Cathelicidins Inhibit Escherichia coli-Induced TLR2 and TLR4 Activation in a Viability-Dependent Manner.

Activation of the immune system needs to be tightly regulated to provide protection against infections and, at the same time, to prevent excessive inflammation to limit collateral damage to the host. This tight regulation includes regulating the activation of TLRs, which are key players in the recognition of invading microbes. A group of short cationic antimicrobial peptides, called cathelicidins, have previously been shown to modulate TLR activation by synthetic or purified TLR ligands and may play an important role in the regulation of inflammation during infections. However, little is known about how these cathelicidins affect TLR activation in the context of complete and viable bacteria. In this article, we show that chicken cathelicidin-2 kills Escherichia coli in an immunogenically silent fashion. Our results show that chicken cathelicidin-2 kills E. coli by permeabilizing the bacterial inner membrane and subsequently binds the outer membrane-derived lipoproteins and LPS to inhibit TLR2 and TLR4 activation, respectively. In addition, other cathelicidins, including human, mouse, pig, and dog cathelicidins, which lack antimicrobial activity under cell culture conditions, only inhibit macrophage activation by nonviable E. coli In total, this study shows that cathelicidins do not affect immune activation by viable bacteria and only inhibit inflammation when bacterial viability is lost. Therefore, cathelicidins provide a novel mechanism by which the immune system can discriminate between viable and nonviable Gram-negative bacteria to tune the immune response, thereby limiting collateral damage to the host and the risk for sepsis.

Specific chaperones for the type VII protein secretion pathway.

Mycobacteria use the dedicated type VII protein secretion systems ESX-1 and ESX-5 to secrete virulence factors across their highly hydrophobic cell envelope. The substrates of these systems include the large mycobacterial PE and PPE protein families, which are named after their characteristic Pro-Glu and Pro-Pro-Glu motifs. Pathogenic mycobacteria secrete large numbers of PE/PPE proteins via the major export pathway, ESX-5. In addition, a few PE/PPE proteins have been shown to be exported by ESX-1. It is not known how ESX-1 and ESX-5 recognize their cognate PE/PPE substrates. In this work, we investigated the function of the cytosolic protein EspG(5), which is essential for ESX-5-mediated secretion in Mycobacterium marinum, but for which the role in secretion is not known. By performing protein co-purifications, we show that EspG(5) interacts with several PPE proteins and a PE/PPE complex that is secreted by ESX-5, but not with the unrelated ESX-5 substrate EsxN or with PE/PPE proteins secreted by ESX-1. Conversely, the ESX-1 paralogue EspG(1) interacted with a PE/PPE couple secreted by ESX-1, but not with PE/PPE substrates of ESX-5. Furthermore, structural analysis of the complex formed by EspG(5) and PE/PPE indicates that these proteins interact in a 1:1:1 ratio. In conclusion, our study shows that EspG(5) and EspG(1) interact specifically with PE/PPE proteins that are secreted via their own ESX systems and suggests that EspG proteins are specific chaperones for the type VII pathway.

Iron nanomedicines induce Toll-like receptor activation, cytokine production and complement activation.

Approximately a dozen of intravenous iron nanomedicines gained marketing authorization in the last two decades. These products are generally considered as safe, but have been associated with an increased risk for hypersensitivity-like reactions of which the underlying mechanisms are unknown. We hypothesized that iron nanomedicines can trigger the innate immune system. We hereto investigated the physico-chemical properties of ferric gluconate, iron sucrose, ferric carboxymaltose and iron isomaltoside 1000 and comparatively studied their interaction with Toll-like receptors, the complement system and peripheral blood mononuclear cells. Two out of four formulations appeared as aggregates by Scanning Transmission Electron Microscopy analysis and were actively taken up by HEK293T- and peripheral blood mononuclear cells in a cholesterol-dependent manner. These formulations triggered in vitro activation of intracellular Toll-like receptors 3, -7 and -9 in a dose- and serum-dependent manner. In parallel experiments, we determined that these compounds activated the complement system. Finally, we found that uptake of aggregation-prone iron nanomedicines by peripheral blood mononuclear cells in whole blood induced production of the proinflammatory cytokine IL-1ß, but not IL-6.

APL-1, an altered peptide ligand derived from human heat-shock protein 60, selectively induces apoptosis in activated CD4+ CD25+ T cells from peripheral blood of rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is a chronic T-cell mediated autoimmune disease that affects primarily the joints. The induction of immune tolerance through antigen-specific therapies for the blockade of pathogenic CD4+ T cells constitutes a current focus of research. In this focus it is attempted to simultaneously activate multiple regulatory mechanisms, such as: apoptosis and regulatory T cells (Tregs). APL-1 is an altered peptide ligand derived from a novel CD4+ T-cell epitope of human heat-shock protein of 60kDa, an autoantigen involved in the pathogenesis of RA. Previously, we have reported that APL-1 induces CD4+ CD25(high)Foxp3+ Tregs in several systems. Here, we investigated the ability of APL-1 in inducing apoptosis in PBMCs from RA patients, who were classified as active or inactive according to their DAS28 score. APL-1 decreased the viability of PBMCs from active but not from inactive patients. DNA fragmentation assays and typical morphological features clearly demonstrated that APL-1 induced apoptosis in these cells. Activated CD4+ CD25+ T cells but not resting CD4+ CD25- T cells were identified as targets of APL-1. Furthermore, CD4+ T-cell responses to APL-1 were found to be dependent on antigen presentation via the HLA-DR molecule. Thus, APL-1 is a regulatory CD4+ T cell epitope which might modulate inflammatory immune responses in PBMCs from RA patients by inducing CD4+ CD25(high)Foxp3+ Tregs and apoptosis in activated CD4+ T cells. These results support further investigation of this candidate drug for the treatment of RA.