The Immunosuppressant Macrolide Tacrolimus Activates Cold-Sensing TRPM8 Channels.

TRPM8 is a polymodal, nonselective cation channel activated by cold temperature and cooling agents that plays a critical role in the detection of environmental cold. We found that TRPM8 is a pharmacological target of tacrolimus (FK506), a macrolide immunosuppressant with several clinical uses, including the treatment of organ rejection following transplants, treatment of atopic dermatitis, and dry eye disease. Tacrolimus is an inhibitor of the phosphatase calcineurin, an action shared with cyclosporine. Tacrolimus activates TRPM8 channels in different species, including humans, and sensitizes their response to cold temperature by inducing a leftward shift in the voltage-dependent activation curve. The effects of tacrolimus on purified TRPM8 in lipid bilayers demonstrates conclusively that it has a direct gating effect. Moreover, the lack of effect of cyclosporine rules out the canonical signaling pathway involving the phosphatase calcineurin. Menthol (TRPM8-Y745H)- and icilin (TRPM8-N799A)-insensitive mutants were also activated by tacrolimus, suggesting a different binding site. In cultured mouse DRG neurons, tacrolimus evokes an increase in intracellular calcium almost exclusively in cold-sensitive neurons, and these responses were drastically blunted in Trpm8 KO mice or after the application of TRPM8 antagonists. Cutaneous and corneal cold thermoreceptor endings are also activated by tacrolimus, and tacrolimus solutions trigger blinking and cold-evoked behaviors. Together, our results identify TRPM8 channels in sensory neurons as molecular targets of the immunosuppressant tacrolimus. The actions of tacrolimus on TRPM8 resemble those of menthol but likely involve interactions with other channel residues.SIGNIFICANCE STATEMENT TRPM8 is a polymodal TRP channel involved in cold temperature sensing, thermoregulation, and cold pain. TRPM8 is also involved in the pathophysiology of dry eye disease, and TRPM8 activation has antiallodynic and antipruritic effects, making it a prime therapeutic target in several cutaneous and neural diseases. We report the direct agonist effect of tacrolimus, a potent natural immunosuppressant with multiple clinical applications, on TRPM8 activity. This interaction represents a novel neuroimmune interface. The identification of a clinically approved drug with agonist activity on TRPM8 channels could be used experimentally to probe the function of TRPM8 in humans. Our findings may explain some of the sensory and anti-inflammatory effects described for this drug in the skin and the eye surface.

Functional and Morphologic Alterations in Mechanical, Polymodal, and Cold Sensory Nerve Fibers of the Cornea Following Photorefractive Keratectomy.

Purpose: To define the characteristics and time course of the morphologic and functional changes experienced by corneal sensory nerves after photorefractive keratectomy (PRK). Methods: Unilateral corneal excimer laser photoablation was performed in 54 anesthetized 3- to 6-month-old mice; 11 naïve animals served as control. Mice were killed 0, 3, 7, 15, and 30 days after PRK. Excised eyes were placed in a recording chamber superfused at 34°C. Electrical nerve impulse activity of single sensory terminals was recorded with a micropipette applied onto the corneal surface. Spontaneous and stimulus-evoked (cold, heat, mechanical, and chemical stimuli) nerve terminal impulse (NTI) activity was analyzed. Corneas were fixed and stained with anti-ß-Tubulin III antibody to measure nerve density and number of epithelial nerve penetration points of regenerating subbasal leashes. Results: Nerve fibers and NTI activity were absent in the injured area between 0 and 7 days after PRK, when sparse regenerating nerve sprouts appear. On day 15, subbasal nerve density reached half the control value and abnormally responding cold-sensitive terminals were recorded inside the lesion. Thirty days after PRK, nerve density was almost restored, active cold thermoreceptors were abundant, and polymodal nociceptor activity first reappeared. Conclusions: Morphologic regeneration of subbasal corneal nerves started shortly after PRK ablation and was substantially completed 30 days later. Functional recovery appears faster in cold terminals than polymodal terminals, possibly reflecting an incomplete damage of the more extensively branched cold-sensitive axon terminals. Evolution of postsurgical discomfort sensations quality may be associated with the variable regeneration pattern of each fiber type.

Functional Properties of Sensory Nerve Terminals of the Mouse Cornea.

Purpose: To define the firing properties of sensory nerve terminals innervating the adult mouse cornea in response to external stimuli of differing modality. Methods: Extracellular electrical activity of single corneal sensory nerve terminals was recorded in excised eyes of C57BL/6J mice. Eyes were placed in a recording chamber and were continuously superfused with warm saline solution. Nerve terminal impulse (NTI) activity was recorded by means of a glass pipette (tip ∼ 50 µm), applied on the corneal surface. Nerve terminal impulse discharges were stored in a computer for offline analysis. Results: Three functionally distinct populations of nerve terminals were identified in the mouse cornea. Pure mechanonociceptor terminals (9.5%) responded phasically and only to mechanical stimuli. Polymodal nociceptor terminals (41.1%) were tonically activated by heat and hyperosmolal solutions (850 mOsm·kg-1), mechanical force, and/or TRPV1 and TRPA1 agonists (capsaicin and allyl isothiocyanate [AITC], respectively). Cold-sensitive terminals (49.4%) responded to cooling. Approximately two-thirds of them fired continuously at 34°C and responded vigorously to small temperature reductions, being classified as high-background activity, low-threshold (HB-LT) cold thermoreceptor terminals. The remaining one-third exhibited very low ongoing activity at 34°C and responded weakly to intense cooling, being named low-background activity, high-threshold (LB-HT) cold thermoreceptor terminals. Conclusions: The mouse cornea is innervated by trigeminal ganglion (TG) neurons that respond to the same stimulus modalities as corneal receptors of other mammalian species. Mechano- and polymodal endings underlie detection of mechanical and chemical noxious stimuli while HB-LT and LB-HT cold thermoreceptors appear to be responsible for basal and irritation-evoked tearing and blinking, respectively.

Polymeric nanocapsules: a potential new therapy for corneal wound healing.

Corneal injuries are one of the most frequently observed ocular diseases, leading to permanent damage and impaired vision if they are not treated properly. In this sense, adequate wound healing after injury is critical for keeping the integrity and structure of the cornea. The goal of this work was to assess the potential of polymeric nanocapsules, either unloaded or loaded with cyclosporine A or vitamin A, alone or in combination with mitomycin C, for the treatment of corneal injuries induced by photorefractive keratectomy surgery. The biopolymers selected for the formation of the nanocapsules were polyarginine and protamine, which are known for their penetration enhancement effect. The results showed that, following topical instillation to a mouse model of corneal injury, all the nanocapsule formulations, either unloaded or loaded with cyclosporine A or vitamin A, were able to stimulate corneal wound healing. In addition, the healing rate observed for the combination of unloaded protamine nanocapsules with mitomycin C was comparable to the one observed for the positive control Cacicol®, a biopolymer known as a corneal wound healing enhancer. Regarding the corneal opacity, the initial grade of corneal haze (>3) induced by the photorefractive keratectomy was more rapidly reduced in the case of the positive control, Cacicol®, than in corneas treated with the nanocapsules. In conclusion, this work shows that drug-free arginine-rich (polyarginine, protamine) nanocapsules exhibit a positive behavior with regard to their potential use for corneal wound healing.