Cutaneous TRPV1+ Neurons Trigger Protective Innate Type 17 Anticipatory Immunity.

Cutaneous TRPV1+ neurons directly sense noxious stimuli, inflammatory cytokines, and pathogen-associated molecules and are required for innate immunity against some skin pathogens. Important unanswered questions are whether TRPV1+ neuron activation in isolation is sufficient to initiate innate immune responses and what is the biological function for TRPV1+ neuron-initiated immune responses. We used TRPV1-Ai32 optogenetic mice and cutaneous light stimulation to activate cutaneous neurons in the absence of tissue damage or pathogen-associated products. We found that TRPV1+ neuron activation was sufficient to elicit a local type 17 immune response that augmented host defense to C. albicans and S. aureus. Moreover, local neuron activation elicited type 17 responses and augmented host defense at adjacent, unstimulated skin through a nerve reflex arc. These data show the sufficiency of TRPV1+ neuron activation for host defense and demonstrate the existence of functional anticipatory innate immunity at sites adjacent to infection that depends on antidromic neuron activation.

Intrathecal cannabinoid-1 receptor agonist prevents referred hyperalgesia in acute acrolein-induced cystitis in rats.

We investigated the capacity of intrathecal arachidonyl-2'-chloroethylamide (ACEA), a cannabinoid-1 receptor (CB1R) agonist, to inhibit referred hyperalgesia and increased bladder contractility resulting from acute acrolein-induced cystitis in rats. 24 female rats were divided into 4 groups: 1) intrathecal vehicle/intravesical saline; 2) intrathecal vehicle/intravesical acrolein; 3) intrathecal ACEA/intravesical saline; and 4) intrathecal ACEA/intravesical acrolein. Bladder catheters were placed 4-6 days prior to the experiment. On the day of the experiment, rats were briefly anesthetized with isoflurane to recover the external end of the cystostomy catheter. After recovery from anesthesia, pre-treatment cystometry was performed, and mechanical sensitivity of the hindpaws was determined. Rats were again briefly anesthetized with isoflurane to inject ACEA or vehicle into the intrathecal space between L5-L6. Beginning 10 minutes after intrathecal injection, saline or acrolein was infused into the bladder for 30 minutes. Post-treatment cystometry and mechanical sensitivity testing were performed. Rats were euthanized, and bladders were collected, weighed, and fixed for histology. The intrathecal vehicle/intravesical acrolein group developed mechanical hyperalgesia with post-treatment mechanical sensitivity of 6 ± 0.3 g compared to pretreatment of 14 ± 0.4 g (p < 0.01). Pre- and post-treatment hind paw mechanical sensitivity was statistically similar in rats that received intrathecal ACEA prior to intravesical infusion of acrolein (15 ± 0.2 g and 14 ± 0.4 g, respectively). Acrolein treatment increased basal bladder pressure and maximal voiding pressure and decreased intercontraction interval and voided volume. However, intrathecal ACEA was ineffective in improving acrolein-related urodynamic changes. In addition, bladder histology demonstrated submucosal and muscularis edema that was similar for all acrolein-treated groups, irrespective of ACEA treatment. Intravesical saline had no effect on results of cystometry or mechanical sensitivity of the hind paws, regardless of intrathecal treatment. Intrathecal ACEA prevented referred hyperalgesia associated with acute acrolein-induced cystitis. However, in this experimental model, ACEA did not ameliorate the associated urodynamic changes. These findings suggest that pain arising from cystitis may be inhibited by activation of spinal CB1R but the acute local response of the bladder appeared to be unaffected by stimulation of spinal CB1R.

Method for screening and MALDI-TOF MS sequencing of encoded combinatorial libraries.

We describe a new method for encoded synthesis, efficient on-resin screening, and rapid unambiguous sequencing of combinatorial peptide libraries. An improved binary tag system for encoding peptide libraries during synthesis was designed to facilitate unequivocal assignment of isobaric residues by MALDI-TOF MS analysis. The improved method for encoded library synthesis was combined with a new versatile on-resin screening strategy that permitted multiple stages and types of screening to be employed successively on one library under mild conditions. The new method facilitated a combinatorial study of transglutaminase (TGase) enzyme substrate peptides, revealing new details of the effect of amino acid composition on TGase substrates. The approach was first demonstrated for an encoded library (130,321 compounds) of lysine pentapeptide substrates of TGase, synthesized using the "split-mix" method. The library was reacted on-resin with TGase enzyme and a soluble desthiobiotin labeled glutamine substrate. Initial screening was performed by adsorbing streptavidin-coated magnetic microparticles onto library beads, followed by magnetic separation. The differential binding affinities of desthiobiotin and biotin for streptavidin were exploited to release the magnetic microparticles and regenerate the desthiobiotin-labeled resin beads for further screening by flow-cytometry-based automated bead sorting, resulting in 345 beads that were sequenced by MALDI-TOF MS analysis. A second library consisted of encoded glutamine hexapeptide substrates, which was reacted on-resin with TGase enzyme and a soluble desthiobiotin-labeled cadaverine. Two-stage screening identified 267 glutamine peptides as TGase-reactive, of which 21 were further analyzed by solution-phase enzyme kinetics. Kinetic results indicated that the peptide PQQQYV from the library has a 68-fold greater substrate specificity than the best known glutamine substrate QQIV. The new encoding and screening strategies described here are expected to be broadly applicable to synthesis and screening of combinatorial peptide libraries in the future.