 0.05; **P < 0.01; ****P < 0.0001.

Figure 4. Correction of GUSB hypomorphism in C3H mice is protective.

(A) Transgenic overexpression of mouse Gusb^b by a pCAGGS-based mammalian expression construct (58). (B) Five founders exhibited elevated GUSB serum activity relative to a β-galactosidase internal control. (C) C3H Gusb^Tg mice developed markedly less severe Lyme arthritis than WT C3H controls (n = 10 for B6 and C3H control groups, n = 22 total Gusb^Tg progeny from 4 different founders, no. 38 was infertile; overall P < 0.001). Significance assessed by 1-way ANOVA and Bonferroni’s post test (ankle swelling) or nonparametric Kruskal-Wallis test and Dunn’s multiple comparison test (histopathology). **P < 0.01; ***P < 0.001.

Figure 5. A conserved role for Gusb^h in rheumatoid arthritis.

(A) 100 μl K/BxN serum was administered to B6 and B6.C3H-Gusb^h congenic mice by intraperitoneal injection on days 0 and 2. Ankle swelling of both rear ankle joints was monitored on days 1, 2, 4, and 7. Congenic mice developed significantly more robust ankle swelling starting on day 4 that was further exacerbated on day 7, and this finding was corroborated by blinded joint histopathology measurements (B) (n = 14 rear ankle joints for each group). Pairwise significance of ankle swelling calculated by Student’s t test. Significance of overall lesion score assessed by Mann-Whitney test. *P < 0.05; **P < 0.01.

Figure 6. GUSB enzymatic hypomorphism is associated with exaggerated accumulation of GAGs in the inflamed joint.

Representative images of Alcian blue–stained rear ankle joint sections from B. burgdorferi–infected GUSB-sufficient (upper panels: A, C, E, and G) or GUSB-deficient (lower panels: B, D, F, and H) strains, or day 7 K/BxN-treated B6 (I) and B6.C3H-Gusb^h (J) congenic mice. Arrowheads indicate position of the cranial tibial tendon sheath. Original magnification, ×4. Scale bars: 500 μm (K) GAG accumulation in the soft tissue and joint/synovial space was scored on a scale of 0–4 (n = 3 to 4 joints per group). Pairwise significance assessed by 1-tailed Mann-Whitney test. *P < 0.05.