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Published as doi: 10.1096/fj.06-7460com.
 (The FASEB Journal. 2007;21:3747-3755.)
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The transient receptor potential vanilloid 1 (TRPV1) receptor protects against the onset of sepsis after endotoxin

Natalie Clark*, Julie Keeble*, Elizabeth S. Fernandes*, Anna Starr*, Lihuan Liang*, David Sugden{dagger}, Patricia de Winter* and Susan D. Brain*,1

* Cardiovascular Division, Waterloo Campus, and

{dagger} Reproduction and Endocrinology Division, Guy’s Campus, King’s College London, London, UK

1Correspondence: Cardiovascular Division, King’s College London, Franklin-Wilkins Bldg., Waterloo Campus, 150 Stamford St., London SE1 9NH, UK. E-mail: sue.brain{at}kcl.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 CONCLUDING REMARKS
 REFERENCES
 
Transient potential vanilloid 1 (TRPV1) receptor is an ion channel receptor primarily localized on sensory nerves and activated by specific stimuli to initiate and amplify pain and inflammation, as typified by murine models of scald and arthritis. Little is known of the role of TRPV1 in sepsis, an infective disease associated with inflammation. Through use of a sublethal murine model of lipopolysaccharide-induced peritoneal sepsis, we provide novel evidence that genetic deletion of TRPV1 leads to an enhanced onset of various pathological components of systemic endotoxemia. Paired studies of TRPV1 knockout (KO) and wild-type mice demonstrate significantly enhanced hypotension (56±2% vs. 38±6% decrease in blood pressure, n=12), hypothermia (13±3% vs. 7±1% decrease in core temperature, n=6), and peritoneal exudate mediator levels (TNF-{alpha}, 0.78±0.2 vs. 0.38±0.1 ng/ml; nitrite, for NO, 35±10 vs. 15±3 µM; n=8) in TRPV1 KO mice, indicating loss of protective effect. Findings correlated with liver edema and raised plasma levels of aspartate aminotransferase in TRPV1 KO mice. These data suggest that TRPV1 may play an important regulatory role in sepsis independent of the major sensory neuropeptide substance P. The findings are relevant to developing strategies that increase the beneficial, and reduce the harmful, components of sepsis to prevent and treat this often fatal condition.—Clark, N., Keeble, J., Fernandes, E. S., Starr, A., Liang, L., Sugden, D., de Winter, P., Brain, S. D. The transient receptor potential vanilloid 1 (TRPV1) receptor protects against the onset of sepsis after endotoxin.


Key Words: mouse sensory nerves • knockout • substance P • capsaicin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 CONCLUDING REMARKS
 REFERENCES
 
THE TRANSIENT RECEPTOR POTENTIAL vanilloid 1 receptor TRPV1 is a nonselective cation channel (1) best known for its localization on a subset of sensory nerve fibers (C and A{delta}) that release the potent vasoactive peptides substance P and CGRP (2) . The chili extract capsaicin and painful heat activate TRPV1, leading to pain and neurogenic inflammation. A growing list of proposed endogenous agonists includes protons (3 , 4) , cannabinoids (5 6 7 8) , arachidonate metabolites (e.g., 20-HETE; refs. 9 , 10 ), and other lipoxygenase products (11) . It is now realized that common signaling mechanisms can enhance activation of TRPV1—for example, through PKC (12) —thus supporting the concept that TRPV1 can act as an integrator of sensory information (13) . There are recent publications of a range of TRPV1 antagonists proposed as drug candidates for novel analgesics and antitussives, but to our knowledge none are appropriate for long-term use in vivo. Furthermore, the traditional method used to probe the role of TRPV1 in vivo, which utilizes neurotoxic doses of capsaicin, affects not only nerves containing TRPV1 receptors but also their contents (14) . Thus, in our opinion TRPV1 knockout (TRPV1 KO) mice offer a distinct experimental advantage when evaluating the role of the TRPV1 in vivo. The TRPV1 receptor is established as being important in mediating pain processing, as confirmed by the first studies of TRPV1 KO mice (15 , 16) as well as more recent studies of disease models of arthritis (17) .

Plasma levels of the major neuropeptides substance P and CGRP are both increased in patients with sepsis, providing evidence that sensory nerves are activated (18 19 20) , with an understanding that substance P, at least, probably plays a proinflammatory role (21) . However, little is known about the role of the TRPV1 receptor in sepsis. Furthermore, Wadachi and Hargreaves (22) recently published the potentially important information that toll-like receptor 4 (TLR4) and CD14 receptors are colocalized with TRPV1 on sensory nerves. These results indicate that bacterial products such as lipopolysaccharide (LPS) may act directly to activate sensory nerves. Certainly there have been links between sensory nerves and infection. Indeed, the experimental removal of sensory nerves was shown many years ago to exacerbate the inflammatory response to infection (23) and, more recently, in a rat lung infection model (24) . However, there has been little progress in understanding the potential involvement of sensory nerves in sepsis, especially as studies involving capsaicin neurotoxic techniques have indicated both detrimental (25) and beneficial roles (26) . We have developed a sublethal murine model of endotoxemia that allowed us to study the onset processes and offers benefits compared with the commonly used mortality studies. This first selective study of the TRPV1 receptor provides clear and surprising evidence that TRPV1 activity is associated with a protective effect in the onset of sepsis, as determined by an enhanced development of the pathological and biomarkers for sepsis in TRPV1 KO compared with wild-type (WT) mice.


   METHODS AND MATERIALS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
RESULTS
 DISCUSSION
 CONCLUDING REMARKS
 REFERENCES
 
All procedures used in this project were conducted in accordance with the Animals (Scientific Procedures) Act of 1986. Mice were kept in a climatically controlled environment and given food and water ad libitum. In all studies, age- and sex-matched mice were utilized at 13–20 wk of age, as in our hands they generated a more consistent response. TRPV1 KO mice donated from Merck Sharp and Dohme (Harlow, UK) were generated by replacing the exon that encodes part of the fifth and the entire sixth transmembrane domain (including the interconnecting p-loop) of the receptor with a neomycin gene, as described by Caterina et al. (15) . Mice were from established WT and TRPV1 KO colonies. For some experiments, WT mice were pretreated with the selective TRPV1 receptor antagonist SB366791 (500 µg/kg, i.p., –30 min; ref. 27 ) or a cocktail (i.v. –5 min) containing effective doses of the tachykinin neurokinin NK1 (SR 140333, 480 nmol/kg), NK2 (SR 48968, 3 mg/kg), and NK3 (SR 142801, 3 mg/kg) antagonists (28 , 29) or their respective vehicle controls before induction of sepsis.

Offspring were genotyped after isolation of genomic DNA from tail tips by proteinase digestion at 55°C overnight, followed by purification with a DNAeasy tissue kit (Qiagen, Valencia, CA, USA). PCR amplication was performed on the genomic DNA using the following primers: TRPV1 (AY445519): forward primer (5'-CGA GGA TGG GAA GAA TAA CTC ACT G-3' (1800–1824), TRPV1 reverse primer (5'-GGA TGA TGA AGA CAG CCT TGA AGT C-3' (1987–19863), neomycin forward primer (5'-TTT TGT CAA GAC CGA CCT GTC C-3') and neomycin reverse primer (5'-CCC TCA GAA GAA CTC GTC AAG AAG-5'). The PCR reaction mix (50 µl) contained: 5 µl genomic DNA, 1 x high-fidelity PCR buffer, dNTP mix (0.4 mM of each nucleotide), the four primers described above (each at 2.5 µM), 2 mM magnesium, 1.25 U platinumTaq DNA polymerase, and 9.8 µl DEPC-treated H2O. The PCR program consisted of 95°C at 14.5 min for initial denaturation of DNA, followed by 35 cycles of: 95°C for 45 s, 60°C for 1 min for annealing of primers, and 72°C for 1 min for elongation. The PCR product size for TRPV1 and neomycin was 188 and 700 bp, respectively; they were visualized after agarose gel (1.8% w/v) electrophoresis and ethidium bromide staining. WT mice produced an amplicon at 188 bp whereas TRPV1KO mice produced an amplicon of 700 bp (Fig. 1 A).


Figure 1
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  		Figure 1. Characterization of TRPV1 KO mice and their response to capsaicin and noxious heat. A) Polymerase chain reaction (PCR) analysis of tail biopsy DNA is shown following gel electrophoresis with TRPV1 and neomycin primers; see Materials and Methods for construct details. The DNA ladder is shown on the left. An absence of the predicted product at 700 bp but the presence of the predicted product at 188 bp in a WT (+/+) mouse and the presence of the predicted product at 700 bp but an absence of the predicted product at 100 bp in a TRPV1 KO (–/–) mouse are shown. B) Effect of topical capsaicin (20 µl of 10 mg/ml) after 1 h and C) noxious heat (55°C for 10 s) on ear edema formation after 30 min in the anesthetized mouse. Values are expressed as mean ± SE (n=6); ***P < 0.001 vs. control WT ear, ###P < 0.001 vs. treated WT ear.

Measurement of plasma extravasation
Plasma extravasation was measured by the extravascular accumulation of i.v. injected 125I-labeled bovine serum albumin (BSA), as described by Cao et al. (30) . Under urethane anesthesia (2.5 g/kg), 125I-BSA (45 kBq) was injected into the tail vein with saline (0.1 ml i.v.). Plasma extravasation after topical application of capsaicin (20 µl of 10 mg/ml) or ethanol vehicle (20 µl) to the ear was allowed for 60 min. In separate experiments, thermal injury was induced in the ear (immersion in water at 55°C for 10 s) or sham in the contralateral ear (immersion in water at room temperature for 10 s) and plasma extravasation was allowed for 30 min. A blood sample (0.5–0.7 ml) was then taken by cardiac puncture and the animal was killed by cervical dislocation. The blood samples were centrifuged at 10,000 rpm for 3 min, after which plasma was taken to measure plasma radioactivity in a gamma counter (1260 Multigamma II; EG & G Wallac, Milton Keynes, UK). The ears were removed and weighed, and their radioactivity was similarly measured. Plasma extravasation in the ears was expressed as microliters of plasma accumulated per gram of tissue.

Measurement of blood pressure
Sepsis was evoked by administration of LPS, serotype 0127:B8, 11.25 million endotoxin units/kg in saline (10 ml/kg) via an i.p. injection to conscious but restrained mice. Blood pressure was assessed noninvasively in conscious restrained mice by the tail cuff technique using the CODA 6 System (Kent Scientific, Torrington, CT, USA), which assesses tail blood pressure by means of volume pressure recording. Blood pressure was measured in a warmed (25°C) room; mice were pretrained on two occasions. The effects of sepsis on blood pressure were assessed 1.5 and 4 h after administration of LPS. Data were assessed as the percent change in mean arterial pressure from baseline measurements taken before administration of LPS; for baseline physiological parameters, see Table 1 .


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  		Table 1. Baseline physiological parameters of WT and TRPV1 KO micea

Mice were anesthetized with 3% isofluorane/3% O2 1.5 or 4 h after administration of LPS; blood (~1 ml) was taken by cardiac puncture into a heparinized syringe, then centrifuged at 10,000 g for 3 min to obtain plasma. To obtain peritoneal exudate lavage fluid (PELF), 0.5 ml of PBS was injected into the peritoneal cavity, carefully mixed, and 0.2 ml of PELF was aspirated. Samples were snap-frozen and stored at –70°C for further analysis.

Measurement of TNF-{alpha} levels
TNF-{alpha} levels in peritoneal exudate lavage fluid and plasma taken 1.5 h after administration of LPS were measured using a murine TNF-{alpha} enzyme-linked immunosorbant assay development kit (Tebu-Bio, Le Perray en Yvelines, France) according to the manufacturer’s recommended protocol. Data are expressed as ng/ml TNF-{alpha}/100 µl of sample.

Assessment of NO levels by measurement of NO2–/NO3–
The NO2–/NO3– content was measured by the Griess assay as an indicator of NO production in plasma and peritoneal exudate lavage fluid. NO3– was reduced to nitrite (NO2–) by incubating 80 µl of sample with 10 µl of 10 U/ml nitrate reductase and 10 µl of 1 mM ß-nicotinamide adenine dinucleotide phosphate (NADPH) for 30 min at 37°C in a 96-well plate. Next, 100 µl of Griess reagent (5% v/v H3PO4 containing 1% w/v sulfanilic acid and 0.1% w/v N-1-napthylethylenediamine) was added and allowed to incubate for 15 min at 37°C. The absorbance at 550 nm was measured immediately using a spectrophotomer (Spectra Max 190; Molecular Devices, Palo Alto, CA, USA). After subtraction of background readings, the absorbance in each sample was compared with those obtained from a sodium nitrite (0–100 µM) standard curve.

Quantification of iNOS mRNA using real-time PCR
Total RNA was extracted from small intestinal smooth muscle cells using Trizol Reagent (Invitrogen, Paisley, UK). Prior to reverse transcription, each RNA sample underwent DNase treatment (RQ1; Promega, Southampton, UK) to eliminate any potential genomic DNA contaminants. DNase-treated RNA (2 µg) then underwent reverse transcription using Moloney murine leukemia virus reverse transcriptase (Promega) and 0.5 µg/ml d(N)10 and d(T)18 primers. iNOS and GAPDH mRNA and 28S rRNA levels were then quantified by SYBR Green real-time polymerase chain reaction (PCR) using a LightCycler (1.2; Roche Applied Science, Indianapolis, IN, USA) under the following thermal cycling parameters: 95°C for 5 min, 30 cycles of denaturation at 95°C for 30 s, annealing at 54°C for iNOS and 28S or 57°C for GAPDH for 30 s, and extension at 72°C for 30 s. The oligonucleotide primers were as follows: iNOS (AF427516): forward primer 5'-CAG CTG GGC TGT ACA AAC CTT-3' (27393–27413), reverse primer 5'-CAT TGG AAG TGA AGC GTT TCG-3' (28182–28162); GAPDH (DQ403054): forward primer 5'-TTC ACC ACC ATG GAG AAG GC-3' (229–248), reverse primer 5'-GGC ATG GAC TGT GGT CAT GA-3' (465–446); 28S (X00525): forward primer 5'-TTG AAA ATC CGG GGG AGA G-3' (2492–2510), reverse primer 5'-ACA TTG TTC CAA CAT GCC AG-3' (2591–2572). iNOS primers were located on different exons and synthesized by Operon (Köln, Germany). Amplification efficiency was 100.5% (iNOS), 94.5% (GAPDH), and 94.5% (28 s). Copy numbers for iNOS and two housekeeping genes (HKG), 28S rRNA and GAPDH, were derived from standard curves of purified cDNA ranging from 108 to 102 copies for 28S and 107 to 101 copies for iNOS and GAPDH. Copy numbers of iNOS were normalized against the geometric mean of the HKG.

Quantification of NOS activity in septic mice using 3H-L-citrulline assay
Four hours after induction of sepsis, lungs were removed, snap-frozen, and stored at –80°C. The next day lungs were defrosted and homogenized (Ultra Turrax; Janke & Kunkel, IKA-Labortechnik, Staufen, Germany) in ice-cold 20 mM Tris-HCl buffer (pH 7.4 containing 2 mM EDTA; w/v=1:5). Homogenates were subsequently centrifuged (8000 g, 4°C, 10 min) and supernatant was extracted. Nitric oxide synthase (NOS) activity in lung tissue was assessed by measuring 3H-L-citrulline formation after incubation of the tissue with 3H-L-arginine (10 µl, 0.1 µCi/µl) in the presence of NADPH (10 µl, 25 mM). All concentrations shown are final concentrations. The supernatant (50 µl) was incubated in duplicate with each treatment group for 15 min at 37°C. Total 3H-L-citrulline production was determined by incubating the reaction mixture with Ca2+ (10 µl, 5 µM). Total NOS-dependent 3H-L-citrulline production was discerned by coincubation with Ca2+ in the presence of an L-NAME (10 µl, 3 mM)/L-NMMA (10 µl, 3 mM) cocktail. Ca2+-independent NOS activity was distinguished by omitting L-NAME/L-NMMA and replacing Ca2+ with EGTA (10 µl, 10 mM). To maintain the same volume of reaction mixture in all samples, Tris HCl/EDTA (20 mM) buffer was used in place of any reagents that were omitted. As an internal control, buffer replaced all reagents in some tubes. The reactions were terminated by adding L-NAME (100 µl, 5 mM) in ice-cold buffer. To separate 3H-L-citrulline from unconverted 3H-L-arginine, Dowex resin (1 ml/sample), which binds the 3H-L-arginine, was mixed with the reaction mixture and left to settle for 15 min; 100 µl of the aqueous phase containing 3H-L-citrulline was then mixed with 5 ml of scintillation fluid (Ultima Gold Cocktail, Packard BioScience, Meriden, CT, USA) and measured by a 1900 TR liquid scintillation analyzer (Packard). Counts were standardized according to protein content (Lowry assay; see ref. 31 ). Results are expressed as pmol L-citrulline/mg protein.

Body temperature
Body temperature was monitored using a rectal probe (Harvard Apparatus, Holliston, MA, USA) just before and 0.5, 1.5, and 4 h after administration of saline vehicle or LPS. Rectal probes were lubricated with petroleum jelly and placed 3 cm into restrained mice.

Assessment of organ damage
Edema formation was monitored by comparing wet and dry weights of whole spleens, right kidneys, and upper liver lobes. Each tissue was taken 4 h after induction of sepsis and weighed (wet). They were then left at 55°C until they reached a constant dry weight. Dysfunction of heart/liver, kidneys, and pancreas was assessed by measuring aspartate aminotransferase, creatinine, and lipase levels, respectively, present in plasma obtained 4 h after administration of saline vehicle or LPS. These studies were out-sourced to Nationwide Laboratories (Lancashire, UK). Heart weight was assessed as heart/body weight ratio, then hearts were fixed in 4% formalin and embedded in paraffin. Five µm-thick tissue sections were cut and stained with hematoxylin-eosin (H&E) for histochemical analysis.

Statistical analysis
All values were expressed as mean ± SE. Results for Fig. 1 and Fig. 2 were analyzed by ANOVA, followed by Bonferroni’s multiple comparison test, and Fig. 3 by unpaired t test on normalized data. Figure 4 and Fig. 5 were analyzed using unpaired t tests. Statistical analyses for real-time PCR data were performed on log2-transformed ratios using a general linear model and Tukey’s post hoc tests at a family error rate of 0.05.


Figure 2
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  		Figure 2. Time-dependent effect of disruption of TRPV1 on blood pressure and temperature after LPS-induced endotoxemia. A) Blood pressure changes and B) temperature changes in response to i.p. vehicle (triangles) or LPS (circles) in WT (open symbols) or TRPV1 KO (filled symbols) mice. Results are shown as % mean change ± SE from baseline (BL). Data expressed as mean *P < 0.05, **P < 0.01, or ***P < 0.001 vs. respective vehicle-treated mouse, #P < 0.05 compared with LPS-treated WT, n = 5–12/group.


Figure 3
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  		Figure 3. TNF-{alpha} and NO levels in peritoneal exudate fluid and plasma. A) NO, measured as nitrite, C) TNF-{alpha} levels in peritoneal exudate lavage fluid (PELF) and B) NO, D) TNF-{alpha} levels in plasma after i.p. vehicle or LPS in WT (open bars) or TRPV1 KO (filled bars) mice. Data are expressed as mean ± SE. *P < 0.05 or **P < 0.01, vs. respective vehicle-treated mouse, #P < 0.05 compared with LPS-treated WT, n = 4–8/group.


Figure 4
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  		Figure 4. Up-regulation and enzymatic activity of iNOS. A) Inducible NOS RNA in small intestine smooth muscle tissue was expressed as iNOS/HKG geomean mRNA copy number. Tissue was obtained in response to i.p. vehicle or LPS in WT (open bars) or TRPV1 KO (filled bars) mice, n = 6/group. B) NOS activity as measured by L-citrulline production is shown in enzyme preparations from lung tissue in the absence and presence of L-NAME/L-NMMA (300 µM of each) or in the presence of the calcium chelating agent EGTA (1000 µM) in lung tissue harvested from WT or TRPV1 KO mice 4 h after i.p. vehicle or LPS, n = 4–6/group. Data expressed as mean *P < 0.05, **P < 0.01, or ***P < 0.001, vs. respective vehicle-treated mouse, ##P < 0.05 vs. Tris-HCl counterparts; {dagger}P < 0.05, {dagger}{dagger}P < 0.01 vs. L-NAME/L-NMMA counterparts.


Figure 5
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  		Figure 5. Measurement of plasma extravasation and markers of organ damage. For plasma extravasation, the wet to dry weight ratio was taken for A) liver, B) kidney, and C) spleen. Serum was measured for markers of organ dysfunction, as shown by % change in LPS response from vehicle response for D) aspartate aminotransferase (AST, liver damage), E) creatinine (kidney damage), and F) lipase (spleen damage) in response to i.p. vehicle or LPS in WT (open columns) or TRPV1 KO (filled columns) mice. Data are expressed as mean ± SE, where P < 0.05 compared with vehicle-treated counterpart. n = 5–7/group.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
 RESULTS
DISCUSSION
 CONCLUDING REMARKS
 REFERENCES
 
TRPV1 deletion is associated with loss of typical neurogenic proinflammatory responses to capsaicin and scald injury but with enhanced hypotension and hypothermia in endotoxemia
The topical application of the TRPV1 agonist capsaicin to the ear induced local edema formation measured as increased plasma extravasation in TRPV1 WT mice but not in TRPV1 KO mice (Fig. 1B ). Plasma extravasation was also observed in TRPV1 WT but not KO mice in response to exposure to 55°C for 10 s (Fig. 1C ). These results demonstrate the established role of TRPV1 in activating proinflammatory pathways in response to capsaicin and the ability of noxious heat to stimulate via the TRPV1 receptor in these mice.

LPS was injected i.p. into WT and TRPV1 KO mice to induce endotoxic shock, with pathological characteristics similar to those observed in clinical sepsis in human patients. We measured the early-onset stages, and blood pressure was reduced by 1.5 h after LPS administration. This reduction was potentiated in TRPV1 KO mice in a highly significant manner compared with WT mice after 4 h (Fig. 2A ). These data are in keeping with the concept that TRPV1 exerts a protective role, such that TRPV1 deletion leads to an accelerated onset of endotoxic shock. Endotoxic shock is associated with fever, and in latter stages with a hypothermic response. We carried out our experiments at room temperature (21 ±1°C). An early hyperthermia was observed in both TRPV1 WT and TRPV1 KO mice 30 min after LPS administration, but a later hypothermic response was significantly enhanced in TRPV1 KO mice (Fig. 2B ).

Increased mediator production in TRPV1 KO mice in sepsis
To characterize tissue inflammation, we examined signal transduction and mediator pathways by measuring both TNF-{alpha} and nitrite (as an indicator of NO levels) levels 4 h after LPS injection. TNF-{alpha} levels were significantly raised 1.5 h after sepsis. Furthermore, TNF-{alpha} levels were enhanced in peritoneal lavage fluid from TRPV1 KO mice compared with WT mice, although levels were raised to similar levels in both WT and TRPV1 KO plasma samples (Fig. 3C, D ). Analysis of nitrite levels revealed trends similar trends to those outlined for TNF-{alpha}. A significant increase in nitrite levels was observed at 4 h in peritoneal lavage fluid from TRPV1 KO mice compared with their WT counterparts, despite similar circulating levels being found in the plasma from both TRPV1 WT and KO mice (Fig. 3A, B ). Analysis of intestinal iNOS up-regulation by RT-PCR and measurement of NOS activity, as determined by the citrulline assay in the lung (little NOS activity could be detected in peritoneal exudates), did not indicate a significant difference in either iNOS expression or NOS activity in septic TRPV1 KO mice compared with septic WT mice, with similar increased levels (see Fig. 4A ). Enzyme activity in the lung was largely calcium independent, as determined by assay of samples in the presence of EGTA, a calcium chelator, indicative of a major role for iNOS (see Fig. 4B ).

In separate experiments, WT mice were pretreated with the selective TRPV1 receptor antagonist SB366791 (0.5 mg/kg, i.p.) (27) or vehicle (1% DMSO in saline) 30 min before LPS injection. Mice treated with SB366791 showed enhanced nitrite levels at 4 h in peritoneal lavage fluid compared with vehicle-treated mice, as follows: LPS + vehicle 31.4 ± 8.3 µM compared with LPS + SB366791 97.5 ± 31.4 µM, mean ± SE, n = 6–7 (P<0.05), providing further evidence of a link between lack of functional TRPV1 receptors and increased peritoneal mediator generation. By comparison, treatment of WT mice with a mix of effective doses of the selective tachykinin NK1, NK2, and NK3 receptor antagonists (28 , 29) had no effect on nitrite production induced by LPS as follows: LPS + vehicle (2.5% ethanol in saline) 16.3 ± 2.8 µM compared with LPS + tachykinin receptor antagonists 14.0 ± 2.0 µM, mean ± SE, n = 6.

TRPV1 protects against liver damage
LPS-induced endotoxemia induced significant liver plasma extravasation in TRPV1 KO mice compared with WT mice, as determined by increases of wet weight over dry weight (Fig. 5A ). Little plasma extravasation was observed in either kidney or spleen (Fig. 5B, C ). Aspartate aminotransferase (AST), creatinine, and lipase levels were assessed in the plasma of TRPV1 WT and KO mice in order to determine liver and/or heart, kidney, and pancreas dysfunction, respectively, and thus provide indications of organ failure. In addition, heart/body weight ratio was similar for WT and TRPV1 KO LPS-treated mice (0.44±0.02% vs. 0.46±0.03, n=5–6). Plasma levels of AST present in vehicle-treated mice were compared with those in respective LPS-treated counterparts, and indicated that levels of AST increased significantly in response to LPS in TRPV1 KO but not WT mice (Fig. 5D ), suggesting that LPS induced significant liver damage in KO but not in WT mice. In addition, a comparison of plasma AST levels observed in response to vehicle compared with LPS in WT and KO mice indicated the possibility that liver and/or heart failure was significantly greater in TRPV1 KO mice than in WT. Plasma levels of creatinine and lipase in vehicle-treated and LPS-treated mice were similar (Fig. 5E, F ), indicating no detectable kidney or pancreatic damage. The rise in AST levels is associated with liver damage. To investigate whether there may also be heart damage, measurement of heart weights and H&E analysis of WT and TRPV1KO hearts were determined, but normal histology was observed (results not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
CONCLUDING REMARKS
 REFERENCES
 
The classic TRPV1 agonist capsaicin induced an acute proinflammatory response in the ear after topical application of capsaicin of acute local plasma extravasation in TRPV1 WT mice. This response was absent in TRPV1 KO mice, as predicted (Fig. 1B ), demonstrating the expected phenotype of TRPV1 KO mice. To address the role of TRPV1 as a biological sensor of noxious heat (>43°C), we examined the response in the ear of the anesthetized mice in a model of thermal injury. Once again, plasma extravasation was observed in TRPV1 WT mice but markedly reduced in TRPV1 KO mice. This novel result provides further evidence for the hypothesis that proinflammatory neuropeptides released after TRPV1 activation are involved in tissue injury associated with burn and thermal injury. Thus, the WT mice used in these studies exhibited normal TRPV1 sensitivity that is not observed with TRPV1 KO mice. Furthermore, the colony was characterized previously for its pain-processing phenotype in this laboratory (17) .

To examine the potential involvement of TRPV1 in sepsis, LPS was injected i.p. into WT and TRPV1 KO mice to induce endotoxic shock. We considered it important to follow the early changes observed after infection in order to determine the influence of TRPV1 on early events. Blood pressure was reduced as soon as 1.5 h after induction of endotoxic shock; this was potentiated in TRPV1 KO mice, with a highly significant difference compared with WT mice after 4 h. These data were the first indication of a vascular protective role of TRPV1 in endotoxic shock.

Iida and colleagues (32) have shown that LPS-induced fever was attenuated in TRPV1 KO mice, possibly through peripheral mechanisms, when studied in a warm (30°C) environment in a model that induces a biphasic fever. This is one of the first in vivo indications that activation of TRPV1 may occur in pathophysiological situations at non-noxious temperatures. Thus, it was important to determine how body temperature was affected in response to LPS in our experiments carried out at 21 ± 1°C temperature. The typical early hyperthermia (in keeping with the early hyperdynamic fever phase observed in human sepsis) was, perhaps surprisingly, observed in both TRPV1 WT and TRPV1 KO mice 30 min after LPS administration, but the subsequent hypothermic response was significantly enhanced in TRPV1 KO mice in a manner analogous (on a time-dependent basis) to the hemodynamic changes. Hypothermia is known to correlate in a highly significant manner with mortality in murine sepsis studies (33) and was used here as a more humane sublethal end point than mortality. The results supported the hypothesis that TRPV1 plays a protective role in sepsis. Evidence indicates that >30% of patients who are nursed in intensive care units in Europe suffer from sepsis and that ~30% of these patients die (34) , similar to statistics in the U.S. This high incidence of sepsis has led us to consider that an increased understanding of the basic mechanisms is essential so that novel therapeutic mechanisms can be developed to increase the beneficial effects and reduce harmful components (35) .

TRPV1 protects against inflammatory mediator production in sepsis
Little is known about innate mechanisms that protect against the onset of sepsis, but it is observed as an immune response, primarily defensive but with escalating inflammation, which becomes directly damaging and includes increased level of mediators (e.g., cytokines and nitric oxide). To dissect the mechanisms responsible for the accelerated onset of sepsis here, we examined signal transduction and mediator pathways by measuring TNF-{alpha} and nitrite (as an indicator of NO levels) 1.5 and 4 h after LPS, respectively. The increased susceptibility to LPS-induced hemodynamic and temperature changes was reflected in the measure of significantly raised TNF-{alpha} levels in TRPV1 KO mice peritoneal lavage fluid, indicating that TRPV1 can protect against an acute increased immune response in the peritoneal cavity. By contrast, TNF-{alpha} levels were found to be raised, but to a similar level, in both WT and TRPV1 KO plasma samples obtained by cardiac puncture. The raised TNF-{alpha} levels in the peritoneal exudate of TRPV1 KO mice were surprising, as TNF-{alpha} levels had been shown to be unaltered in a murine mono-arthritis model, where TRPV1 KO mice demonstrated hypoalgesia (17) , in keeping with the concept that TNF-{alpha} may stimulate the sensory hyperalgesia axis in arthritis but that TRPV1 is not a major modulator of TNF-{alpha} levels. However, the present findings indicate that the local enhanced activation of TRPV1 in the peritoneal cavity acts upstream to directly influence TNF-{alpha} production and, as a consequence, enhance the harmful hemodynamic and hypothermic responses in sepsis. This may be related to known links between abdominal vagal afferents and LPS-induced hypotension (36) . Of note, TNF-{alpha} levels have been shown to be increased after capsaicin pretreatment in a rodent model of allergic inflammation, providing support for the concept that loss of the sensory neuronal component enhances TNF-{alpha} production (37) . Alternatively, recent studies have shown a close link between LPS and sensory nerves. The TLR4 receptor has been shown to be localized on sensory nerves (22) . An early interaction between this receptor and sensory nerves could enhance the consequences of TRPV1 activation, leading to an amplification of sensory nerve activation and release of factors such as CGRP. CGRP has been shown to inhibit release of cytokines from macrophages through an increase of cAMP (38) . Indeed, inflammatory responses are observed to be worsened in CGRP KO mice (39) , just as sepsis is worsened in this study when the TRPV1 receptor is deleted.

TNF-{alpha} up-regulates inducible NOS with a concomitant increase in NO generation, probably from activated macrophages (40) . Analysis of peritoneal fluid revealed a significant increase in nitrite levels at 4 h in TRPV1 KOs compared with WT mice in a manner analogous to that observed for TNF-{alpha}. However, circulating levels were similar in both mouse strains, as determined in plasma, again promoting the concept of a link between mediator generation in the gut and systemic sepsis (36) . A major component of NO was from the iNOS source, but analysis of intestinal iNOS up-regulation by RT-PCR and measurement of iNOS activity, as determined by the citrulline assay in the lung, did not indicate a significant increase in either iNOS expression or activity in the septic TRPV1 KO compared with WT mice. It should be noted that the influence on increased iNOS expression in the gut may have occurred before it was possible to sample. Furthermore, iNOS up-regulation has been shown to vary between tissues and therefore may be differentially influenced in different tissues (40) . Indeed, high levels of iNOS generation in the lung of a murine model where sepsis was induced by i.p. LPS could not be inhibited by TNF-{alpha} antibodies whereas other sources could (40) . There is evidence that vanilloid agonists such as capsaicin can mediate anti-inflammatory mechanisms in macrophages (41 , 42) that include inhibition of iNOS induction (43) , but this has been suggested to possibly be related to antioxidant effects of capsaicin, as no evidence for an involvement of TRPV1 has been found so far. LPS-induced endotoxemia induced significant liver plasma extravasation in TRPV1 KO mice when compared with WT mice, as determined by increases of wet weight over dry weight compared with LPS-treated mice. This also correlated with measure of significant levels of the marker of liver injury AST in LPS-treated TRPV1 KO mice vs. LPS-treated WT mice.


   CONCLUDING REMARKS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 CONCLUDING REMARKS
REFERENCES
 
Acute application of capsaicin, the extract of chili peppers, has been known since the 1960s to induce pain and inflammation, but its chronic application, which desensitizes and removes the whole sensory neurogenic component, is traditionally used experimentally to nonselectively remove the TRPV1-containing nerves and is associated with some pain relief. There is an extensive literature on prohyperalgesic/inflammatory mechanisms. However, little is known of the influence of endogenous TRPV1 receptor activation in systemic diseases. Our study provides a fundamental breakthrough: evidence is provided for a new role for TRPV1 in conferring resistance to the important hypotensive and hypothermic components in endotoxemia. Data indicate that loss of TRPV1 is associated with a vulnerability to septic shock and are supported by findings of increased mediator production in the peritoneal cavity and liver damage. The results highlight the importance of investigating the role of TRPV1 receptors in pathophysiology. We think this is a timely and important finding because, although TRPV1 antagonists are being proposed as therapeutic agents, the results provided here suggest they may have deleterious effects on other biological systems.


   ACKNOWLEDGMENTS
 
We thank the BHF (N.C., P.deW.), BBSRC (A.S., L.L., S.D.B., D.S.), Arthritis Research Campaign (J.E.K.), and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico-CNPq Brazil (ESF) for support.

Received for publication December 21, 2006. Accepted for publication May 10, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 CONCLUDING REMARKS
 REFERENCES
 

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