s2fieldsofstudy: Biology Medicine

text:
Inactivation of the miR-183/96/182 Cluster Decreases the Severity of Pseudomonas aeruginosa-Induced Keratitis.

PURPOSE
The microRNA-183/96/182 cluster (miR-183/96/182) plays important roles in sensory organs. Because the cornea is replete with sensory innervation, we hypothesized that miR-183/96/182 modulates the corneal response to bacterial infection through regulation of neuroimmune interactions.

METHODS
Eight-week-old miR-183/96/182 knockout (ko) mice and their wild-type littermates (wt) were used. The central cornea of anesthetized mice was scarred and infected with Pseudomonas aeruginosa (PA), strain 19660. Corneal disease was graded at 1, 3, and 5 days postinfection (dpi). Corneal RNA was harvested for quantitative RT-PCR. Polymorphonuclear neutrophils (PMN) were enumerated by myeloperoxidase assays; the number of viable bacteria was determined by plate counts, and ELISA assays were performed to determine cytokine protein levels. A macrophage (Mϕ) cell line and elicited peritoneal PMN were used for in vitro functional assays.

RESULTS
MicroRNA-183/96/182 is expressed in the cornea, and in Mϕ and PMN of both mice and humans. Inactivation of miR-183/96/182 resulted in decreased corneal nerve density compared with wt mice. Overexpression of miR-183/96/182 in Mϕ decreased, whereas knockdown or inactivation of miR-183/96/182 in Mϕ and PMN increased their capacity for phagocytosis and intracellular killing of PA. In PA-infected corneas, ko mice showed decreased proinflammatory neuropeptides such as substance P and chemoattractant molecules, MIP-2, MCP1, and ICAM1; decreased number of PMN at 1 and 5 dpi; increased viable bacterial load at 1 dpi, but decreased at 5 dpi; and markedly decreased corneal disease.

CONCLUSIONS
MicroRNA-183/96/182 modulates the corneal response to bacterial infection through its regulation of corneal innervation and innate immunity.

Microbial keratitis is a potentially blinding disease, costing an estimated $175 million in direct health care expenditures in the United States. Pseudomonas aeruginosa (PA) is a gramnegative opportunistic pathogen capable of inducing keratitis and causes one of the most rapidly developing and destructive diseases of the cornea; it remains the most commonly recovered causative organism in contact lens-related disease. 34,35 Currently, bacterial keratitis is mainly treated by topical administration of antibiotics; however, frequent emergence of antibiotic-resistant bacteria poses serious challenges for effective management. 36 Development of alternative treatment depends on discoveries of new molecular mechanisms and therapeutic targets. However, the roles of miRNAs in PA-induced keratitis are still largely unknown. Recently, Yang et al. 37 reported that miR-155 suppresses macrophage (M/)mediated bacterial phagocytosis and intracellular killing; inactivation of miR-155 in M/ mice increased their resistance to PA-induced keratitis, 37 supporting that miRNA plays a role in bacterial keratitis through its modulation of innate immune functions.

Mice
All animals were handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The miR-183C GT/GT mice are on a 129S2/BL6-mixed background 19 and were originally derived from a gene-trap ESC clone. 19,24,25 Male and female, young adult (8-week-old) miR-183C GT/GT mice, their heterozygous (miR-183C GT/þ ) and wildtype (wt) littermates (miR-183C þ/þ ) were used in this study.

Nerve Staining
Corneas were excised posterior to the limbal region and flattened by four evenly spaced cuts from the periphery toward the center. Corneas were fixed in 2% paraformaldehyde (PFA) in 0.1M phosphate buffer (PB), pH 7.4 overnight (O/N) at 48C; then, permeabilized with 0.3% Triton X-100 in PBS for 1 hour at room temperature (RT) and blocked with PBS with 0.3% Triton X-100, 2.5% BSA, and 5% donkey serum (Covance, Battle Creek, MI, USA) for 2 hours. Then, corneas were incubated with rabbit anti-bIII-tubulin antibody (Covance) for 2 days at 48C. After four rinses with PBS plus 0.3% Triton X-100 and 0.05% Tween-20, corneas were incubated with Alexafluor-488-conjugated donkey anti-rabbit secondary antibody (Ther-moFisher, Grand Island, NY, USA) O/N at 48C. Excess antibody was removed with rinses in PBS. Then, the corneas were flattened onto poly-L-lysine-coated slides and coverslipped using Vectashield mounting media (Vector Labs, Burlingame, CA, USA). Controls were treated similarly with omission of primary antibody or addition of nonspecific IgG.

Corneal Infection and Grading
P. aeruginosa infection of the cornea was performed as described before. 58 Briefly, mice were anesthetized with ethyl ether in a well-ventilated hood. The cornea of the left eye was wounded; 5.0 3 10 6 colony forming units (CFU) of PA (strain 19660; ATCC, Manassas, VA, USA) in a 5 lL volume was topically delivered. Corneal disease was graded at 1, 3, and 5 dpi using an established scale 58 : 0, clear or slight opacity, partially or fully covering the pupil; þ1, slight opacity, covering the anterior segment; þ2, dense opacity, partially or fully covering the pupil; þ3, dense opacity, covering the entire anterior segment; and þ4, corneal perforation. Photography with a slit lamp was used to illustrate disease.

RNA Preparation and Real-Time RT-PCR
Total RNA was prepared using the miRVana miRNA isolation kit (Life Technologies, Foster City, CA, USA) for miRNA studies, or the RNeasy (Qiagen, Frederick, MD, USA) and RNA STAT-60 kit (Tel-Test, Friendswood, TX, USA) for mRNA studies as described previously. 17,52,60 Quantitative (q)RT-PCR for miR-NAs was performed using Taqman miRNA primers and RT-PCR kit (Life Technologies) on a CFX Connect Real-time System (Bio-Rad, Hercules, CA, USA) with snRNA U6 as an endogenous control as described before. 17,60 Quantitative RT-PCR assays of protein-coding genes were performed using QuantiFast SYBR Green RT-PCR kit and QuantiTect primers (Qiagen) with 18s rRNA and b-actin as endogenous controls. 17,19,60

X-Gal Staining
Staining with X-gal was performed as described before. 19 6 , 1 mg X-gal/mL) at 378C O/N. After washing with PBS, corneas were observed and photographed under a dissection scope (Accu-scope, Inc., Comack, NY, USA). Then, for whole-mount microscopy, the cornea was flattened by four evenly spaced cuts from the periphery toward the center corneas and mounted on Superfrost plus slides (ThermoFisher); for cross-sections, the cornea was soaked in 20% sucrose in 0.1M PB pH 7.4 O/N and mounted in optimum cutting temperature (OCT; Fisher Healthcare, Houston, TX, USA), 12-lm cryoprotected cross-sections were made and examined using light microscopy (Leica DM4000B; Leica Microsystem, Inc., Buffalo Grove, IL, USA).
For TG X-staining, the whole head of a heterozygous mouse was fixed in 4% PFA in 0.1 M PB, pH 7.4 for 2 hours after the skull was opened. Then the head was sequentially soaked in Buffers A and B, stained in Buffer C and postfixed with 4% PFA as described above. Then, the stained head was carefully dissected under a dissection scope to expose the TG.

Enzyme-Linked Immunosorbent Assay
Protein levels for IL-1b and MIP-2 were tested using ELISA kits (R&D Systems, Minneapolis, MN, USA) as described before. 53,63 Briefly, corneas were homogenized in 1 mL PBS with 0.1% Tween-20 and protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA). An aliquot of each supernatant was assayed in duplicate per the manufacturer's instructions. Sensitivities of the ELISA assays were less than 2.31 pg/mL for IL-1b, and less than 1.5 pg/mL for MIP-2.

Quantitation of Corneal PMN
A myeloperoxidase (MPO) assay was performed as described before. [64][65][66][67] Briefly, corneas were harvested, homogenized in 1 mL potassium phosphate buffer (50 mM, pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide (Sigma-Aldrich Corp., St. Louis, MO, USA) using glass microtissue grinders. The samples were freeze-thawed four times and centrifuged at 21,000g for 10 minutes. To measure MPO, a 100-lL supernatant was added to fresh 2.9 mL o-dianisidine dihydrochloride substrate buffer (16.7 mg/mL; Sigma-Aldrich Corp.) with 0.0005% hydrogen peroxide. The change in absorbance at 460 nm was read every 30 seconds for 5 minutes on a Hekios a spectrophotometer (ThermoFisher). Afterward, units of MPO per cornea were calculated: 1 unit of MPO activity is equivalent to approximately 2 3 10 5 PMN. 68 Quantitation of Viable Bacteria Bacteria were quantitated as described before. [64][65][66][67] Briefly, each cornea was homogenized in 1.0 mL sterile saline containing 0.25% BSA; 0.1 mL corneal homogenate was serially diluted 1:10 in the same solution and selected dilutions were plated in triplicate on Pseudomonas isolation agar (Becton Dickinson, Sparks, MD, USA). Plates were incubated O/N at 378C and the number of viable bacteria counted. Results are reported as log 10 number of CFU/cornea 6 SEM.

In Vitro Transfection and Phagocytosis and Intracellular Killing Assays
A total of 4 3 10 5 RAW264.7 M/ (a cell line derived from the mouse; ATCC) were plated in a 48-well plate and transfected with anti-miR-183/anti-miR-96/anti-miR-182 (10 nM for each) or negative control anti-miR oligonucleotides with scrambled sequences (30 nM; Exiqon, Vedbaek, Denmark) and miRNA mimics of miR-183, miR-96, and miR-182 (10 nM for each) or negative control mimics (30 nM; ThermoFisher) using RNAi-Max Lipofectamine (ThermoFisher) as described before. 69 All experiments were conducted with four replicate wells. Fortyeight hours after transfection, 1 3 10 7 CFU PA (strain 19660)/ well was added. One hour after coculture at 378C, gentamicin (300 lg/mL) was added to kill extracellular bacteria. Thirty minutes after gentamicin treatment, cells were washed with 250 lL Hanks' balanced salt solution (HBSS) twice to remove gentamicin and dead extracellular bacteria. For the phagocytosis assay, cells were lysed in 250 lL ice-cold sterile water. The cell lysate was plated on PA isolation agar plates (Becton Dickinson) and incubated at 378C for 24 hours as described before. 64 Colonies were manually counted; CFU/mL of cell lysate was calculated based on dilution factors. For the intracellular killing assay, fresh media was added to the wells after washing with HBSS to remove gentamicin and dead extracellular bacteria, and then cells were incubated for an additional hour before being washed, lysed, and plated. Killing efficiency was calculated as ([CFU/mL at 1 hour] À [CFU/mL at 2 hour])/(CFU/mL at 1 hour).

Mouse Peritoneal PMN Isolation and Ex Vivo Functional Assay
To harvest peritoneal PMN, mice were injected intraperitoneally with 1 mL 9% casein solution 27 and 3 hours before harvesting; a standard peritoneal PMN isolation protocol was followed as described previously. 64,67-70 A total of 4 3 10 5 PMNs were plated on a 48-well plate and infected with 1 3 10 7 CFU PA (strain 19660). Two hours after infection, gentamicin (300 lg/mL) was added to kill extracellular bacteria. For the phagocytosis assay, 30 minutes after gentamicin treatment, cells were washed, lysed, and plated on PA isolation agar plates as described above. For the intracellular killing assay, cells were incubated for an additional 2 hours after washing; then, cells were lysed and plated. Killing efficiency was calculated as ([CFU/mL at 2 hour] À [CFU/mL at 4 hour])/(CFU/mL at 2 hour).

Statistical Analysis
The difference in clinical score between groups was tested by the Mann-Whitney U test. One-way ANOVA with Bonferroni test (GraphPad Prism; La Jolla, CA, USA) was used to analyze qRT-PCR data of proinflammatory neuropeptides and cytokines. An unpaired, two-tailed Student's t-test was used to determine the significance of qRT-PCR of miRNAs, ELISA, MPO, and plate count data 17,19,60 ; P < 0.05 was considered significant. Each experiment was repeated at least twice to ensure reproducibility and data from a representative experiment are shown. Quantitative data are expressed as the mean 6 SEM.

MicroRNA-183/96/182 is Expressed in the Cornea and TG and Is Inactivated in miR-183C GT/GT Mice
Quantitative RT-PCR analysis showed that all three members of miR-183/96/182 are expressed in the cornea of wt mice (Fig.  1A). Among them, miR-182 and miR-183 showed higher expression than miR-96 (~32-and 4-fold higher, respectively) (Fig. 1A), suggesting that miR-182 and miR-183 play major roles in the cornea. Consistently, miR-183/96/182 is also expressed in human cornea with a similar pattern as in the mouse (Fig.  1B).
In miR-183C GT/GT mice, miR-183/96/182 is inactivated in the cornea. In heterozygous mice, expression of both miR-182 and miR-183 were significantly decreased to approximately one-half of that in wt mice, suggesting a dosage effect because heterozygous mice carry only one wt allele of the miR-183/96/ 182 gene (Fig. 1A).
Because the b-gal cassette in the miR-183C GT allele follows the endogenous expression pattern of miR-183/96/182, 19 to test its spatial expression pattern, we performed X-gal staining in the cornea of miR-183C GT/þ mice. Our results showed that miR-183/96/182 is mainly expressed in the corneal epithelium (Figs. 1C-F). A punctate staining pattern in the corneal epithelium (Fig. 1F) suggested that the expression of miR-183/96/182 may be due to staining of corneal nerve endings.

Inactivation of miR-183/96/182 Resulted in Decreased Nerve Density and Decreased Expression of Proinflammatory Neuropeptides in the Cornea
Sensory innervation of the cornea is from the TG. [38][39][40] To test miR-183/96/182 expression in SN of the TG, we performed X-gal staining on miR-183C GT/þ mouse brain, and showed that miR-183/96/182 is highly expressed in the TG (Figs. 2A, 2B). Quantitative RT-PCR further confirmed its expression in TG (Fig. 2C). Similar to the cornea, miR-182 and miR-183 are predominately expressed in the TG, compared with miR-96 (~16-and 8-fold higher, respectively) (Fig. 2C). MicroRNA-183/96/182 is inactivated in the TG of miR-183C GT/GT mice. In the TG of heterozygous miR-183C GT/þ mice, miR-183 is significantly decreased to approximately one-half of that in wt mice; miR-182 expression is also reduced, although did not reach significance (Fig. 2C). Similarly as in the cornea, these data suggest that miR-183/ 96/182 expression in TG also has a dosage effect based on numbers of wt alleles (Fig. 2C).
To test whether inactivation of miR-183/96/182 has significant impact on corneal innervation, we performed immunofluorescence analysis using an anti-b-III tubulin antibody, a marker for neurons. Our results showed that, in ko animals, corneal nerve density is drastically decreased; the normal whorl-like pattern of the subbasal nerve plexus 39,71-77 is disrupted (Fig. 2D). Local axon enlargements or ''beads,'' which contain high densities of mitochondria and share similarities to nociceptor terminals, 38,39 are decreased in ko compared with wt cornea (Fig. 2D), suggesting that inactivation of miR-183/96/182 has significant consequences on corneal sensory innervation. Consistent with this hypothesis, qRT-PCR using corneal RNA of ko and wt mice showed decreased expression of nociceptor transient receptor potential vanilloid 1 (TRPV1) and SN-released proinflammatory neuropeptides Tac1, the precursor gene for substance P (sP),

Inactivation of miR-183/96/182 Resulted in Significant Decrease of Corneal Inflammatory Response to PA Infection and Severity of PA-Induced Keratitis
Because inactivation of miR-183/96/182 resulted in decreased nerve density and proinflammatory neuropeptides, we hypothesized that this disrupts neuroimmune cross-talk and modulates immune/inflammatory responses in the cornea. To test this hypothesis, the cornea of ko mice and their wt littermates were infected with PA. Our results showed that miR-183/96/182 ko mice had significantly decreased keratitis at 1, 3, and 5 dpi (Fig. 3A). Most of the ko mice had only slight corneal opacity; none of the corneas perforated (Figs. 3A, 3B). Quantitative RT-PCR analysis of the cornea at 5 dpi showed that many proinflammatory factors and their receptors, which have been shown to play important roles in PA-induced keratitis, were decreased in the infected cornea of ko mice (Figs. 3C-J). In wt animals, pattern-recognition receptor Toll-like receptor 4 (TLR4), IL-1b, IL-6, IL-12p40, chemokine (C-C motif) ligand 2 (CCL2) and its receptor CCR2, and intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) were increased in the infected eyes compared with noninfected contralateral eyes (Figs. 3C-J); whereas in ko mice, increased expression of these proinflammatory factors in infected eyes was mitigated (Figs. 3C-J). Expression levels of most of these inflammatory factors were decreased in the infected eyes of ko mice when compared with their wt littermates (Figs. 3C-J). Enzyme-linked immunosorbent assays demonstrated that, at the protein level, IL-1b was significantly decreased at 1 dpi, whereas macrophage inflammatory protein-2 (MIP-2) was decreased at both 1 and 5 dpi (Figs. 4A, 4B). These results suggest that inactivation of miR-183/96/182 resulted in a decreased inflammatory response in infected corneas and contributed to decreased severity of PA-induced keratitis.
Polymorphonuclear neutrophils are the predominant infiltrating immune cells in bacterial keratitis and play important roles in bacterial clearance. [53][54][55][56][57] To begin to understand the pathological processes involved, we first performed MPO assays to quantify PMN in the cornea. Our results showed that PMN in the infected cornea of ko animals were significantly decreased at 1 dpi and slightly more decreased at 5 dpi. This result is consistent with the observation of decreased expression of chemoattractant neuropeptides (e.g., sP) (Fig. 2E) and cytokines (e.g., CCL2 and MIP-2), and leukocyte adhesion molecules, ICAM1 and VCAM1 (Figs. 3G, 3I, 3J, 4B).
To gain further insight into the progression of pathological processes, we quantified viable bacteria in the infected cornea. Intriguingly, our results showed that bacterial load was increased in the infected cornea of ko compared with wt mice at 1 dpi. This observation is consistent with decreased PMN infiltration in ko animals at this time. However, at 5 dpi, bacterial load in the corneas of wt controls was further increased when compared with 1 dpi; whereas the bacterial load in ko animals was decreased at 5 dpi compared with 1 dpi, and was significantly lower than wt controls. These results suggest that, although they had a higher bacterial burden at 1 dpi, bacteria continued to be cleared more efficiently in ko mice than in wt controls.

DISCUSSION
Bacterial keratitis is a potentially blinding corneal infectious disease and PA is the most commonly recovered causative organism in contact lens-related cases. PA-induced keratitis is also one of the most rapidly developing and destructive diseases of the cornea. 34,35 However, the roles of miRNAs in bacterial keratitis are still largely unknown. In this regard, Yang et al. 37 recently provided evidence that miR-155 modulates the   By X-gal staining and qRT-PCR analyses, we demonstrated that miR-183/96/182 is highly expressed in mouse cornea, with miR-182 and miR-183 predominately expressed, suggesting their major roles in the cornea (Fig. 1). Similar to mouse cornea, miR-183/96/182 is also expressed in human cornea, suggesting clinical relevance to the findings in mice. In the cornea, its expression is mostly in the epithelium with a unique punctate pattern, suggesting that its expression in the cornea is likely in the nerve endings (Figs. 1C-E). Consistently, miR-183/ 96/182 is highly expressed in the TG, where the SN to the cornea reside, with similar ratios among the three miRNAs in the cornea ( Figs. 2A-C). Inactivation of miR-183/96/182 resulted in a decrease of corneal nerve density and beaded nociceptor terminals (Fig. 2D) and decreased expression of TRPV1 and proinflammatory neuropeptides, including sP, CGRP, and Cx3cl1 (Figs. 2E-H). It has been shown that sPand CGRP-expressing SN coexpress TRPV1 40,78 ; activation of TRPV1 in sensory nerve endings by capsaicin results in calcium influx-dependent neuropeptide release, including sP and CGRP. 79,80 Therefore, our data suggest that decreased sensory innervation contributes to the decreased release of proinflammatory neuropeptides in the cornea and inflammatory responses. However, we do not exclude the possibility that decreased production of these neuropeptides from other sources (e.g., the corneal epithelium and tears) 38 also contributed to the overall decreased expression of proinflammatory neuropeptides.
Toll-like receptor 4 is a classic member of the pathogenrecognition receptor TLR family and functions as a primary sensor to detect gram-negative bacteria by binding to bacteriaderived lipopolysaccharides (LPS) expressed in bacterial cell walls. 81 As previously described, 82 PA infection induces TLR4 expression in the cornea of wt mice (Fig. 3D); however, intriguingly, in miR-183/96/182 ko mice, PA infection in the cornea did not result in upregulation of TLR4 (Fig. 3C); the level of TLR4 in the infected cornea of ko mice was significantly decreased compared with wt controls (Fig. 3C). Toll-like receptor 4 is expressed in TRPV1-containing trigeminal SN. 83 It has been shown that LPS binding sensitizes TRPV1 and increases neuropeptide release from SN. 84 Therefore, decreased expression of TLR4 in the cornea of miR-183/96/182 ko animals may contribute to decreased neuropeptide release.
The Cx3cl1, also known as fractalkine and a ligand for Cx3cr1, is a neuronally derived proinflammatory neuropeptide. 85 In the peripheral nervous system, dorsal root ganglionreleased Cx3cl1 promotes microglial production of inflammatory factors and contributes to chronic pain. [86][87][88][89][90] In cornea, Cx3cl1/Cx3cr1 signaling mediates direct physical association between corneal nerves and resident M/, which is suggested to act as an indicator of malfunctioning neuroimmune All data are relative expression levels normalized to snRNA U6. nd, not detected. * All data are presented as average (6 SEM). communication in diseases. 44 Decreased expression of Cx3cl1 (Fig. 2G) may have compromised neuroimmune communication in the cornea and contributed to the changes of the overall corneal response to PA infection (Fig. 3). Polymorphonuclear neutrophils are the predominant infiltrating cells in PA keratitis and are essential for microbial clearance. [53][54][55][56][57] Our data showed that the number of PMNs was significantly decreased (by~50%) in the infected cornea of ko mice at 1 dpi (Fig. 4C), resulting in decreased bacterial clearance and increased bacterial load (Fig. 4D). Substance P has been shown to be a potent chemoattractant and to induce leukocyte activation, NF-jB activation, cytokine production, and plasma extravasation. [91][92][93][94][95] Decreased expression of sP (Fig.  2E), together with decreased expression of proinflammatory cytokines, including multiple chemoattractant molecules (e.g., CCL2), also known as monocyte chemoattractant protein (MCP)-1 (Fig. 3G), ICAM1 (Fig. 3I), and MIP-2 (Fig. 4B), may have contributed to decreased recruitment of PMNs, and possibly M/, to the infected cornea. 53,56,[96][97][98] Intriguingly, at 5 dpi, bacterial load in the cornea of ko mice was significantly decreased compared with wt controls (Fig.  4D), whereas the number of PMNs showed no significant difference between ko and wt cornea (Fig. 4C). This cannot be explained by the decreased corneal nerve density and proinflammatory peptides. An alternative hypothesis is that inactivation of miR-183/96/182 has direct impact on PMN function and other components of the innate immune system. Supporting this hypothesis, our studies confirmed that miR-183/96/182 is expressed in M/ and PMNs in both mice and humans ( Fig. 5; Table). More importantly, our in vitro and ex vivo data showed that inactivation or knockdown of miR-183/ 96/182 in M/ and PMNs resulted in a significant increase in their capacity for phagocytosis and intracellular killing of PA ( Fig. 6). Therefore, in spite of the decreased number of PMNs and M/ in the infected cornea, bacteria were efficiently cleared without excessive collateral damage to the cornea, leading to decreased keratitis. Substance P has also been shown to be a potent anti-apoptosis regulator. 99,100 During PA infection, sP delays apoptosis of PMNs 58 ; persistence of PMNs is associated with increased corneal damage and perforation. [53][54][55][56][57][58] Therefore, decreased sP in the infected cornea of the ko animals could result in increased apoptosis of PMNs and contribute to decreased severity of keratitis.
In addition to the innate immune system, emerging evidence suggests that miR-183/96/182 is expressed in the adaptive immune system. [29][30][31] Although in vitro and ex vivo data suggest potential roles of miR-183/96/182 in modulating adaptive immune functions, including clonal expansion of activated Th cells 29 and Th2-induced Treg, 30 other reports 31 and our unpublished data (Xiao C, Xu S, written communication, 2016) do not support significant functions of miR-183/96/ 182 in adaptive immunity in vivo. Further investigation is needed to determine whether inactivation of miR-183/96/182 affects adaptive immunity and whether this impacts the corneal response to PA keratitis.
Overall, our data suggest that miR-183/96/182 is important in the corneal response to PA infection through its regulation of both sensory innervation of the cornea and innate immune functions at multiple levels in the pathogenesis of PA-induced keratitis (Fig. 7). The impact of inactivation of miR-183/96/182 on innate immune functions plays a major role in modulating the corneal response to PA infection and development of keratitis. It will be worthwhile to further tease out the effects of the developmental defects leading to a disrupted pattern of corneal innervation from the effects of gene ablation on innate immunity per se. To our knowledge, our data provide the first evidence that miR-183/96/182 plays an important role in innate immunity and modulates phagocytosis and bacterial killing capacities of both PMNs and M/. This finding has important implications to a wide range of diseases in which innate immunity has a critical role to disease outcome. It is also the first report that an miRNA cluster has a significant functional role in both sensory and innate immune systems. These results lead us to suggest that miR-183/96/182 represents a new therapeutic target for treatment of bacterial keratitis.


extfieldsofstudy: Biology Medicine

text:
Zebrafish heart as a model for human cardiac electrophysiology

ABSTRACT The zebrafish (Danio rerio) has become a popular model for human cardiac diseases and pharmacology including cardiac arrhythmias and its electrophysiological basis. Notably, the phenotype of zebrafish cardiac action potential is similar to the human cardiac action potential in that both have a long plateau phase. Also the major inward and outward current systems are qualitatively similar in zebrafish and human hearts. However, there are also significant differences in ionic current composition between human and zebrafish hearts, and the molecular basis and pharmacological properties of human and zebrafish cardiac ionic currents differ in several ways. Cardiac ionic currents may be produced by non-orthologous genes in zebrafish and humans, and paralogous gene products of some ion channels are expressed in the zebrafish heart. More research on molecular basis of cardiac ion channels, and regulation and drug sensitivity of the cardiac ionic currents are needed to enable rational use of the zebrafish heart as an electrophysiological model for the human heart.

Introduction
Zebrafish, a tropical teleost fish species is increasingly used as a model for human cardiac electrophysiology, arrhythmias and drug screening. 6,12,53,56 The use of zebrafish models is based, besides the several well established technical advantages (optical transparency, large offspring number, rapid development, genetic amenability), on the promise that genetic and molecular mechanisms behind cardiac physiologies are conserved throughout the vertebrate evolution from fishes to humans. 32 Indeed, about 71% of human genes have at least one ortholog in the zebrafish genome. 23 On the other hand evolution has resulted in enormous diversity of life-forms in fishes via genetic adaptation to different habitats. This diversity is largely based on the whole genome duplication early in the teleost lineage and subsequent subfunctionalization, neofunctionalization and loss of genes, and reorganization of gene regulation. 57 As a result, over 3100 human genes have at least 2 orthologues in the zebrafish genome and both species have several thousand unique genes without an ortholog in each other's genome. 23 Therefore, design of experiments and interpretation of the results cannot be based on the assumption that molecular entities and regulatory pathways behind the functions under study are always the same in zebrafish and humans. In order to draw correct conclusions from the zebrafish model to human cardiac electrophysiology, ion channel functions that are shared must be discriminated from those that are derived and novel in zebrafish and humans. 7 The recent analysis of the zebrafish Kir2 channel composition provides a striking example how different the molecular basis of cardiac ionic current (I K1 ) can be in zebrafish and human hearts. 17 This finding warrants a careful examination of other cardiac ionic currents as well. 44 This overview examines the current state of knowledge on the zebrafish cardiac ion channels with relation to the human cardiac ion channels. plateau phase, similar to the human cardiac AP, and consequently a distinct QT-interval in the electrocardiogram (Figs. 1 and 2). 29,63 All phases (0-4) of the cardiac AP, with the exception of the rapid phase-1 repolarization, are present in the zebrafish heart (Fig. 1). 40 The absence of the phase-1 repolarization suggests that the transient outward current (I To ) is tiny or absent in the zebrafish heart. 2 Furthermore, it should be noted, that as an ectotherm the duration of cardiac AP in zebrafish varies with body temperature within its thermal tolerance range between 6.2 C and 41.7 C. 34 In comparison to the hearts of endothermic vertebrates, ion channel function in ectothermic fishes is scaled down to lower temperatures. The duration of zebrafish ventricular AP at 19 C is similar to the human ventricular AP at 37 C, while at the human body temperature the duration of the zebrafish ventricular AP is only about one fifth of the duration of the human cardiac AP (Fig. 1).
Inward rectifier K C current (I K1 ) The cardiac inwardly rectifying K C current (I K1 ) stabilizes the resting membrane potential (phase-4) and is responsible for shaping the initial depolarization and the final phase-3 repolarization of the cardiac AP. 22 A robust background I K1 is present in atrial and ventricular myocytes of the adult zebrafish, and similar to mammalian hearts, the density of I K1 is markedly higher in ventricular than atrial myocytes. 40 There is, however, remarkable differences in cardiac I K1 channel composition between human and zebrafish hearts. The human cardiac I K1 is produced by Kir2.1, Kir2.2 and Kir2.3 channels. Kir2.1 channel forms about 50% of the Kir2 transcripts in the right ventricle, 14 while in the right atrium Kir2.3 is the main Kir2 isoform (56%) with less contributions by Kir2.2 (31%) and Kir2.1 (13%) channels. 14 In the zebrafish heart 6 Kir2 channel isoforms are expressed at the transcript level. In the ventricle an ortholog to the mammalian Kir2.4 channel is the main Kir2 channel isoform constituting 92.9% of the total Kir2 population. In the zebrafish atrium, Kir2.2a and Kir2.4 form 64.7% and 29.3% of the Kir2 transcripts, respectively. 17 Notably Kir2.1a, Kir2.1b and Kir2.3 together form less than 6% and 1% of the Kir2 transcripts in zebrafish atrium and ventricle, respectively. Thus, the Figure 1. Ventricular action potential of the zebrafish heart. (A) Typical action potential of the zebrafish ventricle shows fast upstroke (phase-0), long plateau (phase-2), rapid repolarization (phase-3) and stable resting membrane potential (phase-4), but lacks the fast phase-1 repolarization (arrow), which is typical for the human cardiac action potential (top right). As an ectotherm action potential duration and heart rate in the zebrafish is temperature-dependent within the thermal-tolerance range of the species. (B) Representative microelectrode recordings of ventricular AP at selected temperatures (36 C, 28 C and 19 C) and (C) within the whole range of temperatures between 10 C and 36 C.
Kir2 composition of the zebrafish heart is dominated by an isoform, which in mammals is hardly expressed in cardiac myocytes. 13,24,54 There are also functional differences between zebrafish and mammalian Kir2 channels. The zebrafish Kir2.4 is about 2 orders of magnitude more sensitive to Ba 2C block than its mammalian counterpart. On the other hand zebrafish Kir2.1a is almost an order of magnitude less sensitive to Ba 2C block than the mammalian Kir2.1 channels. 17 Delayed rectifier K C currents (I Kr , I Ks ) The two major repolarizing K C currents, that end the long plateau phase of the human cardiac AP, are I Kr and I Ks . They are active during the phases 2 and 3 of the cardiac AP. I Kr is the main repolarizing current of the human heart, while I Ks comes into a play when more repolarizing reserves are needed, e.g. under b-adrenergic activation and with increasing heart rates. 50 In the human heart the pore forming component of the I Kr channel (human erg1 or herg) is encoded by the KCNH2 gene 49,59 and the functional I Kr channels are heterotetramers of the 2 splice variants of the KCNH2 gene, herg1a and herg1b. 26 I Kr is also the main repolarizing K C current in atrial and ventricular myocytes of the zebrafish heart. 40,60 Interestingly, in the zebrafish heart I Kr is not produced by erg1 channels but by erg2 channels, which are encoded by the zebrafish ortholog to the mammalian KCNH6 gene. 30 In the zebrafish heart 4 erg genes are expressed (KCNH6, KCNH2A, KCNH2B and KCNH7), KCNH6 being clearly the dominant isoform (99.1 § 0.15 and 99.4 § 0.08% of the transcripts in atrium and ventricle, respectively) ( Fig. 3). In mammals erg2 is expressed in the nervous tissue but not in the heart, while in zebrafish erg1 is very weakly expressed in the heart. 30 Thus, the I Kr is generated by non-orthologous genes in human (erg1) and zebrafish (erg2) hearts.
Biophysical properties of the human erg1 and the zebrafish erg2 also differ. Steady-state activation and inactivation of the zebrafish I Kr are ¡15 mV and C23 mV, respectively, different from the respective values of the human I Kr , when measured in the same cellular environment (Xenopus oocytes). Furthermore, the presence of herg1b splice variant in the channel assembly affects drug sensitivity of the human I Kr . 1,27,38 Mutations in the zebrafish erg channel cause similar electrophysiological features that are typical for short QT and long QT syndromes in humans. 3,16 QT-prolonging drugs induce AP prolongation, bradycardia and atrioventricular conduction block in zebrafish embryos. 28,39 Different from the human heart, there is no direct demonstration on the presence of I Ks in the zebrafish cardiac myocytes. 2,40 The mammalian cardiac I Ks is generated by a heteromultimeric assembly of K v 7.1 (KCNQ1 gene) a-subunits and minK (KCNE1 gene) b-subunits. Although I Ks has not been reported for zebrafish cardiac myocytes, 2,40 transcripts of the zebrafish orthologues to the mammalian KCNQ1 and KCNE1 genes are expressed in the zebrafish heart. 62 Experiments with another fish species (Carassius carassius) of the zebrafish family (Cyprinidae) suggest that, when present, the fish cardiac I Ks is mainly produced by homotetramers of K v 7.1 channel without the MinK b-subunit and with distinctly different electrophysiological properties. 20 Unlike the mammalian I Ks , the fish cardiac I Ks has fast activation kinetics, is independent of stimulation frequency, is not augmented by cAMP-dependent pathway and is less sensitive to chromanol 239B block than its mammalian counterpart. 20 Similar to the human heart also other members of the K v 7 channel subfamily (KCNQ2-4) are expressed to some extent in the zebrafish heart. 35,62 Calcium currents Ca 2C currents (I Ca ) maintain the long plateau phase of cardiac APs (phase-2) and provide activator Ca 2C for contraction. Atrial and ventricular myocytes of the zebrafish heart have both T-type (I CaT ) and L-type (I CaL ) Ca 2C currents, 5,8,40,47 while in adult mammalian heart the presence of I CaT is mainly restricted to the sinoatrial node and the conductive pathways. Notably, the density of I Ca,L is markedly larger in zebrafish ventricular myocytes than in human cardiomyocytes. 65 In mammals, there are 4 alpha1 subunits of the Ltype Ca 2C channels (a1S, a1C, a1D, a1F, or Ca v 1.1-4) Ca v 1.2 being the dominant cardiac isoform. 37 In the zebrafish heart ventricular contraction is abolished by mutation in the a-1C subunit (Ca v 1.2) strongly suggesting that I CaL is produced by orthologous genes in humans and zebrafish. In the heart of adult zebrafish transcripts of the a-1D (Ca v 1.3) are also expressed. 52 I CaT is an important component of pacemaker and conductive tissues, but usually absent in atrial and ventricular muscle of adult mammals. In this respect zebrafish is clearly different from humans and other mammals. Two a subunits of T-type Ca 2C channels (Ca v 3.1 and Ca v 3.2) are expressed in mammalian hearts. 11,43 T-type channel composition of the zebrafish has not been studied, but Ni 2C sensitivity suggests that it might be an ortholog to the mammalian Ca v 3.1, 40 while immunofluorescence findings suggest the presence of Ca v 3.2. 2 The diversity of Ca 2C channel a-subunits is, however, much higher in fishes than mammals due to the whole genome duplication in the teleost lineage. Indeed in fugu (Fugu rubribes) 21 Ca 2C channel a-subunits have been found and as many as 16 of them are expressed in the heart. 61

Sodium currents
The voltage-gated Na C channels have a central role in excitability of myocardial cells, because they generate the rapid upstroke of the myocardial AP (phase-0) and determine the velocity of impulse transmission over the heart. 15,46 A fast Na C current (I Na ) exists in atrial and ventricular myocytes of the zebrafish heart. 40,60 The rate of AP upstroke is slower in zebrafish atrial and ventricular muscle in comparison o human heart suggesting that I Na density is significantly lower in the zebrafish heart. 40 In cultured cardiac myocytes of the zebrafish embryos, I Na current density is equal in atrial and ventricular myocytes, but the atrial I Na activates at slightly more negative voltages in comparison to the ventricular I Na . 60 In larval cardiac tissue (72 hpf) of the zebrafish 2 a-subunits of Na C channels (SCN5Laa and SCN5Lab), orthologous to the human cardiac SCN5A, are expressed. 42 However, in the adult zebrafish heart only scn5Lab has been found. 9 In contrast to the mammalian cardiac Na C channel (SCN5A) the Na C channels of the fish heart are about 3 orders of magnitude more sensitive to tetrodotoxin (TTX). 21 This is due to the replacement of non-aromatic cysteine (C401) in the poor loop of the domain-I with an aromatic tyrosine (Y401) in the fish cardiac Na C channels. Since this replacement is also present in both paralogs of the zebrafish cardiac Na C channel, the zebrafish I Na is also highly sensitive to TTX. 9,40,58 Association of the zebrafish SCN5Lab with its beta1subunit increases I Na amplitude when expressed in the CHO cell line. 10 Collectively these findings suggest that the paralogs of the zebrafish cardiac Na C channels are orthologous to the main human cardiac isoform, but different from the mammalian I Na the zebrafish cardiac I Na is TTX-sensitive.

Other ionic currents
Practically nothing is known about the ligand-gated inward rectifiers currents, the ATP-sensitive K C current (I KATP ) and the acetylcholine-activated K C current (I KACh ) of the zebrafish heart. I ATP has been recorded from cardiomyocytes of some fish species and therefore it is likely that similar current exists also in zebrafish cardiac myocytes. Three ATP-sensitive channels have been found in the zebrafish genome (Kir6.1, Kir6.2 and Kir6.3) located in different chromosomes. 64 Synteny data suggests, however, that the Kir6.3 is in fact a paralogue of the Kir6.2 (i.e. Kir6.2b) ( Table 1). In mammals the conductance pore of ATPsensitive channels is produced by Kir6.1 and Kir6.2 channels, which assemble with sulfonylurea receptors to produce fully functional channels. No data is available on the expression of these channels in the zebrafish heart. In the goldfish (Carassius auratus) heart Kir6.1and Kir6.2 channels are expressed.
Acetylcholine induces a large inwardly rectifying K C current in atrial but not in ventricular myocytes of the zebrafish heart. 40 However, the molecular basis of this current has not yet been resolved.
Pacemaker current (I f or I h ) has been demonstrated to be present in cultured cardiac myocytes of embryonic zebrafish as well as in cultured atrial and ventricular myocytes of the adult zebrafish. Down-regulation of the I h by a recessive slo mo mutation (which probably encodes a mitochondrial protein) causes depression of intrinsic heart rate, especially in the embryonic heart. 60 The zebrafish I h consist of slow and fast components suggesting that it is produced by 2 different channels similar to the human heart. Pacemaker tissue of the zebrafish heart demonstrates the presence of HCN4 channels as one of the likely candidates for coding the I h channels. 55

Conclusions and implications
Considering the increasing use of zebrafish as a model for human cardiac electrophysiology, cardiac arrhythmias and drug screening, our knowledge on the zebrafish cardiac ion channels, their biophysical properties, drug sensitivity, molecular basis and regulatory networks is still limited. The current knowledge indicates that human and zebrafish myocytes share several electrical properties, but on the other hand several differences also exist between human and zebrafish cardiac APs, ion channel function and ion channel molecular composition ( Table 2). Zebrafish and human hearts both have a distinct plateau phase. In this respect the zebrafish heart is clearly a better model than the murine heart, which is characterized by a short AP plateau and strong reliance on I To in AP repolarization. 25,41 The ionic current basis of human and zebrafish cardiac APs is similar in that I K1 and I Kr are the major repolarizing currents in both hearts. On the other hand, zebrafish ventricular AP, unlike human cardiac AP, does not have clear phase-1 repolarization suggesting that the transient outward current (I To ) is not expressed in the zebrafish heart. 2 Unlike human heart, atrial and ventricular myocytes of the zebrafish heart have both I CaT and I CaL . I K1 and I Kr are generated by different gene products in humans and zebrafish, and I Ks may be absent from the zebrafish heart.
A number of excellent techniques have been developed for recording electrical and mechanical activity of the zebrafish heart in vivo and in vitro, thereby making this small fish amenable for cardiac studies. 4,31,33,63 Effects of disease causing mutations and pharmaceutical drugs on electrical excitation, rhythm and contraction of the zebrafish heart can be traced with high accuracy, making it putatively an interesting model for human cardiac electrophysiology. Due to the presence of ion channel paralogues in the zebrafish The main ion channel isoforms (if known) are indicated in bold. Ã I Ks current has not been found in the zebrafish heart; ÃÃ I KATP current has not been measured in the zebrafish heart; NK, not known The balance of inward currents: density of I Na low and density of I Ca high in comparison to the human heart genome, differences in ion channel proteins and subunit composition of ion channels between zebrafish and human hearts, zebrafish is unlikely to be a good general-purpose model for the human cardiac electrophysiology. However, similarity of the major repolarizing (I K1 , I Kr ) and depolarizing (I Na , I CaL ) currents, despite of their partially different molecular basis, makes the zebrafish heart an appropriate model for more specific issues of human cardiac excitation. Because various Kir2 channels and different erg channels are mutually similar in function, i.e., all Kir2 channels are strong inward rectifiers and all erg channels show fast inactivation, fast recovery from inactivation and slow deactivation, 36 species-specific differences in channel compositions may not be critical, when studying disease causing mutations or using functional knock-downs and knock-outs. The same applies to the inward currents, I Na and I CaL . However, a prerequisite for these studies is that the ion channel composition of the zebrafish heart under study is known and correct channel paralogs and isoforms are targeted.
The special characteristics of the zebrafish cardiac ionic currents are more problematic in drug screening since different ionic currents, even if qualitatively similar to human counterparts, may show significant quantitative differences in drug affinity due to their different molecular basis. 1,18 Another factor that complicates the use of zebrafish model in selection of candidate molecules for medicinal drug development or in toxicological testing is the total composition of inward and outward currents underlying the cardiac AP, which differs qualitatively (e.g, absence of I To and I Ks ) and quantitatively (I Na , I CaL ) from that of the human heart. In risk/benefit assessment of new drug molecules, more emphasis is currently placed on the potential proarrhythmic effects of pharmaceuticals. According to the current research paradigm of safety pharmacology, proarrhythmic potential of the drugs should be look at the level of multiple ion channels (I Kr , I CaL , I Na , I Ks and I To ) instead of one particular ion channel, since drug molecules may bind to several targets. 45,48 In this experimental approach the total repolarizing reserve of outward currents and the total depolarizing reserves of inward currents is considered decisive in estimating the proarrhythmic liability of pharmaceuticals. 27 Because of the qualitative and quantitative differences in inward and outward currents between zebrafish and human hearts, zebrafish heart and cardiac myocytes are likely to pose problems in nonclinical safety pharmacology and toxicology, especially in regard to arrhythmia liability of drugs.
Clearly, more research on molecular basis of cardiac ion channels, and ion channel regulation and drug sensitivity of cardiac ionic currents are needed to enable rational use of the zebrafish heart as an electrophysiological model for the human heart.

Animals
Zebrafish were reared at C28 C. All experiments were authorized by the National Animal Experimental Board in Finland (permission ESAVI/2832/04.10.07/ 2015).

Action potentials
Ventricular APs of spontaneously beating hearts were recorded with microelectrodes filled with 3M KCl (resistance about 20 mega ohms) in oxygenated external saline solution containing (in mM) NaCl 150, KCl 3.0, MgSO 4 1.2, NaH 2 PO 4 1.2, CaCl 2 2.0, glucose 10.0 and HEPES 10.0 (at pH 7.7). Temperature of the tissue bath was changed at the rate of 3 degrees 10 min ¡1 .

KCNH transcript expression
Atrium and ventricle of the heart were separately snap frozen in liquid nitrogen. Tissues from 5 fishes were pooled for each atrial and ventricular sample (n D 6) and stored at ¡80 C. RNA was extracted by TriReagent (Thermo Scientific) and treated with RNase free DNase to avoid DNA-contamination. First-strand cDNA and -RT-control (reaction containing all other components except the RT-enzyme) were synthesized from each sample using Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Scientific). Each sample was amplified in triplicates using Maxima SYBR Green qPCR Master Mix (Thermo Scientific) ( Table 3) and AriaMx Real Time PCR System (Agilent Technologies) under the cycling parameters: 95 C for 10 min followed by 40 cycles at 94 C for 10 s, 60 C for 20 s and 72 C for 30 s, then 72 C for 5 min. Specificity of amplification was monitored by melting curve analysis from 65 C to 95 C. Transcript levels of KCNH genes were normalized to the expression of DnaJA2 reference gene.

AP
action potential hpf hours post fertilization TTX tetrodotoxin

Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.


text:
How tissue damage MET metabolism: Regulation of the systemic damage response

ABSTRACT Living organisms experience tissue damage from both, the surrounding environment and from inside their bodies. Tissue repair/regeneration is triggered by local tissue injury to restore an injured, or lost, part of the body. Tissue damage results in a series of responses, not only locally but also systemically in distant tissues. In our recent publication, we established a “dual system” that induces spatiotemporal tissue damage simultaneously with gene manipulation in surrounding tissues. With this system, we demonstrated that appropriate regulation of methionine metabolism in the fat body is required for tissue repair in Drosophila wing discs, thus highlighting the importance of systemic damage response (SDR) in tissue repair. This “Extra View” aims to discuss our recent reports that propose methionine metabolism to be an essential part of SDR, together with related topics in several model organisms.

Introduction
Organisms are inevitably affected by their internal and external environments. In multicellular organisms in particular, numerous tissue interactions establish a complex internal environment and are crucial for protection against various stresses. One of the most severe insults to the body is tissue injury. Tissue injury often occurs from the outside because of physical injury. A wound is defined as a disruption of the outer physical barrier, causing not only a local wound response (LWR) but also a systemic wound response (SWR). Green and Ryan observed that when potato or tomato leaves were injured either physically or by herbivore attack, proteinase inhibitors accumulated not only at the site of injury but also in uninjured leaves. 1 Extensive studies in plants have identified systemic defense mechanisms that underlie this wound response. When tomato leaves are injured, the jasmonic acid biosynthetic pathway is locally activated at the wound site. Jasmonic acid then acts as a mobile wound signal and travels to other parts of the body to induce genes that encode plant defensins and anti-microbial peptides, in preparation for the next injury. 2 A systemic response upon tissue injury is not only restricted to the wound but also involves a wide range of tissue damage responses as suggested in various reports, including those discussed below. Thus, we will use the terms systemic damage response (SDR) and local damage response (LDR) in the following sections. SDR is a reasonable homeostatic mechanism for multicellular organisms because the influence of local damage is not always confined to the damaged region (Table 1). For instance, local tissue injury potentially causes infection, body fluid leakage, or chronic pain, which would require the systemic response in an organism. Thus, investigation of SDR is significant for understanding the physiology of multicellular organisms. To elucidate the molecular mechanisms of SDR, it is essential to analyze damaged tissues and responsible tissues separately. Recent development of genetic tools has enabled us to manipulate genes tissue-specifically and tissue-independently. In our recent paper, we established a "dual system" using Drosophila genetics to study tissue repair/regeneration from the viewpoint of SDR. 3 Here, we introduce our recent studies of SDR and methionine metabolism, and discuss related studies and future prospects.
Establishment of a new Drosophila model to study the functional SDR for tissue repair and regeneration The imaginal discs of Drosophila, epithelial sheets fated to become adult organs, are known to have a remarkable ability to repair after tissue damage. [4][5][6] In our study, we established a temporal cell ablation system in imaginal discs by using a temperature-sensitive form of the diphtheria toxin A domain (DtA ts ). 3,7 DtA ts could induce cell death at low temperatures (18 C), but was inactive at high temperatures (29 C) 7,8 , thus enabling us to control the timing of induction of tissue injury by simply placing DtA ts -expressing larvae at 18 C (Fig. 1A). In previous studies, a similar system for spatiotemporal genetic ablation was established, based on the Gal4/UAS/Gal80 ts system. 9,10 The Gal4/UAS system is a budding yeast-based binary expression system for tissue-specific gene manipulation. 11 Gal4 binds to UAS (upstream activating sequence) and induces transcription of downstream target gene. Gal80 ts , a temperature-sensitive Gal4 suppressor, controls the Gal4/ UAS-based gene expression in a temperature dependent manner. 12 Tissue injury can be regulated both spatially and temporally by ectopic expression of cell death-inducing genes using these sophisticated genetic tools. However, simple utilization of Gal80 ts system does not allow the uncoupling of tissue damage induction and the manipulation of genes of interest (i.e. tissue repair genes) using the currently available genetic resources, such as the UAS-RNAi library. Alternative approaches involve using whole body loss-of-function mutations 9,13 , but this cannot tell us specifically where in the organism the gene works. Therefore, the new system is required to test the non cell-autonomous function of the genes/pathways in surrounding tissues.
Drosophila has genetic tools available for tissue-specific gene manipulation with several binary genetic systems in addition to the Gal4/UAS system. Systems such as the LexA 14 or Q systems 15,16 can be utilised independently of Gal4/UAS. The combination of 2 systems makes it possible to control gene A in tissue X while manipulating gene B in tissue Y in the same animal (Fig. 1B). In previous studies, the combination of binary genetic systems have been utilised to screen for functional ligand receptors. For example, the Gal4/ UAS and LexA systems were combined successfully to identify the receptor of Drosophila insulin-like peptide 8 (Dilp8). 17 In our research, we overexpressed DtA ts in imaginal discs to control temporal damage using the Q system, while simultaneously manipulating Gal4/UAS-mediated gene expression in non-ablated tissues (Fig. 1C). Such a "dual system" enables us to search for non-autonomous factors contributing to local tissue repair, even by genome wide RNAi screening, for example.

Involvement of methionine metabolism in SDR
Methionine is an essential amino acid, which must be regularly consumed through our diet. In addition to being a protein constituent, methionine is a precursor of S-adenosylmethionine (SAM), a versatile metabolite required for methylation of many substrates, such as nucleic acids, metabolites, lipids, and proteins, including histones. Methionine, together with ATP, is converted to SAM by a highly conserved enzyme called methionine adenosyltransferase (MAT). In Drosophila, only one SAM synthase (sams) functions to produce SAM ( Fig. 2A). The product of SAMdependent methylation is S-adenosylhomocysteine (SAH), which in turn inhibits methyltransferase activity. Thus, the SAM/SAH ratio can be regarded as a  (18 C) and is inactivated at high temperatures (29 C). Thus, by shifting temperatures, a temporal ablation can be induced. With wing disc specific DtA ts expression, local tissue injury and subsequent tissue repair can be observed in wing discs. The extent of wing regeneration can be estimated by observing the adult wing phenotype. DtA ts : temperaturesensitive form of the diphtheria toxin A domain. (B) Independent tissue-specific gene manipulation by the combination of binary systems. Combination of Gal4/UAS, LexA, or Q system makes it possible to manipulate gene expression in independent tissues. For instance, tissue X specific gene promoter can induce the expression of QF only in tissue X and QF subsequently binds to QUAS, which results in the expression of gene A with no induction of other extrinsic binary system like Gal4/UAS system. Similarly, tissue Y specific gene promoter regulates the expression of gene B through the tissue specific expression of Gal4 and following binding to UAS. (C) Identifying genes required for systemic damage response. Combining a Gal4-based genetic manipulation and a Q-based DtA ts ablation, the systemic factors required for disc repair can be investigated in uninjured and remote tissues. Manipulation of different genes in different tissues can be performed by changing the combination of Gal4 drivers or UAS lines. Together with a UAS-RNAi library, this dual system makes it possible to perform genetic screening for studying the tissue interactions involved in tissue repair. methylation index. SAH is converted to homocysteine (Hcy) by adenosylhomocysteinases (Ahcy). Hcy is either metabolised to cystathionine through the transsulfuration pathway (TSP) via an enzymatic reaction mediated by cystathionine b synthase (CBS), or recycled into methionine. Cystathionine is further converted to cysteine and contributes to redox homeostasis mainly via de novo glutathione (GSH) production. 18,19 A computational estimate suggested that there are approximately 200 putative methyltransferase genes in the human genome. 20 Of these, glycine N-methyltransferase (Gnmt) seems to be the predominant consumer of SAM for converting glycine into sarcosine in liver, a tissue where »85% of methylation reaction takes place. 21 Thus, Gnmt is believed to tightly regulate SAM levels, and the amount of SAM is dramatically increased in Gnmt loss of function mutants. [22][23][24] From transcriptome and metabolome analyses in our previous study, we unexpectedly observed that apoptosis-deficient flies displayed altered methionine metabolism, triggered by necrotic tissue damage in adult wings. 24 Necrotic wings induce Gnmt in the fat body, which contributes to a reduction in SAM and induction of SAH, leading to a decreased methylation index. Gnmt induction in apoptosis-deficient mutants is due to FoxO activation in the fat body, in response to the systemic activation of innate immunity by necrosis in wing epidermal cells. Contrary to our initial assumption that Gnmt induction and a reduced methylation index may contribute to the pathological phenotypes, the decreased SAM levels seem to be an Methionine is converted to SAM by Sams. SAM provides a methyl group for various methyltransferases including Gnmt, and becomes SAH. SAH is further metabolized into homocysteine, which is either regenerated into methionine, or converted into cystathionine through the transsulfuration pathway. SAM also provides an aminopropyl group to synthesize polyamines, such as spermidine and spermine. SAM: Sadenosylmethionine, Sams: SAM synthase, Gnmt: glycine n-methyltransferase, SAH: S-adenosylhomocysteine, CBS: cystathionine b synthase, Ahcy: adenosylhomocysteinases. (B) Enhancement of SAM metabolism in the fat body affects energy wasting and aging. Defects in apoptosis cause necrosis that triggers a systemic immune response including Toll activation in the fat body. dFoxO is activated concomitantly, and induces Gnmt that converts SAM into SAH, possibly functioning as an adaptive response against energy wasting. As aging proceeds, Gnmt is upregulated in the fat body however SAM level is also increased. Enhancing the dFoxO-Gnmt axis rescues agedependent SAM accumulation and extends the lifespan. (C) Fat body methionine metabolism remotely regulates local tissue repair. Local disc injury non-autonomously affects methionine metabolism in the fat body via an unidentified "Help Me" signal. In turn, appropriate regulation of methionine metabolism in the fat body is required for sufficient tissue repair in damaged discs.
adaptive response to suppress the energy wasting in necrotic mutants 24 . FoxO-induced Gnmt is not only observed in necrotic models, but also in various conditions including epidermal physical injury (pricking, Obata and Miura, unpublished), starvation, 24 and aging. 25 Considering that Gnmt is also transcriptionally induced by Nrf2/CncC, 25 a transcriptional factor responsible for either oxidative or xenobiotic stress response, Gnmt-mediated changes in methionine metabolism might be a general mechanism as a part of SDR.
To test whether methionine metabolism is also affected by local tissue damage in our new larval model developed to study systemic control of tissue repair, we quantified the amount of methionine metabolites in the larvae expressing DtA ts in the imaginal discs. As we expected, we found a decrease in methionine and SAM, and an increase in SAH in the fat body after disc ablation, particularly in the early stage of disc repair (6 h after ablation). 3 These data suggest that methionine metabolism in the fat body responds to local tissue damage in wing discs. Notably, Gnmt expression did not change at either the transcript or the protein level following ablation, unlike the necrosis model in adult flies. This implies that SAM consumption by methyltransferases other than Gnmt was up-regulated in the fat body. Instead, DtA ts ablation in wing discs upregulated sams in the fat body, probably as an adaptive response against SAM reduction, which is also similar to the adaptive increase of MAT2A expression observed after partial hepatectomy in the rat. 26 Interestingly, a decline in SAM and an increase in SAH were also observed during liver regeneration. As for rat liver regeneration, hepatic SAM levels fell to about 40% of baseline at 12 and 24 h following partial hepatectomy (PH), which is coincident with an increase in SAH and a decrease in hepatic DNA methylation. 26 This is strikingly similar to the non-autonomous changes seen in the Drosophila fat body during the early stage of disc repair, although mammalian liver cases have been studied in a tissue-autonomous context. The question then arises, how is SAM converted to SAH? It has been reported that methylation of a histone and a cytosolic 20-kDa protein peaked at about 24 h after PH. 27,28 The identity of the 20-kDa protein is still unknown, and its contribution to liver regeneration has not been determined, but it has been suggested that the increased methylation is associated with cell proliferation and transformation. 27,28 It is unclear whether the fat body simply works as a source of SAM for methylation in regenerating discs or if some methylated factors in the fat body directly or indirectly support disc repair. Therefore, the precise identification and analysis of methylation target(s) or other downstream targets of methionine metabolism are required for further understanding of the process.

Functional analysis of methionine metabolism in tissue repair
Functional contributions to tissue repair could be assessed by combining Q-dependent DtA ts -induced wing disc ablation and a fat body Gal4 driver. 3 As we reported, both knockdown and overexpression of gnmt or sams in the fat body impaired the repair of wing discs, while these genetic manipulations had no apparent effects on normal wing development. This indicates that appropriate regulation of methionine metabolism in the fat body is only necessary for disc repair. Additionally, overexpression of a dominant negative form of the insulin receptor (InR DN ) in the fat body did not affect the adult wing morphology. This negates the idea that simple fat body dysfunction causes the impairment of disc repair and sheds light on the importance of methionine metabolism as a systemic modulator of tissue repair. 3 Although it is still unclear how both up-and downregulation of gnmt or sams impairs disc repair, both MAT 1A ¡/¡ and GNMT ¡/¡ mutant mice showed impaired liver regeneration. 29,30 Therefore, the effect of methionine metabolism on tissue repair must be evolutionally conserved. In the case of MAT 1A ¡/¡ mice, S phase hepatocytes were significantly decreased in the injured liver, while hepatic apoptosis was markedly increased in the liver of GNMT ¡/¡ mice. Although it has not been determined whether methionine metabolism metabolites work in damaged or uninjured tissues, further studies will reveal the exact molecular mechanisms that underlie tissue regeneration.

Potential trigger of methionine metabolism changes in SDR
In animals, tissue damage leads to the release of damage-(or danger-) associated molecular patterns (DAMPs) and activation of the innate immune response. 31,32 DAMPs release is often followed by non-apoptotic cellular damage, including plasma membrane rupture and swelling of cells, which are hallmarks of necrosis and associated with pathological conditions such as traumatic injury 33 and spontaneous tumor formation. 34 The larval haemolymph of the apoptosis-deficient mutant contains DAMPs that activate the Toll innate immune pathway. 35 In Drosophila, DAMPs activate a proteolytic cascade of haemolymph to generate the mature form of the Toll ligand, Sp€ atzle (Spz). 36 Spz activates an innate immune reaction not only in blood cells but also in non-circulating cells, such as the fat body. Activated Spz overexpression leads to induced FoxO-Gnmt in adult flies, suggesting that systemic immune activation is sufficient to alter methionine metabolism. 24 However, DtA ts -induced tissue damage in the wing discs did not induce immune activation, at least in the fat body (Kashio, Obata, and Miura, unpublished). This is consistent with the fact that Gnmt expression is not induced in these animals. Thus, additional systemic mediator(s) other than DAMPs, may exist and function as "Help Me" signals from damaged discs to other tissues, which would positively regulate SDR in tissue regeneration.

SDR studies and their relevance to methionine metabolism
The Drosophila model has contributed significantly to studies of SDR. 37 When integumental wounding by simple pricking was performed in adult Drosophila, an inter-tissue reaction was observed in the intestine and neurons. In the intestine, enterocytes, gut epithelial cells, reacted to integumental wounding. The IL-6-like cytokine Upd-3 was induced, and caspases were activated in the enterocytes. When caspase activation was prevented in enterocytes, animals became susceptible to integumental wounding. 38 Another paper demonstrated that wounding of the cuticle induced the production of Upd-2, -3 by haemocytes through JNK activation, which activates JAK/STAT signaling in visceral muscles and intestinal stem cells and regulates gut epithelial renewal. 39 These studies showed that SDR in the gut is required for organismal survival against integumental wounding. Integumental wounding activated the local production of reactive oxygen species (ROS) by dual oxidase (DUOX) or systemic ROS production through activation of pro-phenoloxidase by the haemocyte-derived phenoloxidase-activating enzyme Hayan, a CLIP-domain-containing serine protease. Integumental wounding activated neuronal JNK, and phenoloxydase-mediated ROS was required for this JNK activation. 40 Hayan mutants are susceptible to wound-induced lethality and can be rescued when the neuronal antioxidant pathway is reduced or JNK activity is enhanced in neurons. 40 ROS could be one of the upstream mediators of distant tissue reactions because ROS is also required for caspase activation in enterocytes after wounding. 38 Drosophila metastatic tumors (Ras V12 /scribble ¡/¡ clones in imaginal discs) or neoplastic tumors in scribble ¡/¡ mutants disrupt the basement membrane. 41 As in wounding, JNK and Upd-3 are up-regulated in tumors. Circulating haemocytes adhere to tumors, and Upd-3 is induced in the haemocytes. This represents positive feedback between Upd-3 expression and systemic activation of JAK/STAT signaling. Importantly, the fat body, a tissue distant from the injured site, is also engaged in the amplification loop of JAK/STAT signaling. Haemocytes proliferate via JAK/STAT signaling and attach to tumors to restrict their growth. A pinching method was used to induce physical aseptic tissue damage of the imaginal disc, and haemocyte recruitment to the damage site and amplification of JAK/STAT signaling were initiated. 42 Vidal's group also showed that imaginal disc tumors non-autonomously trigger a tumor suppressor response via Toll activation in the fat body. 43 These studies of SDR in Drosophila reveal bi-/ multi-directional tissue interactions during the tissue damage response and following homeostatic regulation in the host. While it has not been studied whether methionine metabolism is also involved in tumorinducing SDR, some cancers are reported to have a common feature of an absolute requirement for methionine, known as methionine dependency. 44 It would be interesting to determine whether methionine metabolism is altered in tumor-dependent SDR and involved in tumor progression.
Traumatic brain injury (TBI) in Drosophila revealed a secondary, non-mechanical injury in the intestine. 45 TBI increased intestinal permeability, and glucose feeding stimulated intestinal permeability, which enhanced lethality after TBI. Leakage of bacteria from the gut lumen then activated an innate immune response. Although the precise role of this immune response in survival after TBI has not been determined, 46 this observation provides a clear example of TBI-induced SDR in the gut. Intriguingly, the human patient with TBI had altered amounts of methionine and SAM in their blood, 47 which is similar to the case observed in Drosophila disc repair. The direct relevance to methionine metabolism on TBIinduced SDR has not been studied yet, but this suggests the evolutionally conserved significance of methionine metabolism in SDR.

Future directions
Some diseases or metabolic abnormalities cause perturbation of methionine metabolism. In humans, diabetes causes an increase in the amount of Met and SAM. 48 Interestingly, diabetic patients also exhibit impaired wound healing. This wound healing defect is reportedly caused by an impairment in nitric oxide (NO)-mediated endothelial progenitor cells homing to the injured site. 49 It was also reported that 5-methyltetrahydrofolate, the predominant form of dietary folate, regenerated tetrahydrobiopterin (BH 4 ), a cofactor for producing NO with endothelial nitric oxide synthase (eNOS). 50 Although it is unclear whether eNOS function is directly affected by methionine metabolism, it remains likely that a diabetes-induced wound-healing defect is related to disruption of methionine metabolism because methionine metabolism is linked to folate metabolism in mammal. On the contrary, a low nutritional condition induced by a diet lacking in protein also impaired wound healing, but supplementation with Met was able to rescue the formation of connective tissue. 51 It is possible that Met regulates the rate of available protein utilization for connective tissue generation, or it may be utilised as a source of SAM, polyamine, or sulfate at the wound site. This report demonstrated the specific role of methionine metabolism in tissue repair and highlighted the potential of methionine metabolism regulation as a clinical target.
A number of reports have revealed the underlying mechanisms and importance of SDR, but many crucial questions remain unanswered. How is SDR induced? Why is SDR required? What are the mediators among the tissues? One major approach for understanding the mediators is to focus on the secreted factors. In a recent study, in vitro screening was used to show that myristoylated alanine-rich C kinase substrate (MARCKS)-like protein, a secreted factor, is required for the initial cell cycle response during axolotl appendage regeneration. 52 Mutant zebrafish, lacking most haematopoietic tissues, showed a regeneration defect accompanying apoptosis in regenerating fin after amputation. 53 In the tail explant culture, extracts from the bodies of wild-type zebrafish larvae rescued the apoptosis of regenerative cells in the haematopoietic tissue-deficient mutant, implying that diffusible factor(s) from haematopoietic tissue support the survival of regenerative cells. 53 These results prompted the functional screening of secretory factors (i.e., the secretome 54 ) and the use of Drosophila genetics to understand the molecular mechanisms of SDR. Studies that focus on tissue interactions and the internal milieu (i.e., systemic biology) will further reveal the complex systems that multicellular organisms use for survival. Ultimately, such studies will contribute to pharmaceutical progress as well as to understanding the essential homeostatic mechanisms of multicellular organisms.


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NGF-trkA signaling modulates the analgesic effects of prostatic acid phosphatase in resiniferatoxin-induced neuropathy

Background Neuropathic pain in small-fiber neuropathy results from injury to and sensitization of nociceptors. Functional prostatic acid phosphatase (PAP) acts as an analgesic effector. However, the mechanism responsible for the modulation of PAP neuropathology, which leads to loss of the analgesic effect after small-fiber neuropathy, remains unclear. Results We used a resiniferatoxin (RTX)-induced small-fiber neuropathy model to examine whether functional PAP(+) neurons are essential to maintain the analgesic effect. PAP(+) neurons were categorized into small to medium neurons (25th–75th percentile: 17.1–23.7 µm); these neurons were slightly reduced by RTX (p = 0.0003). By contrast, RTX-induced activating transcription factor 3 (ATF3), an injury marker, in PAP(+) neurons (29.0% ± 5.6% vs. 0.2% ± 0.2%, p = 0.0043), indicating PAP neuropathology. Moreover, the high-affinity nerve growth factor (NGF) receptor (trkA) colocalized with PAP and showed similar profiles after RTX-induced neuropathy, and the PAP/trkA ratios correlated with the degree of mechanical allodynia (r = 0.62, p = 0.0062). The NGF inducer 4-methylcatechol (4MC) normalized the analgesic effects of PAP; specifically, it reversed the PAP and trkA profiles and relieved mechanical allodynia. Administering 2.5S NGF showed similar results to those of administering 4MC. This finding suggests that the analgesic effect of functional PAP is mediated by NGF-trkA signaling, which was confirmed by NGF neutralization. Conclusions This study revealed that functional PAP(+) neurons are essential for the analgesic effect, which is mediated by NGF-trkA signaling.

Moreover, small nociceptive DRG neurons, including peptidergic and nonpeptidergic neurons, coexpress PAP, 4 suggesting that PAP profiles correlate with pain modulation. For example, a growing body of evidence has shown that intrathecal injection of recombinant PAP counteracts inflammatory pain 6 and enhances neuropathic pain in PAP-knockout mice. 1,7,8 These findings confirm that PAP acts as an analgesic modulator. PAP expression is higher in small nonpeptidergic DRG than in peptidergic DRG neurons. 4 Previously, we have established a mouse model of small-fiber neuropathy, 9 in which small sensory nerves were specifically depleted by resiniferatoxin (RTX), a capsaicin analog acting on the transient receptor potential vanilloid receptor 1 (TRPV1). [10][11][12] This RTX-induced neuropathy model mimics human small-fiber neuropathy; that is, the model exhibits the degeneration of cutaneous sensory nerves and neuropathic pain. Furthermore, RTXinduced neuropathy with skin denervation markedly increases the expression of activating transcription factor 3 (ATF3), a marker of neuronal injury, in nonpeptidergic P2X3 neurons, which is correlated with neuropathic pain. 10 These observations suggest that PAP exerts analgesic effects for small-fiber neuropathy. Whether the expression profiles of this subpopulation of PAP neurons correlate with injury-induced neuropathic pain should be determined, and the mechanism responsible for modulating the analgesic potential in injuryinduced neuropathic pain should be investigated.
The compound 4-methylcatechol (4MC) enhances the synthesis of neurotrophins such as nerve growth factor (NGF) and promotes skin reinnervation. 13 Cutaneous sensory nerves function as the peripheral receptors of small DRG neurons; thus, inducing ATF3 expression in these neurons caused skin denervation and neuropathic pain in our RTX-induced neuropathy model. 10 NGF is trophic to small DRG neurons and modulates neuropathic pain through its high-affinity receptor, trkA; a reduction in NGF and trkA expression is accompanied by various peripheral neuropathies. 14,15 Whether 4MC modulates PAP expression profiles through NGF-trkA signaling is unclear.
RTX-induced neuropathy is an excellent model for determining how the loss of the analgesic effect of PAP leads to injury-induced neuropathic pain. In the present study, we investigated the intervention of PAP profiles to neuropathic pain and the effect of 4MC on reversing neuropathic pain through NGF-trkA signaling.

Experimental design and groups
This study investigated PAP-trkA signaling-mediated pain behavior in RTX-induced neuropathy by investigating whether PAP neuropathology is correlated with pain development and whether 4MC reverses pain and PAP profiles in RTX-induced neuropathy in an NGF-trkA signaling-dependent manner. Neuropathy was induced by the intraperitoneal (i.p.) administration of a single dose of RTX (50 mg/kg, Sigma, St. Louis, MO) (the RTX group). 9,10,16 Mice received either daily i.p. injections of 4MC (10 mg/kg, Wako, Osaka, Japan) immediately after RTX treatment for seven consecutive days (the 4MC group), 9,13 or an equal volume of vehicle as a control (the vehicle group). To confirm the effect of 4MC, 2.5S NGF and anti-NGF antisera were i.p. administered. Briefly, 2.5S NGF (Alomone Labs, Jerusalem, Israel) was dissolved in Dulbecco's Modified Eagle Medium, and mice received NGF (1 mg/10 g) immediately after RTX treatment (the NGF group). 17 Some mice in the 4MC group received additional anti-NGF antisera (30 mg/kg, Sigma) 18 to neutralize NGF induced by 4MC (the abNGF group). To compare each experimental approach, 2.5S NGF and anti-NGF antisera were administered consecutively for seven days.
After treatment, the mice were housed in plastic cages in a 12-h light/dark cycle and were given access to water and food ad libitum. All procedures were conducted in accordance with ethical guidelines for laboratory animals, 19 and the protocol was approved by the Institutional Animal Care and Use Committee of Kaohsiung Medical University. All experimental procedures were performed carefully, and all efforts were made to minimize animal suffering.

Evaluation of mechanical allodynia
The changes in the mechanical threshold of each group were assessed using the up-and-down method with different calibers of von Frey monofilaments (Somedic Sales AB, Ho¨rby, Sweden) in accordance with our established protocol. 10,16 Briefly, a series of monofilaments was applied to the plantar region of the hindpaw. If paw withdrawal occurred, a monofilament of a smaller caliber was applied. In the absence of paw withdrawal, a monofilament of a larger caliber was applied. Four additional stimuli with monofilaments of various calibers were applied on the basis of the preceding responses, and the mechanical thresholds were calculated using a formula published in a previous study. 20

Double immunofluorescence staining of DRG neurons
Animals were sacrificed by intracardiac perfusion with 0.1 M phosphate buffer (PB) followed by 4% paraformaldehyde (4 P) in 0.1 M PB. After perfusion, the 4th and 5th (L4 and L5, respectively) lumbar DRGs were removed carefully and postfixed in 4P for another 6 h.

Quantification of different phenotypic DRG neurons
To quantify the various phenotypic DRG neurons, each DRG section was systematically photographed at 200 Â under a fluorescence microscope (Axiophot microscope, Zeiss, Oberkochen, Germany) to produce a montage of the entire DRG section according to an established procedure. 9,10,16 To avoid density bias, only neurons with a clear nuclear profile were counted, and only sections containing neuronal ganglia were measured using ImageJ version 1.44d (National Institutes of Health [NIH], Bethesda, MD). For morphometric analysis of PAP neuropathology, the diameters of PAP(þ) neurons and ATF3(þ)/PAP(þ) neurons were measured using Image Pro-Plus (Media Cybernetics, Bethesda, MD) and plotted as a histogram.

Statistical analysis
To minimize individual bias, five to seven mice were included in each group, and the coding information was masked during the behavioral tests and quantification procedures. All data are expressed as mean AE standard derivation (SD) of the mean. A t test was performed to analyze the data following a Gaussian distribution. For data which did not follow a Gaussian distribution, a nonparametric Mann-Whitney test was applied. Results with p < 0.05 were considered statistically significant.
Mice in the RTX group showed mechanical allodynia compared with the vehicle group (747.1 AE 156.0 vs. 299.6 AE 70.6 mg, p ¼ 0.0025) (Figure 3(i)). This behavioral change correlated with the PAP profiles; that is, the densities of PAP(þ) neurons (r ¼ 0.75, p < 0.0001; Figure 3(j)) and the ratios of ATF3(þ)/PAP(þ) neurons (r ¼ À0.60, p ¼ 0.003; Figure 3(k)) were inversely linear to the mechanical thresholds. Collectively, these findings indicate that PAP expression had a strong influence in mediating pain development. To understand the putative mechanisms of pain relief by 4MC, we examined the coexpression of PAP(þ)/ trkA(þ) neurons ( Figure 4). For each group, PAP(þ) neuronal density was similar to trkA(þ) neuronal density (Figure 3(f) vs. 4(k)). The PAP/trkA ratios correlated linearly with the mechanical threshold (r ¼ 0.62, p ¼ 0.0062; Figure 4(m)). To confirm the effect of 4MC, the mice in the NGF group were systemically (i.p.) administered 2.5S NGF. The NGF group exhibited the same effect as the 4MC group; that is, PAP and trkA expression profiles were reversed, ATF3 induction was normalized, and mechanical allodynia relieved. Those findings, however, were not observed in the abNGF group (administered anti-NGF antisera to neutralize NGF) (Figures 3  and 4). These findings suggest that 4MC regulates PAP profiles through NGF-trkA signaling.

Discussion
This is the first report documenting the intervention of PAP and NGF-trkA signaling, which mediates (f) The graph shows the diameter histogram of PAP(þ) neurons, indicating that PAP(þ) neurons are small to medium neurons. Bar, 50 mm. **p < 0.01 neuropathic pain. We speculate one possible mechanism: neuropathic pain in RTX-induced small-fiber neuropathy develops because of PAP neuropathology and the decrease in NGF-trkA signaling.
PAP downregulation was correlated with pain perception and molecular significance of PAP neuropathology RTX is specifically neurotoxic to TRPV1(þ) neurons; it also induces small-fiber neuropathy, such as skin denervation, which results in thermal hypoalgesia, 9 smalldiameter neuron injury, 10 and peripheral sensitization of P2X3 purinoceptors, leading to mechanical allodynia. 16 ATF3 is both an injury marker and a marker of pain; for example, the ratios of P2X3(þ)/ATF3(þ) neurons correlate linearly with the degree of neuropathic pain. 10 PAP is susceptible to RTX because of the high ratios of PAP(þ)/P2X3(þ) and PAP(þ)/peripherin(þ) neurons. Thus, injured PAP neurons are critical for modulating neuropathic pain; that is, the ratios of ATF3(þ)/PAP(þ) neurons are inversely linear to the mechanical threshold.
The balance of extracellular purine nucleotides such as adenine nucleotides is maintained by functional membrane-bound ectonucleotidase activity, which responded for the cellular pathophysiological homeostasis. [21][22][23][24] The potential role of PAP downregulation in nociceptive transmission is unclear. The ectonucleotidase activity of PAP may facilitate the hydrolysis of AMP to adenosine, which acts as an analgesic ligand. 6,7 PAP downregulation leads to the imbalance of AMP/adenosine ratios, inhibiting the analgesic effect of adenosine. Taken together, these findings suggest the neuronal soma underlies the irritation and/ or injury coincident with the metabolic imbalance of extracellular nucleotides, resulting in the loss of the analgesic effect of PAP. Additional studies should investigate the effects of altered purine metabolism to clarify the cellular responses to neuronal soma irritation. A previous study demonstrated that PAP-mediated neuropathic pain was TRPV1-dependent. 8 In our RTX-induced neuropathy model, TRPV1 was completely depleted, 9,10 and the present study provides evidence of an alternative nociceptive pathway to the TRPV1-dependent nociceptive pathway; the loss of the analgesic effect of PAP is mediated by neuronal soma injury and/or irritation. Furthermore, pain development correlated with trkA profiles; for example, the ratios of PAP(þ)/trkA(þ) neurons were linearly correlated with the degree of mechanical allodynia.

Amelioration of neuropathic pain by 4MC through NGF-trkA signaling
This report demonstrates the amelioration of neuropathic pain by 4MC through the intervention of PAP and NGF-trkA signaling. In addition to previous studies demonstrating that 4MC promotes skin reinnervation, 9,13 the present study demonstrated that 4MC could facilitate the recovery irritated and/or injured small-diameter neuronal soma, as indicated by the reduced ratios of ATF3(þ)/PAP(þ) neurons. Conversely, NGF-sensitized sensory nerves resulted in pain perception 25 and targeting of NGF-trkA signaling 26 is a new direction for pain therapy. 27,28 Coincidentally, several studies have suggested that the interaction of NGF and TRPV1 is critical for pain transmission. 17,[29][30][31][32] The present study shows contrary outcomes; for example, 4MC-promoted NGF played the role of a modulator in the recovery of PAP and PAP(þ)/ trkA(þ) neuronal profiles coinciding with the normalization of mechanical allodynia. 4MC appears to have a pleiotropic effect in modulating neuronal activity; for example, it also increases the synthesis of the brain-derived neurotrophic factor (BDNF) 33,34 and enhances the phosphorylation of the trk family and activation of the MAPK/ERK cascade. 35 Regarding the targeted signaling pathway of 4MC in RTX-induced neuropathy, BDNF modulates the function of the motor 36,37 and myelinated sensory neurons, 37 but no effect was observed in this current neuropathic model because (1) RTX specifically affects smalldiameter sensory neurons with no pathologic alternations in the motor and myelinated sensory neurons, 9 and (2) 4MC has no effect on trkB and/or trkC signaling because there are no changes in BDNF and neurotrophic 3 (NT3) expression in the peripheral tissues, as determined in our previous study. 13 Regarding the possible mechanism of the effect of 4MC on pain-relief in RTX-induced neuropathy, this report provides immunohistochemical evidence of the potential effect of 4MC, as demonstrated by the PAP/trkA ratios of approximately 70%. Collectively, these data reinforce our speculations: 4MC regulates PAP pathology through NGF-trkA signaling, which was confirmed through alternative NGF administration and NGF neutralization experiments. Combining with the 4MC reduced the ATF3(þ)/PAP(þ) neurons; this study suggests that 4MC has a pleiotropic effect on both the recovery of irritated and/or injured neuronal soma and restores NGF-trkA signaling. In summary, ATF3 might be an upstream molecule initiating the loss of the analgesic effect of PAP, and PAP is a downstream molecule of NGF-trkA signaling, restoring the analgesic effect of PAP in a trkA-dependent manner ( Figure 5).

Conclusion
For pain development, this study provides evidence of an alternative pathway to conventional small nociceptor sensitization-induced neuropathic pain; the loss of analgesic effect of PAP is mediated by neuronal soma irritation and/or injury. Alternatively, restoring the analgesic ligand may be a new direction for targeting painful neuropathy, that is, maintenance of steady-static adenosine content and functional PAP ectonucleotidase activity at the neuronal soma level.

Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants from the National Science Council (NSC100-2320-B-037-018 and NSC102-2320-B-037-009); Ministry of Science and Technology (MOST103-2320-B-037-015-MY3), Taiwan; and Kaohsiung Medical University Hospital (KMUH104-4M19).