The Lyme disease vaccine is based on the outer-surface lipoprotein (OspA) of the pathogen Borrelia burgdorferi, and 95% of vaccine recipients develop substantial titers of antibodies against OspA. Here, we identified seven individuals with very low antibody titers after vaccination (low responders). The macrophages of low responders produced less tumor necrosis factor-alpha and interleukin-6 after OspA stimulation and had lower cell-surface expression of Toll-like receptor (TLR) 1 as compared to normal cells, but normal expression of TLR2. TLRs activate innate responses to pathogens, and TLR2 recognizes lipoproteins and peptidoglycan (PGN). After OspA immunization, mice genetically deficient in either TLR2 (TLR2(-/-)) or TLR1 (TLR1(-/-)) produced low titers of antibodies against OspA. Notably, macrophages from TLR2(-/-) mice were unresponsive to OspA and PGN, whereas those from TLR1(-/-) mice responded normally to PGN but not to OspA. These data indicate that TLR1 and TLR2 are required for lipoprotein recognition and that defects in the TLR1/2 signaling pathway may account for human hyporesponsiveness to OspA vaccination.
Tick saliva has pleiotropic properties that facilitate persistence of the arthropod upon the host. We now describe a feeding-inducible protein in Ixodes scapularis saliva, Salp15, that inhibits CD4(+) T cell activation. The mechanism involves the repression of calcium fluxes triggered by TCR ligation and results in lower production of interleukin-2. Salp15 also inhibits the development of CD4(+) T cell-mediated immune responses in vivo, demonstrating the functional importance of this protein. Salp15 provides a molecular basis for understanding the immunosuppressive activity of I. scapularis saliva and vector-host interactions.
The effects of aging on human TLR function remain incompletely understood. We assessed TLR function and expression in peripheral blood monocytes from 159 subjects in 2 age categories, 21–30 and >65 years of age, using a multivariable mixed effect model. Using flow cytometry to assess TLR-induced cytokine production, we observed a substantial, highly significant defect in TLR1/2-induced TNF-α (p = 0.0003) and IL-6 (p < 0.0001) production, in older adults compared with young controls. In contrast to findings in aged mice, other TLR (including TLR2/6)-induced cytokine production appeared largely intact. These differences were highly significant even after correcting for covariates including gender, race, medications, and comorbidities. This defect in TLR1/2 signaling may result from alterations in baseline TLR1 surface expression, which was decreased by 36% in older adults (p < 0.0001), whereas TLR2 surface expression was unaffected by aging. Production of IL-6 (p < 0.0001) and TNF-α (p = 0.003) after stimulation by N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-Cys-[S]-Ser1-[S]-Lys(4) trihydrochloride was strongly associated with TLR1 surface expression. Diminished TLR1/2 signaling may contribute to the increased infection-related morbidity and mortality and the impaired vaccine responses observed in aging humans.
The tick Ixodes scapularis is an efficient vector for microbes, including the Lyme disease agent Borrelia burgdorferi. Ticks engorging on vertebrates induce recruitment of inflammatory cells to the bite site. For efficient transmission to the vector, pathogens have to traffic through this complex feeding site while avoiding the deleterious effects of immune cells. We show that a tick protein, Salp25D, plays a critical role-in the mammalian host-for acquisition of Borrelia burgdorferi by the vector. Silencing salp25D in tick salivary glands impaired spirochete acquisition by ticks engorging on B. burgdorferi-infected mice. Immunizing mice against Salp25D also decreased Borrelia acquisition by I. scapularis. Salp25D detoxified reactive oxygen species at the vector-pathogen-host interface, thereby providing a survival advantage to B. burgdorferi at the tick feeding site in mice. These data demonstrate that pathogens can exploit arthropod molecules to defuse mammalian responses in order to successfully enter the vector.
Salp15 is an Ixodes scapularis salivary protein that inhibits CD4+ T cell activation through the repression of TCR ligation-triggered calcium fluxes and IL-2 production. We show in this study that Salp15 binds specifically to the CD4 coreceptor on mammalian host T cells. Salp15 specifically associates through its C-terminal residues with the outermost two extracellular domains of CD4. Upon binding to CD4, Salp15 inhibits the subsequent TCR ligation-induced T cell signaling at the earliest steps including tyrosine phosphorylation of the Src kinase Lck, downstream effector proteins, and lipid raft reorganization. These results provide a molecular basis to understanding the immunosuppressive activity of Salp15 and its specificity for CD4+ T cells.
Lyme disease and human granulocytic ehrlichiosis (HGE) are tick-borne illnesses caused by Borrelia burgdorferi and the agent of HGE, respectively. We investigated the influence of dual infection with B. burgdorferi and the HGE agent on the course of murine Lyme arthritis and granulocytic ehrlichiosis. Coinfection resulted in increased levels of both pathogens and more severe Lyme arthritis compared with those in mice infected with B. burgdorferi alone. The increase in bacterial burden during dual infection was associated with enhanced acquisition of both organisms by larval ticks that were allowed to engorge upon infected mice. Coinfection also resulted in diminished interleukin-12 (IL-12), gamma interferon (IFN-␥), and tumor necrosis factor alpha levels and elevated IL-6 levels in murine sera. During dual infection, IFN-␥ receptor expression on macrophages was also reduced, implying a decrease in phagocyte activation. These results suggest that coinfection of mice with B. burgdorferi and the HGE agent modulates host immune responses, resulting in increased bacterial burden, Lyme arthritis, and pathogen transmission to the vector.
SummaryAnaplasma phagocytophilum, an obligate intracellular pathogen that persists within polymorphonuclear leucocytes, is the second most common tick-borne agent in North America. We now show that infection of a promyelocytic cell line and neutrophils with A. phagocytophilum results in pathogen-specific tyrosine phosphorylation of ROCK1. Phosphorylation is associated with PSGL-1 and Syk, because PSGL-1 blocking antibodies and siRNA targeting Syk interfere with ROCK1 phosphorylation in A. phagocytophiluminfected cells. Knockdown of either Syk or ROCK1 also markedly impaired A. phagocytophilum infection. These data demonstrate a role for A. phagocytophilum-mediated ROCK1 phosphorylation in infection, and suggests that inhibiting this pathway may lead to new, non-antibiotic strategies to treat human granulocytic anaplasmosis.
The enzyme that transfers D-xylose from UDP-xylose to the -linked mannose of plant N-linked oligosaccharides was purified about 51,000-fold to apparent homogeneity from soybean microsomes. On SDS gels, two proteins of 56 and 59 kDa were detected and both were labeled to the same extent by the photoaffinity label
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