Nicolas W. J. Schröder

Heterozygous Arg753Gln Polymorphism of Human TLR-2 Impairs Immune Activation by Borrelia burgdorferi and Protects from Late Stage Lyme Disease

Lyme disease (LD) is caused by Borrelia burgdorferi and displays different stages, including localized, early disseminated, and persistent infection, all of which are associated with profound inflammatory reactions in the host. Induction of proinflammatory cytokines by B. burgdorferi is mainly mediated by outer surface proteins interacting with TLR-2/TLR-1 heterodimers. In this study, we show that TNF-α induction by Borrelia lysate was impaired in heterozygous TLR-2 knockout mice, while reactivity to lipoteichoic acid, another TLR-2 ligand signaling via TLR-2/TLR-6 heterodimers, was unaffected. Blood from individuals heterozygous for the TLR-2 polymorphism Arg753Gln was tested for cytokine release upon stimulation with Borrelia lysate, and induction of TNF-α and IFN-γ was significantly lower as compared with individuals not exhibiting this variation. Overexpression of TLR-2 carrying the Arg753Gln polymorphism in HEK 293 cells led to a significantly stronger impairment of activation by TLR-2/TLR-1 ligands as compared with TLR-2/TLR-6 ligands. To study whether heterozygosity for the Arg753Gln variant of TLR-2 influenced susceptibility for LD, we analyzed 155 patients for this polymorphism. The Arg753Gln variant occurs at a significantly lower frequency in LD patients as compared with matched controls (5.8 vs 13.5%, odds ratio 0.393, 95% confidence interval 0.17–0.89, p = 0.033), with an even more pronounced difference when late stage disease was observed (2.3 vs 12.5%, odds ratio 0.163, 95% confidence interval 0.04–0.76, p = 0.018). These data suggest that Arg753Gln may protect from the development of late stage LD due to a reduced signaling via TLR-2/TLR-1.

Heterozygous Toll-Like Receptor 4 Polymorphism Does Not Influence Lipopolysaccharide-Induced Cytokine Release in Human Whole Blood

The heterozygous Asp299Gly mutation of the toll-like receptor (TLR) 4, the key receptor for lipopolysaccharide (LPS), has been associated with attenuated inflammatory responses. When 160 healthy volunteers (9% heterozygous and 0.6% homozygous) were genotyped and their LPS-inducible cytokine release was assessed in an ex vivo whole blood test, the responses of heterozygotes did not differ significantly from those of wild-type carriers for any of the cytokines (tumor necrosis factor-alpha, interleukin [IL]-1beta, IL-6, interferon-gamma, and granulocyte colony-stimulating factor) or eicosanoids measured or for serum cytokines and C-reactive protein. Ten heterozygous subjects and 12 wild-type control subjects responded similarly to a graded series of LPS and Escherichia coli concentrations, excluding the possibility that allele-specific differences are evident only at low stimulus concentrations or in response to whole pathogens. These data demonstrate that the heterozygous Asp299Gly polymorphism does not exhibit a functional defect in cytokine release after the stimulation of blood monocytes.

Heterozygous Toll-Like Receptor 2 Polymorphism Does Not Affect Lipoteichoic Acid-Induced Chemokine and Inflammatory Responses

ABSTRACT While transfection of tlr2 conveyed responsiveness to lipoteichoic acid (LTA), the Arg753Gln polymorphic gene could not. LTA induced a stronger chemokine and anti-inflammatory response than lipopolysaccharides did. Blood from heterozygous polymorphic and wild-type donors reacted uniformly to LTA and Staphylococcus aureus. Thus, one functional allele for Toll-like receptor 2 suffices for full cytokine response.

Acylated cholesteryl galactoside as a novel immunogenic motif in Borrelia burgdorferi sensu stricto.

Borrelia burgdorferi sensu lato is the causing agent of Lyme disease, an infectious disease frequently occurring in the United States, Europe, and Northern Asia. Currently, diagnosis of and vaccination strategies against this pathogen are exclusively based on proteinaceous structures. Here we report on a novel class of immunogenic glycolipids purified from B. burgdorferi sensu stricto B31. Employing a butanol/water extraction procedure with subsequent Bligh/Dyer extraction of the organic phase, thin layer chromatography analysis revealed the presence of three distinct glycolipids, which were chemically analyzed employing combined gas-liquid chromatography/mass spectroscopy, matrix-assisted laser desorption/ionization mass spectrometry, and NMR. We identified acylated cholesteryl galactoside (ACG) next to cholesteryl galactoside and α-monogalactosyl-diacylglycerol. After extensive purification, the glycolipids investigated failed to cause proinflammatory responses in human cells transfected with human toll-like receptor (TLR)-2 or -4. However, we observed a marked recognition of ACG by sera derived from patients suffering from Lyme disease. These data indicate that newly described ACG is involved in developing host immunity during Lyme disease and thus may be useful for diagnosis and vaccination.

Immune responses induced by spirochetal outer membrane lipoproteins and glycolipids.

The class of Spirochetes comprises a wide array of clinically important pathogens, including Treponema pallidum causing syphilis as well as Borrelia burgdorferi, the agent of Lyme disease (LD). Diseases caused by spirochetes are characterized by specific sequelae of host reactions, and also by characteristic antibody response patterns. Over the last decades, research on the interaction of spirochetes with the hosts immune system had a strong emphasis on outer membrane lipoproteins. In fact, these structures have been convincingly shown to activate immune cells via CD14 and Toll-like receptor (TLR)-2, and recent data also indicate an interaction with lipopolysaccharide (LPS)-binding protein (LBP). In particular, the interaction of B. burgdorferi with TLR-2 could not only be demonstrated in mice, but was also supported by data showing that genetic variants of TLR-2 in humans influenced the clinical course of LD. However, there is increasing evidence that next to lipoproteins, glycolipids may also play an important role in responses of the immune system towards spirochetes. Diacylglycerol-containing glycolipids exhibiting similarities with lipoteichoic acid (LTA) of Gram-positive bacteria have been demonstrated in various Treponema species, whereas LPS-like glycolipids have been shown to be present in Leptospira. Treponema glycolipids, comparably to lipoproteins and LTA, interact with LBP, CD14 and TLRs. In contrast, complex glycolipids of high molecular weight could not be demonstrated in Borrelia, whereas these bacteria exhibit a number of unique low molecular weight glycolipids. Some of these glycolipids cause strong immediate immune responses, while others appear to be potent antigens for induction of an adaptive immune response. This review summarizes data obtained so far on amphiphilic and hydrophobic molecules from spirochetes regarding structure and influence on innate as well as adaptive immune responses.

Acylated Cholesteryl Galactosides Are Specific Antigens of Borrelia Causing Lyme Disease and Frequently Induce Antibodies in Late Stages of Disease

Borrelia burgdorferi sensu lato is the causative agent of Lyme disease (LD), an infectious disease occurring in North America, Europe, and Asia in different clinical stages. B. burgdorferi sensu lato encompasses at least 12 species, with B. burgdorferi sensu stricto, B. garinii, and B. afzelii being of highest clinical importance. Immunologic testing for LD as well as recent vaccination strategies exclusively refer to proteinaceous antigens. However, B. burgdorferi sensu stricto exhibits glycolipid antigens, including 6-O-acylated cholesteryl β-d-galactopyranoside (ACGal), and first the data indicated that this compound may act as an immunogen. Here we investigated whether B. garinii and B. afzelii also possess this antigen, and whether antibodies directed against these compounds are abundant among patients suffering from different stages of LD. Gas-liquid chromatography/mass spectroscopy and NMR spectroscopy showed that both B. garinii and B. afzelii exhibit ACGal in high quantities. In contrast, B. hermsii causing relapsing fever features 6-O-acylated cholesteryl β-d-glucopyranoside (ACGlc). Sera derived from patients diagnosed for LD contained antibodies against ACGal, with 80% of patients suffering from late stage disease exhibiting this feature. Antibodies reacted with ACGal from all three B. burgdorferi species tested, but not with ACGlc from B. hermsii. These data show that ACGal is present in all clinically important B. burgdorferi species, and that specific antibodies against this compound are frequently found during LD. ACGal may thus be an interesting tool for improving diagnostics as well as for novel vaccination strategies.

Chemoenzymatic Synthesis of a Glycolipid Library and Elucidation of the Antigenic Epitope for Construction of a Vaccine Against Lyme Disease

Lyme disease (LD) is the most common tick-borne disease in Europe, North America, and Asia. The etiologic agents of LD are spirochetes of the group Borrelia burgdorferi sensu lato, which possess a lipid content of 25-30% of the dry weight. The major glycolipid cholesteryl 6-O-acyl-beta-D-galactopyranoside (ACGal), present in B. burgdorferi sensu stricto, B. afzelii, and B. garinii, is a specific and highly prevalent antigen frequently recognized by antibodies in late-stage LD. Here we report a convenient route for the chemical synthesis of ACGal by employing a combination of chemical synthesis steps with enzymatic transformations. This synthesized molecule was compared with bacterial extracts by immunoblots with patient sera, confirming the preserved antigenicity. Next, a glycolipid library derived from the native molecules with variations in the fatty acyl moiety and derivatives in which the cholesterol has been replaced was designed and synthesized. The chemical structures were confirmed by 1D and 2D NMR spectroscopy and mass spectrometry. The native and synthetic glycolipids were utilized in immunoblots to determine the epitope recognized by antibodies in patient sera. By this method we could demonstrate that galactose, cholesterol, and a fatty acid with a minimal chain length of four carbon atoms comprises the essential structure for recognition by antibodies. Finally, this finding allowed the synthesis of a functionalized ACGal with an omega-mercapto group at the fatty acid and a facile protection and deprotection strategy. This antigenic hapten can be conjugated to a carrier protein to effect immunization against Lyme disease.

Non-enhancing relapse of a primary CNS lymphoma with multiple diffusion-restricted lesions

Primary CNS lymphoma (PCNSL) and its variant primary intraocular lymphoma (PIOL) are rare forms of extranodal non-Hodgkin’s lymphoma confined to the CNS including the retina and the optical nerve; histologically, most cases are diffuse large B cell lymphomas. PCNSL in immunocompetent patients display typical radiological features on MRI, i.e. intensely and homogeneously enhancing lesions with moderate edema. Here, we report a 52-year-old male with a history of a PIOL and two consecutive intracerebral relapses who presented with dysarthria, dysphagia, and gait ataxia. Gadolinium-enhanced T1 scans were unremarkable but multiple lesions with restricted water diffusivity were seen on diffusion-weighted imaging. Relapse of his PCNSL was secured histologically only on autopsy. The possible etiology of the diffusion-restricted lesions is discussed.

Acylated cholesteryl galactosides are ubiquitous glycolipid antigens among Borrelia burgdorferi sensu lato

Lyme disease (LD) is the most common tick-borne disease in the Northern hemisphere. It is caused by Borrelia burgdorferi sensu lato, in particular, B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii. However, other genospecies have been implicated as causative factors of LD as well. Borrelia burgdorferi exhibits numerous immunogenic lipoproteins, but due to strong heterogeneity, the use of these proteins for serodiagnosis and vaccination is hampered. We and others have identified acylated cholesteryl galactosides (ACGal) as a novel glycolipid present in B. burgdorferi sensu stricto, B. afzelii, and B. garinii. ACGal is a strong antigen and the majority of patients display anti-ACGal antibodies in the chronic stages of LD. However, it is unknown whether ACGal is present in other presumably pathogenic B. burgdorferi genospecies. Therefore, we performed an analysis of the total lipid extracts of a wide spectrum of genospecies of B. burgdorferi sensu lato using thin-layer chromatography as well as Western blot and dot-blot assays. We show that ACGal is present in substantial quantities in all B. burgdorferi genospecies tested. Therefore, this molecule might improve the serological detection of rarely pathogenic genospecies, and may be used as a protective vaccine regardless of the prevailing genospecies.

Pipestem capillaries in necrotizing myopathy revisited

Pipestem-capillaries in necrotizing myopathy, have been reported as a feature of a distinct type of myopathy. Here, we analyze four muscle biopsy specimens from patients exhibiting endomysial fibrosis associated with pipestem capillaries using histological and electronmicroscopic techniques. However, only one case displayed all of the originally described features, including necrotic fibres, capillary thickening and lack of a significant lymphocytic inflammation, while one case exhibited striking capillary pathology with minimal necrosis and absence of inflammation, and the other two cases were accompanied by additional pathological features. These data support the existence of a microangiopathy with pipestem capillaries as a characteristic and distinct histopathological pattern, and indicate that it occurs in the context of a variety of muscular disorders broader than initially suspected. Furthermore, we show that the pipestem-capillary associated decrease in fibre size is at least in part a result of hypoxic changes.