Dysregulation of the initial, innate immune response to bacterial infection may lead to septic shock and death. Toll-like receptors (TLRs) play a crucial role in this innate immune response, and yet the regulatory mechanisms controlling microbial-induced TLR triggering are still to be fully understood. We have therefore sought specific regulatory mechanisms that may modulate TLR signaling. In this study, we tested for the possible existence of a functionally active soluble form of TLR2. We demonstrated the existence of natural soluble forms of TLR2 (sTLR2), which we show to be capable of modulating cell activation. We found that blood monocytes released sTLR2 constitutively and that the kinetics of sTLR2 release increased upon cell activation. Analysis of cells expressing the human TLR2 cDNA or its c-myc-tagged version indicated that sTLR2 resulted from the posttranslational modification of the TLR2 protein in an intracellular compartment. Moreover, an intracellular pool of sTLR2 is maintained. sTLR2 was found naturally expressed in breast milk and plasma. Milk sTLR2 levels mirrored those of the TLR coreceptor soluble CD14. Depletion of sTLR2 from serum resulted in an increased cellular response to bacterial lipopeptide. Notably, serum sTLR2 was lower in tuberculosis patients. Coimmunoprecipitation experiments and computational molecular docking studies showed an interaction between sTLR2 and soluble CD14 in plasma and milk. These findings suggest the existence of a novel and specific innate immune mechanism regulating microbial-induced TLR triggering, and may lead to new therapeutics for the prevention and/or treatment of severe infectious diseases.

Little is known about innate immunity to bacteria after birth in the hitherto sterile fetal intestine. Breast-feeding has long been associated with a lower incidence of gastrointestinal infections and inflammatory and allergic diseases. We found in human breast milk a 48-kD polypeptide, which we confirmed by mass spectrometry and sequencing to be a soluble form of the bacterial pattern recognition receptor CD14 (sCD14). Milk sCD14 (m-sCD14) concentrations were up to 20-fold higher than serum sCD14 from nonpregnant, pregnant, or lactating women. In contrast, lipopolysaccharide (LPS)-binding protein was at very low levels. Mammary epithelial cells produced 48-kD sCD14. m-sCD14 mediated activation by LPS and whole bacteria of CD14 negative cells, including intestinal epithelial cells, resulting in release of innate immune response molecules. m-sCD14 was undetectable in the infant formulas and commercial (cows') milk tested, although it was present in bovine colostrum. These findings indicate a sentinel role for sCD14 in human milk during bacterial colonization of the gut, and suggest that m-sCD14 may be involved in modulating local innate and adaptive immune responses, thus controlling homeostasis in the neonatal intestine.

Background and purpose:  Oily extracts of Sichuan and Melegueta peppers evoke pungent sensations mediated by different alkylamides [mainly hydroxy‐α‐sanshool (α‐SOH)] and hydroxyarylalkanones (6‐shogaol and 6‐paradol). We assessed how transient receptor potential ankyrin 1 (TRPA1) and TRP vanilloid 1 (TRPV1), two chemosensory ion channels, participate in these pungent sensations.Experimental approach:  The structure–activity relationships of these molecules on TRPA1 and TRPV1 was measured by testing natural and synthetic analogues using calcium and voltage imaging on dissociated dorsal root ganglia neurons and human embryonic kidney 293 cells expressing the wild‐type channels or specific cysteine mutants using glutathione trapping as a model to probe TRPA1 activation. In addition, using Trpv1 knockout mice, the compounds' aversive responses were measured in a taste brief‐access test.Key results:  For TRPA1 activation, the cis C6 double bond in the polyenic chain of α‐SOH was critical, whereas no structural specificity was required for activation of TRPV1. Both 6‐shogaol and 6‐paradol were found to activate TRPV1 and TRPA1 channels, whereas linalool, an abundant terpene in Sichuan pepper, activated TRPA1 but not TRPV1 channels. Alkylamides and 6‐shogaol act on TRPA1 by covalent bonding whereas none of these compounds activated TRPV1 through such interactions. Finally, TRPV1 mutant mice retained sensitivity to 6‐shogaol but were not responsive to α‐SOH.Conclusions and implications:  The pungent nature of components of Sichuan and Melegueta peppers was mediated via interactions with TRPA1 and TRPV1 channels and may explain the aversive properties of these compounds.

Serpins form a large class of protease inhibitors involved in regulation of a wide spectrum of physiological processes. Recently identified prokaryotic members of this protein family may provide a key to the evolutionary origins of the unique serpin fold and the associated inhibitory mechanism. We performed a biochemical characterization of a serpin from Bifidobacterium longum, an anaerobic Gram-positive bacterium that naturally colonizes human gastrointestinal tract. The B. longum serpin was shown to efficiently inhibit eukaryotic elastase-like proteases with a stoichiometry of inhibition close to 1. Porcine pancreatic elastase and human neutrophil elastase were inhibited with the second order association constants of 4.7 × 10⁴ m^-1 s^-1 and 2.1 × 10⁴ m^-1 s^-1, respectively. The B. longum serpin is expected to be active in the gastrointestinal tract, because incubation of the purified recombinant serpin with mouse feces produces a stable covalent serpin-protease adduct readily detectable by SDS-PAGE. Bifidobacteria may encounter both pancreatic elastase and neutrophil elastase in their natural habitat and protection against exogenous proteolysis may play an important role in the interaction between these commensal bacteria and their host.