Anneke Oei

The relapsing fever spirochete Borrelia miyamotoi is cultivable in a modified Kelly-Pettenkofer medium, and is resistant to human complement

BackgroundBorrelia miyamotoi is a relapsing fever spirochete found in Ixodes ticks in North America, Europe, and Asia, and has recently been found to be invasive in humans. Cultivation of this spirochete has not yet been described, but is important for patient diagnostics and scientific purposes. Host specificity of Borrelia species is dependent on resistance to host complement (serum resistance), and since B. miyamotoi has been identified as a human pathogen we were interested whether B. miyamotoi is resistant to human complement.MethodsWe inoculated B. miyamotoi strains LB-2001 and HT31 in modified-Kelly-Pettenkofer medium with 10% fetal calf serum (MKP-F), and used standard non-laborious Borrelia culture methods to culture the spirochetes. Next, we assessed serum sensitivity by a direct killing assay and a growth inhibition assay.ResultsWe were able to passage B. miyamotoi over 10 times using a standard culture method in MKP-F medium, and found B. miyamotoi to be resistant to human complement. In contrast to B. miyamotoi, Borrelia anserina - a relapsing fever spirochete unrelated to human infection- was serum sensitive.ConclusionsUsing a variation on MKP medium we were able to culture B. miyamotoi, opening the door to in vitro research into this spirochete. In addition, we describe that B. miyamotoi is resistant to human complement, which might play an important role in pathogenesis. We have also found B. anserina to be sensitive to human complement, which might explain why it is not related to human infection. Summarizing, we describe a novel culture method for B. miyamotoi and show it is resistant to human complement.

The urokinase receptor (uPAR) facilitates clearance of Borrelia burgdorferi

The causative agent of Lyme borreliosis, the spirochete Borrelia burgdorferi, has been shown to induce expression of the urokinase receptor (uPAR); however, the role of uPAR in the immune response against Borrelia has never been investigated. uPAR not only acts as a proteinase receptor, but can also, dependently or independently of ligation to uPA, directly affect leukocyte function. We here demonstrate that uPAR is upregulated on murine and human leukocytes upon exposure to B. burgdorferi both in vitro as well as in vivo. Notably, B. burgdorferi-inoculated C57BL/6 uPAR knock-out mice harbored significantly higher Borrelia numbers compared to WT controls. This was associated with impaired phagocytotic capacity of B. burgdorferi by uPAR knock-out leukocytes in vitro. B. burgdorferi numbers in vivo, and phagocytotic capacity in vitro, were unaltered in uPA, tPA (low fibrinolytic activity) and PAI-1 (high fibrinolytic activity) knock-out mice compared to WT controls. Strikingly, in uPAR knock-out mice partially backcrossed to a B. burgdorferi susceptible C3H/HeN background, higher B. burgdorferi numbers were associated with more severe carditis and increased local TLR2 and IL-1β mRNA expression. In conclusion, in B. burgdorferi infection, uPAR is required for phagocytosis and adequate eradication of the spirochete from the heart by a mechanism that is independent of binding of uPAR to uPA or its role in the fibrinolytic system.

Genetic deletion of dectin-1 does not affect the course of murine experimental colitis

BackgroundIt is believed that inflammatory bowel diseases (IBD) result from an imbalance in the intestinal immune response towards the luminal microbiome. Dectin-1 is a widely expressed pattern recognition receptor that recognizes fungi and upon recognition it mediates cytokine responses and skewing of the adaptive immune system. Hence, dectin-1 may be involved in the pathogenesis of IBD.MethodsWe assessed the responses of dectin-1 deficient macrophages to the intestinal microbiota and determined the course of acute DSS and chronic Helicobacter hepaticus induced colitis in dectin-1 deficient mice.ResultsWe show that the mouse intestinal microbiota contains fungi and the cytokine responses towards this microbiota were significantly reduced in dectin-1 deficient macrophages. However, in two different colitis models no significant differences in the course of inflammation were found in dectin-1 deficient mice compared to wild type mice.ConclusionsTogether our data suggest that, although at the immune cell level there is a difference in response towards the intestinal flora in dectin-1 deficient macrophages, during intestinal inflammation this response seems to be redundant since dectin-1 deficiency in mice does not affect intestinal inflammation in experimental colitis.

Ixodes scapularis dystroglycan-like protein promotes Borrelia burgdorferi migration from the gut

The causative agent of Lyme borreliosis, Borrelia burgdorferi, is transmitted by Ixodes ticks. During tick feeding, B. burgdorferi migrates from the tick gut to the salivary glands from where transmission to the host occurs. B. burgdorferi-interacting tick proteins might serve as vaccine targets to thwart B. burgdorferi transmission. A previous screening for B. burgdorferi-interacting Ixodes scapularis gut proteins identified an I. scapularis putative dystroglycan protein (ISCW015049). Here, we describe the ISCW015049’s protein structure and its cellular location in the tick gut in relation to B. burgdorferi migration. Secondly, in vivo B. burgdorferi–tick attachment murine models were performed to study the role of ISCW015049 during B. burgdorferi migration and transmission. In silico analysis confirmed that ISCW015049 is similar to dystroglycan and was named I. scapularis dystroglycan-like protein (ISDLP). Confocal microscopy of gut tissue showed that ISDLP is expressed on the surface of gut cells, is upregulated during tick feeding, and is expressed significantly higher in infected ticks compared to uninfected ticks. Inhibition of ISDLP by RNA interference (RNAi) resulted in lower B. burgdorferi transmission to mice. In conclusion, we have identified a dystroglycan-like protein in I. scapularis gut that can bind to B. burgdorferi and promotes B. burgdorferi migration from the tick gut.Key messagesB. burgdorferi exploits tick proteins to orchestrate its transmission to the host.B. burgdorferi is able bind to an I. scapularis dystroglycan-like protein (ISDLP).Inhibition of ISDLP in ticks results in lower B. burgdorferi transmission to mice.ISDLP is a potential target to prevent Lyme borreliosis.

Borrelia burgdorferi clinical isolates induce human innate immune responses that are not dependent on genotype

Borrelia burgdorferi can be categorized based on restriction fragment length polymorphism analysis into ribosomal spacer type (RST) 1, 2 and 3. A correlation between RST type and invasiveness of Borrelia isolates has been demonstrated in clinical studies and experimental models, and RST 1 isolates are more likely to cause disseminated disease than RST 3 isolates. We hypothesized that this could partially be due to increased susceptibility of RST 3 isolates to killing by the innate immune system early in infection. Thus, we investigated the interaction of five RST 1 and five RST 3 isolates with various components of the human innate immune system in vitro. RST 3 isolates induced significantly greater upregulation of activation markers in monocyte-derived dendritic cells compared to RST 1 isolates at a low multiplicity of infection. However, RST 1 isolates stimulated greater interleukin-6 production. At a high multiplicity of infection no differences in dendritic cell activation or cytokine production were observed. In addition, we observed no differences in the ability of RST 1 and RST 3 isolates to activate monocytes or neutrophils and all strains were phagocytosed at a comparable rate. Finally, all isolates tested were equally resistant to complement-mediated killing, as determined by dark-field microscopy and a growth inhibition assay. In conclusion, we demonstrate that the RST 1 and 3 isolates showed no distinction in their susceptibility to the various components of the human immune system studied here, suggesting that other factors are responsible for their differential invasiveness.

Borrelia burgdorferi Induces TLR2-Mediated Migration of Activated Dendritic Cells in an Ex Vivo Human Skin Model.

Borrelia burgdorferi is transmitted into the skin of the host where it encounters and interacts with two dendritic cell (DC) subsets; Langerhans cells (LCs) and dermal DCs (DDCs). These cells recognize pathogens via pattern recognition receptors, mature and migrate out of the skin into draining lymph nodes, where they orchestrate adaptive immune responses. In order to investigate the response of skin DCs during the early immunopathogenesis of Lyme borreliosis, we injected B. burgdorferi intradermally into full-thickness human skin and studied the migration of DCs out of the skin, the activation profile and phenotype of migrated cells. We found a significant increase in the migration of LCs and DDCs in response to B. burgdorferi. Notably, migration was prevented by blocking TLR2. DCs migrated from skin inoculated with higher numbers of spirochetes expressed significantly higher levels of CD83 and produced pro-inflammatory cytokines. No difference was observed in the expression of HLA-DR, CD86, CD38, or CCR7. To conclude, we have established an ex vivo human skin model to study DC-B. burgdorferi interactions. Using this model, we have demonstrated that B. burgdorferi-induced DC migration is mediated by TLR2. Our findings underscore the utility of this model as a valuable tool to study immunity to spirochetal infections.

Rapid outer-surface protein C DNA tattoo vaccination protects against Borrelia afzelii infection.

Borrelia afzelii is the predominant Borrelia species causing Lyme borreliosis in Europe. Currently there is no human vaccine against Lyme borreliosis, and most research focuses on recombinant protein vaccines against Borrelia burgdorferi sensu stricto. DNA tattooing is a novel vaccination method that can be applied in a rapid vaccination schedule. We vaccinated C3H/HeN mice with B. afzelii strain PKo OspC (outer-surface protein C) using a codon-optimized DNA vaccine tattoo and compared this with recombinant protein vaccination in a 0–2–4 week vaccination schedule. We also assessed protection by DNA tattoo in a 0–3–6 day schedule. DNA tattoo and recombinant OspC vaccination induced comparable total IgG responses, with a lower IgG1/IgG2a ratio after DNA tattoo. Two weeks after syringe-challenge with 5 × 105 B. afzelii spirochetes most vaccinated mice had negative B. afzelii tissue DNA loads and all were culture negative. Furthermore, DNA tattoo vaccination in a 0–3–6 day regimen also resulted in negative Borrelia loads and cultures after challenge. To conclude, DNA vaccination by tattoo was fully protective against B. afzelii challenge in mice in a rapid vaccination protocol, and induces a favorable humoral immunity compared to recombinant protein vaccination. Rapid DNA tattoo is a promising vaccination strategy against spirochetes.

In Vitro Susceptibility of the Relapsing-Fever Spirochete Borrelia miyamotoi to Antimicrobial Agents

ABSTRACT Hard-tick-borne relapsing fever (HTBRF) is an emerging infectious disease throughout the temperate zone caused by the relapsing-fever spirochete Borrelia miyamotoi. Antibiotic treatment of HTBRF is empirically based on the treatment of Lyme borreliosis; however, the antibiotic susceptibility of B. miyamotoi has not been studied to date. Thus, we set out to determine the in vitro antimicrobial susceptibility of B.miyamotoi. A microdilution method with 96-well microtiter plates was used to determine the antibiotic susceptibilities of two B.miyamotoi strains isolated on two different continents (Asia and North America), two Borrelia burgdorferisensu lato strains, and one Borrelia hermsii isolate for purposes of comparison. The MIC and minimal bactericidal concentration (MBC) were determined by both microscopy and colorimetric assays. We were able to show that relative to the B. burgdorferi sensu lato isolates, both B.miyamotoi strains and B. hermsii demonstrated greater susceptibility to doxycycline and azithromycin, equal susceptibility to ceftriaxone, and resistance to amoxicillin in vitro. The MIC and MBC of amoxicillin for B. miyamotoi evaluated by microscopy were 16 to 32 mg/liter and 32 to 128 mg/liter, respectively. Since B. miyamotoi is susceptible to doxycycline, azithromycin, and ceftriaxone in vitro, our data suggest that these antibiotics can be used for the treatment of HTBRF. Oral amoxicillin is currently used as an alternative for the treatment of HTBRF; however, since we found that the B. miyamotoi strains tested were resistant to amoxicillin in vitro, this issue warrants further study.

MRP8/14 does not contribute to dissemination or inflammation in a murine model of Lyme borreliosis

Myeloid-related protein (MRP)8 and MRP14 form a complex (MRP8/14) that is released by activated neutrophils and monocytes during infection. MRP8/14 has been shown to have bacteriostatic activity in vitro against Borrelia burgdorferi, the spirochete that causes Lyme borreliosis. Furthermore, levels of MRP8/14 have been shown to be elevated in the joints of patients with Lyme arthritis. We hypothesized that MRP8/14 has a protective effect during B. burgdorferi infection. To determine the role of MRP8/14 in the immune response to B. burgdorferi, we studied the course of B. burgdorferi infection in wildtype (wt) and mrp14-/- mice. In addition, we studied the response of leukocytes from mice lacking MRP8/14 to B. burgdorferi ex vivo. We demonstrated similar levels of B. burgdorferi dissemination, cytokine and immunoglobulin production in infected wt and mrp14-/- mice after 21 days. Neutrophils and monocytes lacking MRP8/14 were undiminished in their ability to become activated or phagocytose B. burgdorferi. In conclusion, we did not find a central role of MRP8/14 in the immune response against B. burgdorferi. As the levels of MRP8/14 in the serum of infected mice were low, we speculate that MRP8/14 is not released in levels great enough to influence the course of B. burgdorferi infection.