Beate Berghöfer

IFN-alpha exercises multiple immune modulatory and antiviral activities and has been suggested to play a critical role in the pathogenesis of systemic lupus erythematosus (SLE). Plasmacytoid dendritic cells (pDCs) release IFN-alpha upon TLR7 and TLR9 ligation. With respect to the nine times higher incidence of SLE in women and the clinical use of synthetic TLR ligands as novel immune adjuvants, we analyzed IFN-alpha and TNF-alpha production in healthy human individuals. Blood samples were incubated with synthetic TLR7 and TLR9 ligands. In three independent groups (n(1) = 120, n(2) = 101, and n(3) = 123), analysis revealed a capacity of female PBLs to produce significantly higher IFN-alpha levels after TLR7 stimulation (p(1) < 0.0000001, p(2) < 0.0000001, and p(3) < 0.0001) compared with male PBLs. In contrast, no sex differences were evident after TLR9 stimulation. TNF-alpha production after TLR7 stimulation and also total pDC numbers were not different between females and males. X-inactivation escape of the TLR7 gene was investigated in monoclonal B cell lines and, independently, in pDCs after cell sorting and single-cell picking, indicating regular silencing of one TLR7 allele in females. Additionally, exogenous 17beta-estrogen and estrogen receptor antagonism did not indicate a significant role on TLR7-induced IFN-alpha production. Our data reveal for the first time a profound sex-dependent pathway of TLR7-induced IFN-alpha with higher production in females. These findings may explain the higher prevalence of SLE in females and the reported decreased therapeutic efficacy of synthetic TLR7 ligands in male individuals.

The Marburg virus (MARV) envelope consists of a lipid membrane and two major proteins, the matrix protein VP40 and the glycoprotein GP. Both proteins use different intracellular transport pathways: GP utilizes the exocytotic pathway, while VP40 is transported through the retrograde late endosomal pathway. It is currently unknown where the proteins combine to form the viral envelope. In the present study, we identified the intracellular site where the two major envelope proteins of MARV come together as peripheral multivesicular bodies (MVBs). Upon coexpression with VP40, GP is redistributed from the trans-Golgi network into the VP40-containing MVBs. Ultrastructural analysis of MVBs suggested that they provide the platform for the formation of membrane structures that bud as virus-like particles from the cell surface. The virus-like particles contain both VP40 and GP. Single expression of GP also resulted in the release of particles, which are round or pleomorphic. Single expression of VP40 led to the release of filamentous structures that closely resemble viral particles and contain traces of endosomal marker proteins. This finding indicated a central role of VP40 in the formation of the filamentous structure of MARV particles, which is similar to the role of the related Ebola virusVP40. In MARV-infected cells, VP40 and GP are colocalized in peripheral MVBs as well. Moreover, intracellular budding of progeny virions into MVBs was frequently detected. Taken together, these results demonstrate an intracellular intersection between GP and VP40 pathways and suggest a crucial role of the late endosomal compartment for the formation of the viral envelope.

VP40, the matrix protein of Marburg virus, is a peripheral membrane protein that has been shown to associate with membranes of multivesicular bodies (MVBs) (L. Kolesnikova, H. Bugany, H.-D. Klenk, and S. Becker, J. Virol. 76:1825-1838, 2002). The present study revealed that VP40 is bound to cellular membranes rapidly after synthesis. Time course studies were performed to trace the distribution of VP40 during the course of expression. First, VP40 was homogenously distributed throughout the cytoplasm, although the majority of protein (70%) was already membrane associated. Next, VP40 accumulated in MVBs and in tubular protrusions emerging from MVBs. Finally, VP40 appeared in a patch-like pattern beneath the plasma membrane. These morphological results were supported by iodixanol density gradient analyses. The majority of VP40-positive membranes were first detected comigrating with small vesicles. VP40 was then shifted to fractions containing endosomal marker proteins, and later, to fractions containing plasma membrane marker proteins. Blocking of protein synthesis by use of cycloheximide at the time when VP40 was mainly associated with the small vesicles did not prevent the redistribution of VP40 to the late endosomes and further to the plasma membrane. The inhibition of intracellular vesicular trafficking by monensin significantly reduced the appearance of VP40 at the plasma membrane. In conclusion, we suggest that the transport of the Marburg virus matrix protein VP40 involves its accumulation in MVBs followed by the redistribution of VP40-enriched membrane clusters to the plasma membrane.

BACKGROUND: Toll-like receptor 9 (TLR9) is a pattern-recognition receptor that detects unmethylated CpG motifs prevalent in bacterial and viral DNA. TLR9 stimulation is a key event after bacterial infection, triggering innate immunity and T-helper type 1 skewed adaptive immunity. Synthetic CpG-oligodeoxynucleotides (CpG-ODNs) represent a promising and novel class of immune adjuvants for allergy treatment, vaccination, and cancer therapy. However, common functional TLR9 gene variants could interfere with the clinical utilization of CpG-ODN in immunotherapy. Recently, a possible association of TLR9 polymorphism C-1237T with asthma has been reported. OBJECTIVE: The aim of the present study was to investigate whether TLR9 polymorphisms or haplotypes have functional relevance and are associated with atopy. METHODS: We genotyped five common TLR9 single-nucleotide polymorphisms (SNPs) in promoter, exon, and intron regions of the gene in 527 healthy blood donors, and estimated four common haplotypes. The total IgE and specific IgE levels against the most common aeroallergens were measured (n=303). IFN-alpha production by plasmacytoid dendritic cells (pDCs) was analysed after stimulation with TLR9 ligand CpG-ODN (n=220). RESULTS: No significant influence of common TLR9 polymorphisms and haplotypes on the total and specific IgE levels was found. Functional analysis of CpG-ODN-induced IFN-alpha did not indicate a significant role for common TLR9 gene polymorphisms in TLR9 function. CONCLUSION: We conclude that common genetic differences in the TLR9 gene exert no major influence on allergy susceptibility, and are unlikely to have on impact on clinical application of CpG-ODNs.

Plasmacytoid dendritic cells (pDCs) are unique with respect to their capacity to produce unsurpassed amounts of IFN-alpha and coexpress TLR7 and TLR9, mediating IFN-alpha production. Although TLRs are critical receptors of innate immunity, little is known about the immunological effects of TLR7/TLR9 costimulation. We have analyzed the effects of TLR7/TLR9 costimulation on IFN-alpha production by leukocytes and pDCs. Our experiments revealed that both synthetic (resiquimod and loxoribine) and natural (ssRNA40) TLR7 ligands abrogate CpG-A- and CpG-C-oligodeoxynucleotide (ODN)-induced IFN-alpha production by human leukocytes. Because TLR7 ligands themselves represent important IFN-alpha inducers, we demonstrated that substimulatory TLR7 ligand concentrations significantly inhibited CpG-A-induced IFN-alpha. Delayed addition of TLR7 ligands still resulted in complete suppression of CpG-A-ODN-induced IFN-alpha production, suggesting that the inhibition is unlikely to be caused by a kinetic uptake advantage. Unlike for CpG-A and CpG-C, TLR7 ligands did not inhibit CpG-B-ODN-induced IFN-alpha production. Experiments with purified human pDCs demonstrated that the inhibitory effects of TLR7/TLR9 costimulation were mediated directly by pDCs. Suppression of IFN-alpha production was not related to increased cell death and was also detectable in enriched mouse pDCs. Analyses of pDCs suggested that the TLR7 signal regulates the outcome of TLR7 ligand/CpG-A-ODN costimulation and can either inhibit (IFN-alpha) or promote (IL-8/CD40) cytokine and surface marker expression. Our data reveal for the first time a strong inhibitory effect of TLR7 stimulation on IFN-alpha production induced by CpG-A- and CpG-C-ODNs. These findings provide novel insight into the effects of TLR7/TLR9 costimulation and may support the development of novel TLR9 inhibitors.