Single-cell transcriptome analysis of the early immune response in the lymph nodes of Borrelia burgdorferi-infected mice.

Lyme borreliosis is a disease caused by Borrelia burgdorferi sensu lato bacteria. Borrelia burgdorferi is known to induce prolonged extrafollicular immune responses and abnormal germinal centre formation. The infection fails to generate a neutralizing type of immunity, eventually establishing a persistent infection. Here, we performed single-cell RNA sequencing to characterize the immune landscape of lymph node lymphocytes during the early Borrelia burgdorferi infection in a murine model. Our results indicate key features of an extrafollicular immune response four days after Borrelia burgdorferi infection, including notable B cell proliferation, immunoglobulin class switching to IgG3 and IgG2b isotypes, plasmablast differentiation, and the presence of extrafollicular B cells identified through immunohistochemistry. Additionally, we found infection-derived upregulation of suppressor of cytokine signalling genes Socs1 and Socs3, along with downregulation of genes associated with MHC II antigen presentation in B cells. Our results support the central role of B cells in the immune response of a Borrelia burgdorferi infection, and provide cues of mechanisms behind the determination between extrafollicular and germinal centre responses during Borrelia burgdorferi infection.

The effects of Borrelia infection on its wintering rodent host.

In seasonal environments, appropriate adaptations are crucial for organisms to maximize their fitness. For instance, in many species, the immune function has been noticed to decrease during winter, which is assumed to be an adaptation to the season's limited food availability. Consequences of an infection on the health and survival of the host organism could thus be more severe in winter than in summer. Here, we experimentally investigated the effect of a zoonotic, endemic pathogen, Borrelia afzelii infection on the survival and body condition in its host, the bank vole (Myodes glareolus), during late autumn-early winter under semi-natural field conditions in 11 large outdoor enclosures. To test the interaction of Borrelia infection and energetic condition, four populations received supplementary nutrition, while remaining seven populations exploited only natural food sources. Supplementary food during winter increased the body mass independent of the infection status, however, Borrelia afzelii infection did not cause severe increase in the host mortality or affect the host body condition in the late autumn-early winter. While our study suggests that no severe effects are caused by B. afzelii infection on bank vole, further studies are warranted to identify any potentially smaller effects the pathogen may cause on the host fitness over the period of whole winter.

Borrelia burgdorferi specific serum and cerebrospinal fluid antibodies in Lyme neuroborreliosis.

We used definite Lyme neuroborreliosis (LNB) adult patient acute and convalescent phase serum (n = 63 and 61, respectively) and cerebrospinal fluid (CSF; acute n = 63, 3 weeks timepoint n = 41) samples to characterize Borrelia burgdorferi specific antibody responses in patient subgroups categorized by demographics, infection manifestation and phase, infecting B. burgdorferi genospecies, received antibiotic treatments, and treatment outcome. B. burgdorferi antibodies were analyzed using 4 different assays incorporating a large array of antigens. We observed that B. burgdorferi specific serum antibodies show a universal, antigen independent declining trend after antibiotic treatment of LNB at 1 year. Antibodies declined similarly among women and men over time, and the decline was independent of patient age. The antibody responses were independent of the predominant LNB manifestation, treatment received by the patient, infecting B. burgdorferi genospecies, or the subjective improvement experienced by the patients. Finally, the antibody specificities in CSF reflected the specificities observed in serum samples.

Clinical performance and analytical accuracy of a C6 peptide-based point-of-care lateral flow immunoassay in Lyme borreliosis serology.

We evaluated the analytical accuracy and the clinical performance of a ReaScan+ C6 LYME IgG point-of-care immunoassay (Reagena; index test). Analytical accuracy was evaluated in comparison to a C6 Lyme ELISA™ reference method (Oxford Immunotec) with retrospectively identified serum and CSF samples. The clinical performance was evaluated by using Lyme borreliosis patient and control subject serum and CSF samples. The study was conducted by following the 2015 Standards for Reporting of Diagnostic Accuracy Studies procedure. The sensitivity and specificity of the index test with serum samples were 83% and 91.6%, respectively, when C6 Lyme ELISA™ was used as a reference. The clinical sensitivity of the index test was 97.2%/96.8% for identifying Borrelia specific antibodies in definite/possible Lyme neuroborreliosis. With CSF samples, the clinical sensitivity was 97.2% for definite and 87.1% for possible Lyme neuroborreliosis. The clinical specificity of the assay was 96.1% with serum and 100% with CSF samples.

Conserved lysine residues in decorin binding proteins of Borrelia garinii are critical in adhesion to human brain microvascular endothelial cells.

Lyme borreliosis is a tick-borne disease caused by Borrelia burgdorferi sensu lato spirochetes (Lyme borreliae). When the disease affects the central nervous system, it is referred to as neuroborreliosis. In Europe, neuroborreliosis is most often caused by Borrelia garinii. Although it is known that in the host Lyme borreliae spread from the tick bite site to distant tissues via the blood vasculature, the adherence of Lyme borreliae to human brain microvascular endothelial cells has not been studied before. Decorin binding proteins are adhesins expressed on Lyme borreliae. They mediate the adhesion of Lyme borreliae to decorin and biglycan, and the lysine residues located in the binding site of decorin binding proteins are important to the binding activity. In this study, we show that lysine residues located in the canonical binding site can also be found in decorin binding proteins of Borrelia garinii, and that these lysines contribute to biglycan and decorin binding. Most importantly, we show that the lysine residues are crucial for the binding of Lyme borreliae to decorin and biglycan expressing human brain microvascular endothelial cells, which in turn suggests that they are involved in the pathogenesis of neuroborreliosis.

Oral Doxycycline Compared to Intravenous Ceftriaxone in the Treatment of Lyme Neuroborreliosis: A Multicenter, Equivalence, Randomized, Open-label Trial.

BACKGROUND: Lyme neuroborreliosis (LNB) is often treated with intravenous ceftriaxone even if doxycycline is suggested to be noninferior to ceftriaxone. We evaluated the efficacy of oral doxycycline in comparison to ceftriaxone in the treatment of LNB. METHODS: Patients with neurological symptoms suggestive of LNB without other obvious reasons were recruited. The inclusion criteria were (1) production of Borrelia burgdorferi-specific antibodies in cerebrospinal fluid (CSF) or serum; (2) B. burgdorferi DNA in the CSF; or (3) an erythema migrans during the past 3 months. Participants were randomized in a 1:1 ratio to receive either oral doxycycline 100 mg twice daily for 4 weeks, or intravenous ceftriaxone 2 g daily for 3 weeks. The participants described their subjective condition with a visual analogue scale (VAS) from 0 to 10 (0 = normal; 10 = worst) before the treatment, and 4 and 12 months after the treatment. The primary outcome was the change in the VAS score at 12 months. RESULTS: Between 14 September 2012 and 28 December 2017, 210 adults with suspected LNB were assigned to receive doxycycline (n = 104) or ceftriaxone (n = 106). The per-protocol analysis comprised 82 patients with doxycycline and 84 patients with ceftriaxone. The mean change in the VAS score was -3.9 in the doxycycline group and -3.8 in the ceftriaxone group (mean difference, 0.17 [95% confidence interval, -.59 to .92], which is within the prespecified equivalence margins of -1 to 1 units). Participants in both groups improved equally. CONCLUSIONS: Oral doxycycline is equally effective as intravenous ceftriaxone in the treatment of LNB. CLINICAL TRIALS REGISTRATION: NCT01635530 and EudraCT 2012-000313-37.

C6 peptide enzyme immunoassay in Lyme borreliosis serology.

The cut-off values used in C6 peptide-based enzyme immunoassay (EIA), a widely used test in Lyme borreliosis (LB) serology, have not been thoroughly analysed. The objective of the study was to examine the performance of the C6 EIA, and to determine optimal cut-off values for the test. The analysed data contained results of 1368 serum samples. C6 EIA index values were compared statistically with the immunoblot (IB) test results. The identified cut-off values were further tested in a well-defined LB patient cohort. Cut-off value 1.6 appeared to be optimal when C6 EIA was used as a stand-alone test. When using C6 EIA as the first-tier test, the optimal cut-off values were 0.9 and 2.4 for negative and positive results. When C6 EIA was used as a second-tier test, samples yielding C6 index values ≥3.0 could be considered positive. The identified cut-off values had also a high sensitivity to identify seropositivity among definite LB patients. The identified cut-off values refine the role of C6 EIA in LB serology. Importantly, the use of C6 EIA leads to a reduction in the number of samples that need to be analysed using an IB, thus also reducing the costs. Two alternative workflows for LB serology including the C6 EIA are suggested.

Structural and Biomolecular Analyses of Borrelia burgdorferi BmpD Reveal a Substrate-Binding Protein of an ABC-Type Nucleoside Transporter Family.

Borrelia burgdorferisensu lato, the causative agent of tick-borne Lyme borreliosis (LB), has a limited metabolic capacity and needs to acquire nutrients, such as amino acids, fatty acids, and nucleic acids, from the host environment. Using X-ray crystallography, liquid chromatography-mass spectrometry, microscale thermophoresis, and cellular localization studies, we show that basic membrane protein D (BmpD) is a periplasmic substrate-binding protein of an ABC transporter system binding to purine nucleosides. Nucleosides are essential for bacterial survival in the host organism, and these studies suggest a key role for BmpD in the purine salvage pathway of B. burgdorferi sensu lato Because B. burgdorferisensu lato lacks the enzymes required for de novo purine synthesis, BmpD may play a vital role in ensuring access to the purines needed to sustain an infection in the host. Furthermore, we show that, although human LB patients develop anti-BmpD antibodies, immunization of mice with BmpD does not confer protection against B. burgdorferi sensu lato infection.

Borrelia burgdorferi Infection in Biglycan Knockout Mice.

BACKGROUND: Borrelia burgdorferi sensu lato spirochetes (Borrelia) causing Lyme borreliosis are able to disseminate from the initial entry site to distant organs in the host. Outer-surface adhesins are crucial in the bacterial dissemination and adhesion to various tissues. Two well-characterized Borrelia adhesins, decorin-binding proteins A and B, have been shown to bind to 2 host receptors, decorin and biglycan. However, the role of biglycan in Borrelia infection has not been characterized in vivo. METHODS: We infected biglycan knockout (KO) and wild-type (WT) C3H mice with strains representing 3 Borrelia genospecies, Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii. The infection was monitored by measuring joint swelling, Borrelia culture, polymerase chain reaction analysis, and serologic analysis. The host immune responses were analyzed by histological scoring of the inflammation in tissues and by cytokine profiling. RESULTS: B. burgdorferi sensu stricto and B. garinii established long-term infection in mice of both genotypes, while B. afzelii failed to disseminate in KO mice. Further, the B. burgdorferi sensu stricto-infected KO mice had persistent inflammation in the joints. CONCLUSIONS: The dissemination and tissue colonization of Borrelia and the inflammatory response of the host differ in a mouse biglycan expression- and Borrelia genospecies-dependent manner.

Point-of-care testing for CXCL13 in Lyme neuroborreliosis.

Cerebrospinal fluid chemokine (C-X-C motif) ligand 13 (CXCL13) is a marker for Lyme neuroborreliosis (LNB). CXCL13 lateral flow immunoassay (LFA) was compared with CXCL13 ELISA. CXCL13 LFA results correlated strongly with CXCL13 ELISA results. CXCL13 LFA is a rapid and easy-to-perform test, which is suitable for routine point-of-care diagnostics of suspected LNB patients.

Targeting of vascular adhesion protein-1 by positron emission tomography visualizes sites of inflammation in Borrelia burgdorferi-infected mice.

BACKGROUND: In the present study, we sought to evaluate the feasibility of targeting vascular adhesion protein-1 (VAP-1) by positron emission tomography (PET) for the longitudinal quantitative assessment of Borrelia burgdorferi infection-induced inflammation in mice. METHODS: Mice with B. burgdorferi infection-induced arthritis were studied. During a 7-week follow-up period, the progression of arthritis was monitored weekly with 68Ga-DOTA-Siglec-9 PET/computed tomography (CT) and measurement of tibiotarsal joint swellings. A subgroup of infected mice was treated with ceftriaxone. Finally, histopathological assessment of joint inflammation was performed and VAP-1 expression in joints were determined. RESULTS: Explicit joint swelling and 68Ga-DOTA-Siglec-9 uptake could be demonstrated in the affected joints from B. burgdorferi-infected mice. By contrast, no obvious accumulation of 68Ga-DOTA-Siglec-9 was detected in joints of uninfected mice. The maximum swelling and highest uptake in the affected joints were observed 4 weeks after the infection. 68Ga-DOTA-Siglec-9 uptake in joints correlated with joint swelling (P < 0.0001) and histopathological scoring of inflammation (P = 0.020). Despite short-term antibiotic treatment, the arthritis persisted, and the PET signal remained as high as in nontreated mice. Immunohistochemistry revealed strong-to-moderate expression of VAP-1 in the synovium of B. burgdorferi-infected mice, while only weak expression of VAP-1 was detected in uninfected mice. CONCLUSIONS: The present study showed that 68Ga-DOTA-Siglec-9 can detect B. burgdorferi infection-induced arthritis in mice. Furthermore, longitudinal PET/CT imaging allowed monitoring of arthritis development over time.

Cerebrospinal fluid cytokines in Lyme neuroborreliosis.

BACKGROUND: Lyme neuroborreliosis (LNB) is one of the manifestations of Lyme disease. Although it is known that immune reaction of LNB patients is dominated by Th1 and Th2 responses and patients have elevated numbers of B cells in their cerebrospinal fluid (CSF), not all the cells involved in inflammation and cytokine secretion have been characterized. The current diagnostics of LNB is based on intrathecal production of antibodies. In recent years, the measurement of chemokine CXCL13 concentration from the CSF has been introduced as a new promising diagnostic tool for LNB to complement the antibody-based diagnostic methods. A few other cytokines have also been analyzed as possible diagnostic markers. However, multiplex analyses simultaneously evaluating the concentrations of a large number of different cytokines in the CSF of LNB patients have been lacking thus far. Extensive cytokine profiling CSF samples of LNB patients would also help in understanding the complex immunopathogenesis of LNB. METHODS: CSF samples were analyzed from 43 LNB patients, 19 controls, 18 tick-borne encephalitis patients, and 31 multiple sclerosis patients. In addition, CSF samples from 23 LNB patients obtained after the antibiotic treatment were examined. Altogether, the concentrations of 49 different cytokines were determined from all of the samples. The concentrations of 48 different cytokines were analyzed by magnetic bead suspension array using the Bio-Plex Pro Human Cytokine 21- and 27-plex panels, and the concentration of CXCL13 was analyzed by an ELISA based method. RESULTS: Distinct cytokine profiles which were able to distinguish LNB patients from controls, tick-borne encephalitis patients, multiple sclerosis patients, and LNB patients treated with antibiotics were identified. LNB patients had elevated concentrations of all major T helper cell type cytokines (Th1, Th2, Th9, Th17, and Treg) in their CSF. CONCLUSIONS: Despite the great differences in the CSF cytokine profiles of different patient groups, CXCL13 still remained as the best marker for LNB. However, IL-1ra might also be helpful as a marker for the antibiotic treatment response. Concerning the immunopathogenesis, this is the first report suggesting the involvement of Th9 cells in the immune response of LNB.

Flow-Tolerant Adhesion of a Bacterial Pathogen to Human Endothelial Cells Through Interaction With Biglycan.

BACKGROUND: Bacterial pathogens causing systemic infections disseminate from the initial infection focus to the target organs usually through the blood vasculature. To be able to colonize various organs, bacteria need to adhere to the endothelial cells of the vascular wall, and the adhesion must be strong enough to resist the shear force of the blood flow.Borrelia burgdorferi sensu lato spirochetes, the causative agents of the tick-borne disease Lyme borreliosis, disseminate hematogenously from the tick bite site to the joints, the heart, and the central nervous system of the patient. METHODS: We used both wild-type and genetically modified B. burgdorferi s. l. bacteria, recombinant borrelia adhesins, and an array of adhesion assays carried out both under stationary and flow conditions to investigate the molecular mechanisms of borrelial adhesion to human endothelial cells. RESULTS: Borrelia garinii, a member of the B. burgdorferi s. l. complex, adhered to biglycan expressed by human endothelial cells in a flow-tolerant manner. The adhesion was mediated by the decorin-binding protein A (DbpA) and DbpB surface molecules of B. garinii. CONCLUSIONS: The proteoglycan biglycan is a receptor molecule for flow-resistant adhesion of the bacterial pathogen B. garinii on human endothelial cells.