The syndrome of periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) is the most common periodic fever disease in children. However, the pathogenesis is unknown. Using a systems biology approach we analyzed blood samples from PFAPA patients whose genetic testing excluded hereditary periodic fevers (HPFs), and from healthy children and pediatric HPF patients. Gene expression profiling could clearly distinguish PFAPA flares from asymptomatic intervals, HPF flares, and healthy controls. During PFAPA attacks, complement ( C1QB, C2, SERPING1 ), IL-1–related ( IL-1B, IL-1RN, CASP1, IL18RAP ), and IFN-induced ( AIM2 , IP-10/CXCL10 ) genes were significantly overexpressed, but T cell-associated transcripts ( CD3, CD8B ) were down-regulated. On the protein level, PFAPA flares were accompanied by significantly increased serum levels of chemokines for activated T lymphocytes (IP-10/CXCL10, MIG/CXCL9), G-CSF, and proinflammatory cytokines (IL-18, IL-6). PFAPA flares also manifested a relative lymphopenia. Activated CD4 + /CD25 + T-lymphocyte counts correlated negatively with serum concentrations of IP-10/CXCL10, whereas CD4 + /HLA-DR + T lymphocyte counts correlated positively with serum concentrations of the counterregulatory IL-1 receptor antagonist. Based on the evidence for IL-1β activation in PFAPA flares, we treated five PFAPA patients with a recombinant IL-1 receptor antagonist. All patients showed a prompt clinical and IP-10/CXCL10 response. Our data suggest an environmentally triggered activation of complement and IL-1β/-18 during PFAPA flares, with induction of Th1-chemokines and subsequent retention of activated T cells in peripheral tissues. IL-1 inhibition may thus be beneficial for treatment of PFAPA attacks, with IP-10/CXCL10 serving as a potential biomarker.
It is widely believed that innate immune responses to Borrelia burgdorferi (Bb) are primarily triggered by the spirochete's outer membrane lipoproteins signaling through cell surface TLR1/2. We recently challenged this notion by demonstrating that phagocytosis of live Bb by peripheral blood mononuclear cells (PBMCs) elicited greater production of proinflammatory cytokines than did equivalent bacterial lysates. Using whole genome microarrays, we show herein that, compared to lysates, live spirochetes elicited a more intense and much broader transcriptional response involving genes associated with diverse cellular processes; among these were IFN-β and a number of interferon-stimulated genes (ISGs), which are not known to result from TLR2 signaling. Using isolated monocytes, we demonstrated that cell activation signals elicited by live Bb result from cell surface interactions and uptake and degradation of organisms within phagosomes. As with PBCMs, live Bb induced markedly greater transcription and secretion of TNF-α, IL-6, IL-10 and IL-1β in monocytes than did lysates. Secreted IL-18, which, like IL-1β, also requires cleavage by activated caspase-1, was generated only in response to live Bb. Pro-inflammatory cytokine production by TLR2-deficient murine macrophages was only moderately diminished in response to live Bb but was drastically impaired against lysates; TLR2 deficiency had no significant effect on uptake and degradation of spirochetes. As with PBMCs, live Bb was a much more potent inducer of IFN-β and ISGs in isolated monocytes than were lysates or a synthetic TLR2 agonist. Collectively, our results indicate that the enhanced innate immune responses of monocytes following phagocytosis of live Bb have both TLR2-dependent and -independent components and that the latter induce transcription of type I IFNs and ISGs.
Treponema pallidum reacts poorly with the antibodies present in rabbit and human syphilitic sera, a property attributed to the paucity of proteins in its outer membrane. To better understand the basis for the syphilis spirochete's "stealth pathogenicity," we used a dual-label, 3-step amplified assay in which treponemes encapsulated in gel microdroplets were probed with syphilitic sera in parallel with anti-FlaA antibodies. A small (approximately 5 to 10%) but reproducible fraction of intact treponemes bound IgG and/or IgM antibodies. Three lines of evidence supported the notion that the surface antigens were likely -barrel-forming outer membrane proteins (OMPs): (i) surface labeling with anti-lipoidal (VDRL) antibodies was not observed, (ii) immunoblot analysis confirmed prior results showing that T. pallidum glycolipids are not immunoreactive, and (iii) labeling of intact organisms was not appreciably affected by proteinase K (PK) treatment. With this method, we also demonstrate that TprK (TP0897), an extensively studied candidate OMP, and TP0136, a lipoprotein recently reported to be surface exposed, are both periplasmic. Consistent with the immunolabeling studies, TprK was also found to lack amphiphilicity, a characteristic property of -barrel-forming proteins. Using a consensus computational framework that combined subcellular localization and -barrel structural prediction tools, we generated ranked groups of candidate rare OMPs, the predicted T. pallidum outer membrane proteome (OMPeome), which we postulate includes the surface-exposed molecules detected by our enhanced gel microdroplet assay. In addition to underscoring the syphilis spirochete's remarkably poor surface antigenicity, our findings help to explain the complex and shifting balance between pathogen and host defenses that characterizes syphilitic infection.
The endosomal Toll-like receptors (TLRs) TLR3, TLR7, TLR8 and TLR9 are important in sensing foreign nucleic acids encountered by phagocytes. Because TLR8 was initially thought to be non-functional in mice, less is known about TLR8 than the genetically and functionally related TLR7. Originally associated with the recognition of single-stranded RNA of viral origin, there is now evidence that human TLR8 is also able to sense bacterial RNA released within phagosomal vacuoles, inducing the production of both nuclear factor (NF)-κB-dependent cytokines and type I interferons (IFNs), such as IFN-β. The functions of TLR8 extend beyond the recognition of foreign pathogens and include cross-talk with other endosomal TLRs, a process that may also have a role in the generation of autoimmunity.
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