Severe Lyme arthritis: Gagging on GAGs

Janis Weis' group has been mapping genetic variants that make laboratory mice prone to severe Lyme arthritis.  One of these variants is described in a paper that appeared in The Journal of Clinical Investigation earlier this year.  The affected gene encodes the enzyme β-glucuronidase, which carries out a critical function in the lysosome. β-glucuronidase cooperates with other degradative enzymes in the lysosome to break down glycosaminoglycans (GAGs) into their individual sugar units, which are then removed from the lysosome and reused by the cells.  GAGs are long chains of specific disaccharides located on the cell surface and within the extracellular matrix.  GAGs are covalently (in proteoglycans) or noncovalently attached to proteins.  GAGs are always being degraded and resynthesized by cells. Blocking any of the enzymes involved in GAG breakdown causes accumulation of GAG fragments, which are potentially detrimental to health.  In humans, certain mutations in the β-glucuronidase gene lead to a rare condition called Sly syndrome.

Large amounts of GAGs are found in the joints, where they serve an important mechanical function.  GAGs carry a high density of negative charge due to the presence of acidic sugars such as glucuronic acid, the target of β-glucuronidase, and the sulfate groups attached to most types of GAGs.  The negative charge attracts cations, which in turn attract large numbers of water molecules.  The water within GAGs acts as a cushion that allows the joints to withstand large compressive forces.

The key to the study was having strains of mice that differed in their susceptibility to Lyme arthritis. The C3H mouse strain develops severe joint inflammation during B. burgdorferi infection. On the other hand, the B6 strain develops mild joint inflammation when infected. Weis' group had earlier narrowed the locations of the genetic variations accounting for the different susceptibilities to several distinct segments within the mouse genome.  They used the traditional techniques of mouse genetics, which involved numerous matings involving the C3H and B6 strains and their progeny (see this review for details).  The authors focused on one of the segments, and with help from mouse genome sequence data that became available, they found a nucleotide difference within the Gusb (β-glucuronidase) gene that changed a single amino acid in the enzyme.

The investigators found that β-glucuronidase activity was mildly reduced in the infected C3H strain relative to the B6 strain.  Staining tissue sections of infected mice with Alcian blue, a dye attracted to polyanions, revealed accumulation of GAGs in the joint tissues of infected C3H mice but not infected B6 mice, lending further support to the lesion in Gusb being responsible for severe inflammation.  When a functional copy of the β-glucuronidase gene was stitched into the genome of C3H mice, B. burgdorferi infection no longer caused joint inflammation.

Does the same process occur in humans with Lyme arthritis?  One hint that β-glucuronidase influences the course of Lyme arthritis is the finding from other labs that found that the concentration of the enzyme in joint fluid is higher in patients with Lyme arthritis than it is in healthy uninfected individuals, although how the high enzyme levels are mechanistically linked to arthritis remains unexplained.

So how does β-glucuronidase deficiency lead to severe Lyme arthritis?  One possibility raised by the authors is that GAG fragments worsen tissue inflammation by stimulating Toll-like receptors, as shown in other studies (see this paper for an example).

The findings may also tell us something about rheumatoid arthritis (RA).  The B6 strain ends up with a form of RA following injection with certain autoantibodies.  One of Weis' mouse crosses generated a B6 strain with its Gusb gene and flanking regions swapped for the same region of the C3H strain.  This strain developed severe arthritis when injected with the same autoantibodies and when infected with B. burgdorferi.  Therefore, the pathologic processes leading to Lyme arthritis and RA share common steps, at least in laboratory mice.  In humans, RA patients, like those with Lyme arthritis, have high levels of β-glucuronidase levels in their joint fluid.

The search for host factors affecting the development of Lyme arthritis goes on.  Weis' group continue to identify genetic variants responsible for severe Lyme arthritis.


References

Bramwell KK, Ma Y, Weis JH, Chen X, Zachary JF, Teuscher C, & Weis JJ (2014). Lysosomal β-glucuronidase regulates Lyme and rheumatoid arthritis severity. The Journal of Clinical Investigation, 124 (1), 311-320 PMID: 24334460

Bramwell KK, Teuscher C, & Weis JJ (2014). Forward genetic approaches for elucidation of novel regulators of Lyme arthritis severity. Frontiers in Cellular and Infection Microbiology, 4 PMID: 24926442

Pancewicz S, Popko J, Rutkowski R, Knaś M, Grygorczuk S, Guszczyn T, Bruczko M, Szajda S, Zajkowska J, Kondrusik M, Sierakowski S, & Zwierz K (2009). Activity of lysosomal exoglycosidases in serum and synovial fluid in patients with chronic Lyme and rheumatoid arthritis. Scandinavian Journal of Infectious Diseases, 41 (8), 584-589 PMID: 19513935

Jiang D, Liang J, Fan J, Yu S, Chen S, Luo Y, Prestwich GD, Mascarenhas MM, Garg HG, Quinn DA, Homer RJ, Goldstein DR, Bucala R, Lee PJ, Medzhitov R, & Noble PW (2005). Regulation of lung injury and repair by Toll-like receptors and hyaluronan. Nature Medicine, 11 (11), 1173-1179 PMID: 16244651

Video microscopy of ticks acquiring the Lyme disease spirochete from mice

The bite of an infected Ixodes hard tick transmits the Lyme disease spirochete, Borrelia burgdorferi, to humans.  Ticks acquire B. burgdorferi by feeding on reservoir hosts colonized with the spirochete.  Reservoir hosts include small mammals such as the white-footed mouse, the main reservoir of B. burgdorferi in the northeastern United States.

A study that just came out in The Yale Journal of Biology and Medicine is accompanied by videos of Borrelia burgdorferi being transmitted between mouse and tick.¹  The authors prepared the mice by infecting them with B. burgdorferi genetically modified to express green fluorescent protein.  After waiting two weeks to allow the spirochetes to disseminate, they placed one hungry Ixodes scapularis tick onto an ear of each infected mouse.

Figure 1 from Bockenstedt et al., 2014¹.  A feeding tick (arrow) attached to the ear of a mouse.  The tick is engorged with blood.

Nymphal ticks feed for an average of 2.5 to 8 days.  The meal starts with the tick inserting its barbed feeding apparatus into the skin.  (For a close-up view of this process, head over to the blog Phenomena: Not Exactly Rocket Science.)  The tick releases saliva through the feeding canal into the skin.  Tick saliva contains substances that damage host tissue surrounding the feeding apparatus and pharmacologic agents that inhibit clotting and engage the immune system. Intervals of salivation alternate with ingestion of blood, tissue fluid, and lymph that pool at the feeding site.²

The authors examined the feeding site by two photon intravital microscopy to observe what was happening to the spirochetes. They saw spirochetes in the dermis moving towards the feeding apparatus and disappearing as they presumably got sucked into the feeding canal.  One such spirochete is digitally colored in red in the video below, which was shot 48 hours into the blood meal.  (One hour of video footage was compressed into 30 seconds.)  The feeding apparatus is the green structure near the top of the viewing field.  Previous studies have suggested that ticks acquire B. burgdorferi from skin, not from blood.³  The videos from this study provide support for this notion, according to the authors.

Are the spirochetes mere passengers that get caught in the flow of fluid being drawn into the feeding canal, or are they active participants?  The authors argue for an active role for the spirochetes:

Spirochete movement is unlikely to be due simply to the mechanical flux of tissue fluid as the tick feeds because close examination of individual spirochetes that move toward the hypostome reveals both the oscillating movements that we observe in the absence of tick feeding as well as directional translocation.

References

1.  Bockenstedt LK, Gonzalez D, Mao J, Li M, Belperron AA, & Haberman A (2014). What ticks do under your skin: two-photon intravital imaging of Ixodes scapularis feeding in the presence of the Lyme disease spirochete. The Yale Journal of Biology and Medicine, 87 (1), 3-13 PMID: 24600332

2.   Anderson JF, & Magnarelli LA (2008). Biology of ticks. Infectious Disease Clinics of North America, 22 (2) PMID: 18452797

3.  Nakayama Y, & Spielman A (1989). Ingestion of Lyme disease spirochetes by ticks feeding on infected hosts. The Journal of infectious diseases, 160 (1), 166-7 PMID: 2732513