A Murine Model of Lyme Disease Demonstrates That Borrelia burgdorferi Colonizes the Dura Mater and Induces Inflammation in the Central Nervous System

Lyme disease, which is caused by infection with Borrelia burgdorferi and related species, can lead to inflammatory pathologies affecting the joints, heart, and nervous systems including the central nervous system (CNS). Inbred laboratory mice are effective models for characterizing B. burgdorferi infection kinetics and host immune responses in joints and heart tissues; however, similar studies are lacking in the CNS of these animals. Here we characterize the kinetics of B. burgdorferi colonization and associated immune responses in the CNS of infected C3H mice during early and subacute infection. B. burgdorferi colonized the dura mater following needle or tick challenge, and induced expression of inflammatory cytokines and a robust IFN response as well as histopathological changes. A sterile IFN response in the absence of B. burgdorferi or inflammatory cytokines was unique to the brain parenchyma, and could provide insights into the mechanism of inflammatory CNS pathology associated with this important pathogen

DNA Methylation by Restriction Modification Systems Affects the Global Transcriptome Profile in \u3cem\u3eBorrelia burgdorferi\u3c/em\u3e

Prokaryote restriction modification (RM) systems serve to protect bacteria from potentially detrimental foreign DNA. Recent evidence suggests that DNA methylation by the methyltransferase (MTase) components of RM systems can also have effects on transcriptome profiles. The type strain of the causative agent of Lyme disease, Borrelia burgdorferi B31, possesses two RM systems with N6-methyladenosine (m6A) MTase activity, which are encoded by the bbe02 gene located on linear plasmid lp25 and bbq67 on lp56. The specific recognition and/or methylation sequences had not been identified for either of these B. burgdorferi MTases, and it was not previously known whether these RM systems influence transcript levels. In the current study, single-molecule real-time sequencing was utilized to map genome-wide m6A sites and to identify consensus modified motifs in wild-type B. burgdorferi as well as MTase mutants lacking either the bbe02 gene alone or both bbe02 and bbq67 genes. Four novel conserved m6A motifs were identified and were fully attributable to the presence of specific MTases. Whole-genome transcriptome changes were observed in conjunction with the loss of MTase enzymes, indicating that DNA methylation by the RM systems has effects on gene expression. Genes with altered transcription in MTase mutants include those involved in vertebrate host colonization (e.g., rpoS regulon) and acquisition by/transmission from the tick vector (e.g., rrp1 and pdeB). The results of this study provide a comprehensive view of the DNA methylation pattern in B. burgdorferi, and the accompanying gene expression profiles add to the emerging body of research on RM systems and gene regulation in bacteria

Borrelia burgdorferi Adhere to Blood Vessels in the Dura Mater and are Associated with Increased Meningeal T Cells during Murine Disseminated Borreliosis

Borrelia burgdorferi, the causative agent of Lyme disease, is a vector-borne bacterial infection that is transmitted through the bite of an infected tick. If not treated with antibiotics during the early stages of infection, disseminated infection can spread to the central nervous system (CNS). In non-human primates (NHPs) it has been demonstrated that the leptomeninges are among the tissues colonized by B. burgdorferi spirochetes. Although the NHP model parallels aspects of human borreliosis, a small rodent model would be ideal to study the trafficking of spirochetes and immune cells into the CNS. Here we show that during early and late disseminated infection, B. burgdorferi infects the meninges of intradermally infected mice, and is associated with concurrent increases in meningeal T cells. We found that the dura mater was consistently culture positive for spirochetes in transcardially perfused mice, independent of the strain of B. burgdorferi used. Within the dura mater, spirochetes were preferentially located in vascular regions, but were also present in perivascular, and extravascular regions, as late as 75 days post-infection. At the same end-point, we observed significant increases in the number of CD3+ T cells within the pia and dura mater, as compared to controls. Flow cytometric analysis of leukocytes isolated from the dura mater revealed that CD3+ cell populations were comprised of both CD4 and CD8 T cells. Overall, our data demonstrate that similarly to infection in peripheral tissues, spirochetes adhere to the dura mater during disseminated infection, and are associated with increases in the number of meningeal T cells. Collectively, our results demonstrate that there are aspects of B. burgdorferi meningeal infection that can be modelled in laboratory mice, suggesting that mice may be useful for elucidating mechanisms of meningeal pathogenesis by B. burgdorferi

Morphological and functional analysis of cardiomyocytes differentiated from adipose-derived human adult mesenchymal stem cells

We recently published a highly reproducible protocol that permitted to isolate, culture and expand human Multipotent Adult Stem Cells (hMASCs) from human liver, heart and bone marrow (Beltrami et al., 2007). Although their high proliferative rate and differentiation ability make MASC dependable candidates for regenerative medicine applications, they do not represent a convenient source of cells for the inherent limits dictated by their scarce accessibility. Looking for more convenient alternatives, we applied the same isolation protocol to obtain MASCs from adipose tissue. Adipose Tissuederived MASCs (AT‐MASCs) can represent a valid substitute to Heart‐derived MASCs (H‐MASCs) because readily accessible, highly proliferative and widely multipotent, being able to differentiate into mesenchymal and non‐ mesenchymal lineages. In addition, the number of cells obtained by lipoaspirates is usually largely sufficient for many clinical uses. With the aim to explore possible therapeutic applications of MASCs in cardiac regenerative medicine, we compared ATMASCs and H‐MASCs for their in vitro myogenic differentiation potential. Several induction protocols were applied, and in order to compare their efficiency we evaluated the degree of myogenic differentiation under different morphological, functional and electrophysiological aspects

A murine model of Lyme disease demonstrates that Borrelia burgdorferi colonizes the dura mater and induces inflammation in the central nervous system.

Lyme disease, which is caused by infection with Borrelia burgdorferi and related species, can lead to inflammatory pathologies affecting the joints, heart, and nervous systems including the central nervous system (CNS). Inbred laboratory mice have been used to define the kinetics of B. burgdorferi infection and host immune responses in joints and heart, however similar studies are lacking in the CNS of these animals. A tractable animal model for investigating host-Borrelia interactions in the CNS is key to understanding the mechanisms of CNS pathogenesis. Therefore, we characterized the kinetics of B. burgdorferi colonization and associated immune responses in the CNS of mice during early and subacute infection. Using fluorescence-immunohistochemistry, intravital microscopy, bacterial culture, and quantitative PCR, we found B. burgdorferi routinely colonized the dura mater of C3H mice, with peak spirochete burden at day 7 post-infection. Dura mater colonization was observed for several Lyme disease agents including B. burgdorferi, B. garinii, and B. mayonii. RNA-sequencing and quantitative RT-PCR showed that B. burgdorferi infection was associated with increased expression of inflammatory cytokines and a robust interferon (IFN) response in the dura mater. Histopathologic changes including leukocytic infiltrates and vascular changes were also observed in the meninges of infected animals. In contrast to the meninges, we did not detect B. burgdorferi, infiltrating leukocytes, or large-scale changes in cytokine profiles in the cerebral cortex or hippocampus during infection; however, both brain regions demonstrated similar changes in expression of IFN-stimulated genes as observed in peripheral tissues and meninges. Taken together, B. burgdorferi is capable of colonizing the meninges in laboratory mice, and induces localized inflammation similar to peripheral tissues. A sterile IFN response in the absence of B. burgdorferi or inflammatory cytokines is unique to the brain parenchyma, and provides insight into the potential mechanisms of CNS pathology associated with this important pathogen

Evaluation of the Importance of VlsE Antigenic Variation for the Enzootic Cycle of Borrelia burgdorferi

Efficient acquisition and transmission of Borrelia burgdorferi by the tick vector, and the ability to persistently infect both vector and host, are important elements for the life cycle of the Lyme disease pathogen. Previous work has provided strong evidence implicating the significance of the vls locus for B. burgdorferi persistence. However, studies involving vls mutant clones have thus far only utilized in vitro-grown or host-adapted spirochetes and laboratory strains of mice. Additionally, the effects of vls mutation on tick acquisition and transmission has not yet been tested. Thus, the importance of VlsE antigenic variation for persistent infection of the natural reservoir host, and for the B. burgdorferi enzootic life cycle in general, has not been examined to date. In the current work, Ixodes scapularis and Peromyscus maniculatus were infected with different vls mutant clones to study the importance of the vls locus for the enzootic cycle of the Lyme disease pathogen. The findings highlight the significance of the vls system for long-term infection of the natural reservoir host, and show that VlsE antigenic variability is advantageous for efficient tick acquisition of B. burgdorferi from the mammalian reservoir. The data also indicate that the adaptation state of infecting spirochetes influences B. burgdorferi avoidance from host antibodies, which may be in part due to its respective VlsE expression levels. Overall, the current findings provide the most direct evidence on the importance of VlsE for the enzootic cycle of Lyme disease spirochetes, and underscore the significance of VlsE antigenic variation for maintaining B. burgdorferi in nature

Doxycycline induced gene expression changes associated with protein translation.

(A) Fold change versus expression strength for all detectable genes after 3 or 24 hours doxycycline treatment compared to untreated controls. Red (increased) and blue (decreased) dots represent genes with significantly different levels in treated vs. control bacteria (α = 0.05, log2(fold-change) > 1). Yellow dots represent significantly different expression (α = 0.05) without meeting our fold-change cutoff for differential expression (“sigNC”). Gray dots represent genes that were not significantly different between treatment and control bacteria (“NS”). Numbers of significantly upregulated (up) and downregulated (down) genes are shown as proportions of all detectable genes. (B) Clusters of Orthologous Genes (COG) pathways displayed as proportion of all detectable genes (“Total”) compared to differentially expressed genes after 3h or 24h of doxycycline treatment [47]. (C) Stacked bar graph showing the number of increased (red) and decreased (blue) genes in each COG pathway at 3h and 24h timepoints. Percentage of genes in each pathway that were differentially expressed is stated within each bar. Note: Unclassified and general function prediction not shown.</p

T cells in dura mater are CD4 and CD8 T cells.

<p>(A-C) Representative flow cytometric density plots showing gating strategy for the identification of CD4+ and CD8+ T cell subsets in the spleen (A) and dura mater (B-C). (A) CD3+ T cells (left), and CD4+ / CD8+ subsets (right) in splenocytes of control mice. (B/C) CD3+ T cells and CD4+/CD8+ subsets in dura mater of (B) control mice, and (C) <i>B</i>. <i>burgdorferi</i> infected mice. Prior to analysis all samples were gated on singlets using FSC-A vs FSC-H, and live cells were identified using Aqua amine reactive dye.</p

<i>B</i>. <i>burgdorferi</i> in dura mater during late disseminated infection.

<p>(A-C) Representative images of <i>B</i>. <i>burgdorferi</i> (Bb), blood vessels (CD31), and nucleated cells (DAPI or TOPRO-3) in regions of the dura mater, 75 dpi. (A) Confocal images described from left to right. <i>B</i>. <i>burgdorferi</i> (Bb) shown in 488 channel; CD31+ blood vessels in 555 channel; TOPRO-3+ nucleated cells in 633 channel; merged image showing <i>B</i>. <i>burgdorferi</i> in association with a blood vessel. (B) Epifluorescence image of <i>B</i>. <i>burgdorferi</i> in perivascular region near a blood vessel. (C) Confocal image of <i>B</i>. <i>burgdorferi</i> in extravascular region of dura mater. (D) Cumulative sum of spirochete locations in 3 dura mater samples; differences in (D) were not statistically significant. (E) 100x magnification epifluorescence image showing blood vessels (CD31) in the dura mater, but not within the region where the spirochete shown in (C) was detected (dashed circle). (F) 400x magnification of circled region in (E) showing spirochete (Bb).</p