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Published ahead of print on January 18, 2007, doi:10.1164/rccm.200608-1103OC
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American Journal of Respiratory and Critical Care Medicine Vol 175. pp. 595-603, (2007)
© 2007 American Thoracic Society
doi: 10.1164/rccm.200608-1103OC

Original Article

Selective NOD1 Agonists Cause Shock and Organ Injury/Dysfunction In Vivo

Neil Cartwright1,*, Oliver Murch2,*, Shaun K. McMaster1, Mark J. Paul-Clark1, David A. van Heel3, Bernhard Ryffel4, Valerie F. J. Quesniaux4, Timothy W. Evans1, Christoph Thiemermann2,* and Jane A. Mitchell1,*

1 Department of Critical Care, National Heart and Lung Institute, Imperial College, London, United Kingdom; 2 The William Harvey Research Institute, and 3 Institute of Cell and Molecular Science, Queen Mary's School of Medicine and Dentistry, Barts and The London, London, United Kingdom; and 4 Transogenose Institute, Orleans, France

Correspondence and requests for reprints should be addressed to Professor Jane A. Mitchell, Ph.D., Department of Critical Care, National Heart and Lung Institute, Imperial College, Dovehouse Street, London SW3 6LY, UK. E-mail: j.a.mitchell{at}imperial.ac.uk


   ABSTRACT
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 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: NLRs (nucleotide oligomerisation domain [NOD] proteins containing a leucine-rich repeat) are cytosolic pattern recognition receptors. NOD1 senses diaminopimelic acid–containing peptidoglycan present in gram-negative bacteria, whereas NOD2 senses the muramyl dipeptide (MDP) present in most organisms. Bacteria are the most common cause of septic shock, which is characterized clinically by hypotension resistant to vasopressor agents. In animal models, gram-negative septic shock is mimicked by lipopolysaccharide (LPS), which signals through Toll-like receptor 4 (TLR4) and its adaptor MyD88. The role of NLRs in the pathophysiology of septic shock is not known.

Objectives: To compare the effects of selective NOD1 agonists with LPS in vivo.

Methods: Vascular smooth muscle cells or whole aortas from wild-type or genetically modified mice were stimulated in vitro with agonists of NOD1 (FK565) or NOD2 (MDP). Vasoconstriction was measured using wire myography. Nitric oxide (NO) formation was measured using Griess reaction and NO synthase-II protein by Western blotting. In vivo, blood pressure, heart rate, and urine output were measured in sham-, LPS-, or FK565-treated animals. Biomarkers of end-organ injury, coagulation activation, NO, and cytokines were measured in plasma.

Main Results: FK565, but not MDP, induced NO synthase-II protein/activity in vascular smooth muscle and vascular hyporeactivity to pressor agents. FK565 had no effect on vessels from NOD1–/– mice, but was active in vessels from TLR4–/–, TLR2–/–, or MyD88–/– mice. FK565 induced hypotension, increased heart rate, and caused multiple (renal, liver) injury and dysfunction in vivo.

Conclusions: Activation of NOD1 induces shock and multiple organ injury/dysfunction.

Key Words: peptidoglycan • lipopolysaccharides • receptors • pattern recognition



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
There is little information on the role of NOD proteins in contributing to the pathophysiology of sepsis-induced organ dysfunction.

What This Study Adds to the Field
Activation of NOD1 induces shock and multiple organ injury/dysfunction.

 
There are an estimated 750,000 cases of sepsis in the United States each year, an incidence that is likely to increase (1, 2). Sepsis and its sequela, septic shock, carry a mortality rate of up to 64% (3). Although there has been a fall in mortality rates over the last 30 years, a rise in incidence means the total number of deaths has increased (4). The pathophysiology of sepsis is complex and the patient population heterogeneous, confounding progress in the search for new treatments (5). Pattern recognition receptors (PRRs) are germline-encoded receptors that sense specific pathogen–associated molecular patterns (PAMPs) and activate inflammation. PRRs include transmembrane Toll-like receptors (TLRs) and cytosolic nucleotide oligomerization domain (NOD) proteins containing a leucine-rich repeat (NLRs) (6).

TLR4 senses lipopolysaccharide (LPS), the cell wall component of gram-negative bacteria. When given in vivo to animals, LPS produces signs indistinguishable from septic shock (7), and in vitro, induces nitric oxide synthase II (NOSII) in murine vessels in a TLR4-dependent manner (8, 9). Studies of polymorphisms in humans indicate TLRs and related signaling proteins play an essential role in sepsis, and may also explain the variable response to infection seen among individuals (10, 11). Furthermore, TLR4 antagonists are being developed (12), although their role in sepsis has yet to be assessed. There is also evidence that peptidoglycan (PepG) from gram-positive organisms causes inflammation (13, 14) and acts in synergy with lipoteichoic acid, another wall fragment of gram-positive bacteria, to induce systemic inflammation, shock, and multiple organ injury/dysfunction (15). Until recently, it was believed that TLR2 was the receptor for PepG, but this has been disproved (16). Indeed, NLRs have been shown to be PRRs for bacterial PepG in that NOD1 senses diaminopimelic acid (DAP)–containing PepG from mainly gram-negative bacteria (17) and NOD2 senses the muramyl dipeptide (MDP) present in most bacterial PepG (18). FK565 is a synthetic agonist of NOD1 (19) and synthetic MDP was used here as an agonist of NOD2 (20). Both NOD1 and NOD2 signal via the adaptor protein apoptosis-regulatory protein kinase [RICK; also known as Rip2 or CARDIAK (caspase recruitment domain containing interleukin-1beta converting enzyme associated kinase)]. RICK and NOD1 or NOD2 both contain caspase recruitment domains that interact to form a signaling platform. The ensuing signaling cascade leads to the activation of nuclear factor (NF)-{kappa}B (21). Synthetic NOD1 agonists stimulate chemokine production and neutrophil recruitment in vivo (22), but alone are ineffective in activating monocytic cells (23). The role of NLRs in sepsis is not known. We therefore explored the hypothesis that NOD1 represents an important receptor in the development of gram-negative septic shock.


   METHODS
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 ABSTRACT
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 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
NOD Agonists
The NOD1 agonist FK565 was obtained from Fujisawa Pharmaceutical, Japan, and the NOD2 agonist MDP-Lys18 from Nopia, Daiichi Pharmaceutical, Japan.

Cell Culture and Western Blotting
Rat arterial vascular smooth muscle cells were cultured and NOSII expression was assessed by Western blotting as previously described (24). Briefly, cells were treated with FK565 (10 nM), MDP-Lys18 (10 nM), or medium alone (control) for 48 hours, before lysis and protein separation by gel electrophoresis. NOSII was detected using an antibody specific to rat NOSII.

Organ Culture
Culture of murine aortas was performed as described previously (9). Aortas from C57BL6, NOSII–/– (25), NOD1–/–, TLR4–/–, or TLR2–/– mice were cultured for 24 hours in medium alone (control) or in medium containing FK565 (100 nM), FK156 (1 µM), or MDP-Lys18 (1 µM).

Myography
Aortic rings from organ culture or ex vivo were mounted on a wire myograph in physiologic saline solution containing L-arginine (10–3 M) at 37°C, bubbled with 95% O2/5% CO2. The vessel length was accurately measured and a standard start-up procedure was used (26). Vessels were contracted using a cumulative dose of phenylephrine (10–8 – 3 x 10–6 M) and contractile force was measured. Acetylcholine (10–5 M) was added to the maximally contracted vessels. After washing, L-NAME (10–3 M) was added to the vessels on the myograph 30 minutes before repeating the phenylephrine dose–response measuring force.

In Vivo Hemodynamic Measurements
Twenty-nine male Wistar rats were used maintained and managed experimentally according to U.K. Home Office guidelines (Scientific Procedures Act 1986). Animals were anesthetized with sodium thiopentone and instrumented as previously described (27). Blood pressure was monitored via a carotid cannula using a pressure transducer and data acquisition system. A jugular venous cannula was inserted for intravenous drug administration and a suprapubic catheter inserted to quantify urine output. A tracheostomy was performed to protect the airway of the anesthetized animals, which breathed spontaneously throughout the experiment. Mean arterial pressure and heart rate were calculated. Arterial blood samples were drawn from the carotid cannula at 90 minutes into heparinized tubes and at 5 hours into heparinized and serum tubes. Plasma and serum were isolated from the respective samples using a centrifuge and stored at –80°C. Plasma biochemical markers, urea, creatinine, creatine kinase (CK), aspartate amino transferase (AST), alanine aminotransferase (ALT), bilirubin, lipase, and amylase, were determined from the serum samples. The 90-minute plasma sample was used to determine tumor necrosis factor (TNF)–{alpha} levels. The 5-hour plasma sample was used to determine TNF-{alpha}, IL-1beta, IL-6, IL-10, thrombin–antithrombin III complex (TAT), and nitrate concentrations. Plasma concentrations of cytokines and TAT were determined by ELISA (28).

Measurement of Nitric Oxide
Nitric oxide (NO) was measured by the cumulative formation of nitrite in culture medium by the Griess reaction (29). Nitrate in rat plasma was enzymatically converted to nitrite. Subsequently, total nitrite in the serum was assayed by the Griess reaction (30).

Histology and Determination of Myeloperoxidase Activity in Rat Lungs
Lungs were taken from the rats at the end of the experiment and samples were frozen in liquid nitrogen or fixed in formalin solution. The fixed samples were sectioned and stained with hematoxylin and eosin for histologic examination. The frozen samples were used for determination of myeloperoxidase (MPO) activity (31).

All statistical anaylyses were performed using Prism version 4.0c for Macintosh (Graphpad Software Inc., San Diego, CA); p < 0.05 was considered significant.

Additional details of the methods used are provided in the online supplement.


   RESULTS
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 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hemodynamic Effects of FK565 or LPS In Vivo
Intravenous administration of LPS (1 mg/kg) did not significantly alter blood pressure when compared with sham-operated control rats (two-way analysis of variance [ANOVA]; n = 7, p > 0.05). LPS (6 mg/kg intravenously) induced significant hypotension within 1 hour of administration (two-way ANOVA, Bonferroni posttest; n = 7, p < 0.01), which recovered initially before the animals again became significantly hypotensive at 5 hours (p < 0.01). FK565 (1 mg/kg) induced significant hypotension after 3 hours of intravenous administration, which lasted until the end of the observation period (two-way ANOVA, Bonferroni posttest; n = 8, p < 0.01). Furthermore, the mean arterial blood pressure of the FK565-treated rats was significantly lower than that of those treated with 6 mg/kg LPS between 3 and 5 hours after administration (two-way ANOVA, p < 0.01) (Figure 1A).


Figure 1
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  		Figure 1. Hemodynamic effects after intravenous injection of FK565 or LPS in rats. (A) Mean arterial blood pressure (MAP) was significantly reduced by FK565 at 3–5 hours (p < 0.05) and by LPS (6 mg) after both 1 hour (p < 0.05) and 5 hours (p < 0.05). LPS (1 mg/kg) did not significantly affect MAP. (B) Heart rate was significantly increased in animals receiving LPS (1 or 6 mg/kg) or FK565 from 2–5 hours (p < 0.05). A, B: Solid squares, control; open squares, FK565; solid inverted triangles, LPS 1 mg/kg; solid diamonds, LPS 6 mg/kg. *p < 0.05, analysis by two-way analysis of variance (ANOVA) with Bonferroni posttest analysis.

 
When compared with sham-operated control rats, intravenous LPS (1 mg/kg or 6 mg/kg) caused significant tachycardia from 3 to 5 hours after administration (two-way ANOVA with Bonferroni posttests, p < 0.05). FK565 (1 mg/kg) also caused tachycardia between 3 and 5 hours after administration (two-way ANOVA with Bonferroni posttests, p < 0.05). There was no significant difference between the heart rates of rats receiving LPS (1 or 6 mg/kg) and those receiving FK565 (1 mg/kg) (Figure 1B).

Effects of FK565 or LPS on End-organ Function and Injury
Compared with sham-operated animals, administration of both FK565 (1 mg/kg) and LPS (6 mg/kg only) resulted in significant increases in serum urea and creatinine levels indicating renal dysfunction (one-way ANOVA, p < 0.05). Rats receiving low-dose LPS (1 mg/kg) had elevated levels of urea (one-way ANOVA, p < 0.05), but not creatinine (Figures 2A and 2B). CK was elevated at both doses of LPS (1 and 6 mg/kg; one-way ANOVA, p < 0.05), but was not significantly elevated in FK565-treated animals (one-way ANOVA, p > 0.05) (Figure 2C). AST and ALT were elevated in rats treated with LPS (6 mg/kg) (one-way ANOVA, p < 0.05), but not in those receiving FK565 (1 mg/kg) or LPS (1 mg/kg) (Figures 2D and 2E). Serum levels of amylase, a marker of pancreatic injury, were elevated after both doses of LPS (1 and 6 mg/kg; one-way ANOVA, p < 0.05), but not after FK565 (1 mg/kg) (Figure 2F). Finally, TAT, an indicator of the level of activation of the coagulation system, was significantly elevated by FK565 (1 mg/kg) and high-dose (6 mg/kg; one-way ANOVA, p < 0.01), but not low-dose (1 mg/kg) LPS (Figure 2G).


Figure 2
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  		Figure 2. Alterations in the serum levels of (A) urea, (B) creatinine, (C) creatinine kinase (CK), (D) aspartate aminotransferase (AST), (E) alanine aminotransferase (ALT), (F) amylase, and (G) thrombin–antithrombin III complex (TAT) after sham operation or exposure to FK565 (1 mg/kg) or LPS (1 or 6 mg/kg). *p < 0.05 and **p < 0.01 by ANOVA when comparing treated animals to sham-operated control animals with Dunnett's post hoc test.

 
Effects of FK565 or LPS on Neutrophil Recruitment to the Lung
There was significantly increased MPO activity measured in the lungs of rats treated with 1 mg/kg LPS (one-way ANOVA, p < 0.05) and 6 mg/kg LPS (one-way ANOVA, p < 0.01). However, lungs from rats treated with FK565 did not show greater MPO activity than in lungs from sham-operated control rats (Figure 3E). This finding was also supported by histologic examination of the lungs. Lungs from sham-operated control rats and rats treated with FK565 had normal histologic appearances, whereas lungs from both LPS groups showed interstitial edema and infiltration by inflammatory cells (Figures 3A–3D).


Figure 3
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  		Figure 3. Lung histology (hematoxylin–eosin staining) at 5 hours. (A) Sham group: no inflammation. (B) LPS 1-mg/kg group: inflammatory cell infiltration is observed, especially neutrophils in the interstitium and airspace of the lung. Interstitial edema and vascular congestion are observed. (C) LPS 6-mg/kg group: profound inflammatory cell infiltration is observed, especially neutrophils in the interstitium and airspace of the lung. Marked interstitial edema and vascular congestion are observed. (D) FK565 1-µg/kg group: no inflammatory change is observed compared with sham-treated animals. Original magnification, x26. (E) Myeloperoxidase (MPO) levels, a marker for neutrophils, is elevated in lungs from rats given LPS (1 and 6 mg/kg) but not in rats given FK565 (1 mg/kg). *p < 0.05, **p < 0.01. Analysis by one-way ANOVA with Dunnett's multiple-comparison posttest.

 
Effects of FK565 or LPS on Cytokine Production In Vivo
At 90 minutes, serum levels of TNF were elevated at both doses of LPS (one-way ANOVA, p < 0.01) (1 and 6 mg/kg). LPS (6 mg/kg) caused a greater increase in TNF than LPS (1 mg/kg). By contrast, FK565 did not significantly increase TNF levels at 90 minutes (Figure 4A). However, there was a modest but significant increase of TNF at 5 hours in the FK565-treated rats (one-way ANOVA, p < 0.01). At the 5-hour time point, both LPS groups showed lower levels of TNF than at 90 minutes, although the 6-mg/kg group still had significantly raised TNF levels (one-way ANOVA, p < 0.05) (Figure 4B). Serum IL-6 and IL-1beta were raised in the LPS (6 mg/kg) and FK565 (1 mg/kg) groups (one-way ANOVA, p < 0.01), but not in the LPS (1 mg/kg) group (Figures 4C and 4E, respectively). Serum IL-10 was elevated in all groups when compared with sham-operated control rats (one-way ANOVA, p < 0.05) (Figure 4D).


Figure 4
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  		Figure 4. Alterations in the serum levels of (A) tumor necrosis factor (TNF) at 90 minutes, and (B) TNF, (C) IL-6, (D) IL-10, and (E) IL-1beta at 5 hours. *p < 0.05, **p < 0.01 by one-way ANOVA using Dunnett's multiple-comparison posttest.

 
Effects of In Vivo FK565 or LPS on Vascular Contractility Ex Vivo
Aortas taken from rats after sham operation were responsive to phenylephrine (Figure 5A). Aortas from rats treated with LPS (1 or 6 mg/kg) were hyporesponsive to phenylephrine (two-way ANOVA, p < 0.01 in both instances). Similarly, aortas from FK565-treated rats were hyporesponsive to phenylephrine (one-way ANOVA, p < 0.01) when compared with control rats (Figure 5A). The vascular hyporeactivity afforded by both FK565 (Figure 5B) and LPS (data not shown) was reversible with L-NAME (10–3 M). When compared with control rats, the serum of the LPS-treated (1 or 6 mg/kg) or FK565-treated (1 mg/ kg) rats showed increased levels of nitrite/nitrate (one-way ANOVA, p < 0.01; Figure 5C), indicating increased NO formation. The increase in plasma nitrite/nitrate in animals that had received either LPS (1 or 6 mg/kg) or FK565 (1 mg/kg) was not significantly different.


Figure 5
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  		Figure 5. Aortas were mounted on the myograph ex vivo after 5 hours of sham operation (control) or exposure to LPS or FK565. (A) Vessels were hyporesponsive to phenylephrine after exposure to 1 or 5 mg/kg of LPS or 1 mg/kg FK565 when compared with control responses (n = 6, p < 0.001 in all cases). (B) Hyporesponsiveness to phenylephrine induced by in vivo exposure to FK565 was reversible by L-NAME (10–3 M; n = 6, p < 0.001). (C) Levels of serum nitrite after nitrate reductase step, assessed by the Griess reaction. **p < 0.01 by one-way ANOVA using Dunnett's multiple-comparison posttest.

 
Effects of FK565 or MDP-Lys18 on Vascular Contractile Function In Vitro
Incubation of aortas from wild-type mice with FK565 (100 nM) induced vascular hyporeactivity to phenylephrine (two-way ANOVA, p < 0.001) (Figure 6A). The hyporeactivity was significantly, although not fully, reversed by the NOS inhibitor L-NAME (10–3 M; two-way ANOVA, p < 0.001) (Figure 6D). However, vessels from NOSII–/– mice incubated with FK565 contracted slightly more to phenylephrine than did control-treated vessels (100 nM; two-way ANOVA, p = 0.0033) (Figure 6C). Blood vessels from NOD1–/– mice also did not develop vascular hyporeactivity to phenylephrine when incubated with FK565 (100 nM; two-way ANOVA, p = 0.08) (Figure 6B). MDP-Lys18 (1 µM) had no effect on the contractility of aortas from wild-type mice (two-way ANOVA, p = 0.3523) (Figure 6E).


Figure 6
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  		Figure 6. Effect of culture in vitro with FK565 or MDP-Lys18 on contractility of murine aortas. Control responses were obtained from vessels cultured in media alone. (A) After culture for 24 hours with FK565 (100 nM) vessels from wild-type mice were hyporesponsive to phenylephrine (n = 5, p < 0.001). (B) Vessels from NOD1–/– mice were unaffected by culture with FK565 (n = 5, p = 0.1926). (C) After incubation with FK565 vessels from NOSII–/– mice displayed a small increase in contractility (n = 5, p = 0.033). (D) The hypocontractility induced by FK565 in wild-type vessels was significantly reversed by L-NAME (n = 5, p < 0.001). (E) MDP-Lys18 (1 µM) had no significant effect on contractility of wild-type vessels after 24-hour culture in vitro (n = 6, p = 0.3523). Analysis by two-way ANOVA. NOD = nucleotide oligomerization domain; NOS = nitric oxide synthase II.

 
FK565 (100 nM) induced hyporesponsiveness to phenylephrine in aortas from TLR2–/– (two-way ANOVA, p < 0.001; Figure 7A), TLR4–/– (two-way ANOVA, p < 0.001; Figure 7B), and MyD88–/– (two-way ANOVA, p < 0.001; Figure 7C) animals. In all instances, this was partially reversible with L-NAME (data not shown).


Figure 7
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  		Figure 7. Effect of culture in vitro with FK565 on Toll-like receptor (TLR) or MyD88 knockout murine aortas. Control responses were those obtained in vessels cultured in media alone. (A) After culture for 24 hours with FK565 (100 nM), vessels from TLR2–/– mice were hyporesponsive to phenylephrine (n = 5, p < 0.001). (B) Vessels from TLR4–/– mice were also hyporesponsive to phenylephrine after culture with FK565 (n = 5, p < 0.001). (C) Vessels from MyD88–/– mice were hyporesponsive after culture with FK565 (n = 4, p < 0.001). Analysis by two-way ANOVA.

 
Effects of NOSII Activity and Expression in Vascular Smooth Muscle Cells
Under control culture conditions, vascular smooth muscle cells did not express NOSII protein. However, there was an increase in NOSII expression in vascular smooth muscle cells after incubation with FK565 for 24 hours (Figure 8A). In line with this, nitrite levels in the medium of vascular smooth muscle cells at 24 hours were increased in a concentration-dependent manner by the addition of 1 to 10 nM of FK565 to the culture medium (Figure 8B). By contrast, the selective NOD2 ligand MDP-Lys18 did not increase NOSII expression (Figure 8A) or nitrite levels (Figure 8C).


Figure 8
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  		Figure 8. The effects of FK565 and MDP-Lys18 on cultured vascular smooth muscle cells. (A) Western blotting for NOSII protein, showing that FK565 (10 nM) induced NOSII expression in cultured vascular smooth muscle cells at 48 hours. By contrast, MDP-Lys18 (10 nM) does not induce NOSII. (B) FK565 (0.1–10 nM) induced significant increases in the release of NO from vascular smooth muscle cells at 10 nM (n = 3, p < 0.05) (measured by the oxidation product nitrite by Griess assay). (C) MDP-Lys18 (0.1–10 nM) did not cause significant release of NO. **p < 0.01 by Kruskal-Wallis with Dunn's posttest analysis.

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
These data show that FK565, a NOD1 agonist, induces the features of septic shock in rats. Furthermore, this action was specific to NOD1, because all effects on blood vessels are blocked in the NOD1–/– mice, with no contribution (or contaminating factors) acting via TLR2 or TLR4 sensing. Furthermore, signaling of FK565 was MyD88 independent in that the actions of TLR4 and TLR2 agonists in blood vessels are MyD88 dependent (8). FK565 contains the chemical motif iE-DAP, the minimum essential structure for recognition by NOD1 (17), which is found in gram-negative bacterial PepG. Although it is usually locked into the larger PepG structure, smaller molecules containing the iE-DAP portion are released during bacterial growth and replication through the action of bacterial amidases, and by the host responses or beta-lactamase antibiotics. The route that these molecules enter the cells to reach the cytosolic NOD1 receptor has not been identified, although human peptide transporter 1 (HPepT1) represents a possible intracellular transport pathway for PepG fragments (32).

The mechanism via which FK565 causes shock is not fully elucidated. However, we show here that FK565 is sensed directly by vascular smooth muscle cells in culture and by whole blood vessels in vitro, leading to the expression of NOSII. Vascular smooth muscle appears to mediate the vascular dysfunction. However, in vivo, NOD1 is likely to be an important receptor in endothelial cells. The excess NO produced by NOSII, which acts as a potent vasodilator, is an important mediator of septic shock in rodent models (33), although other vasoactive mediators have been implicated in the development of septic shock (34). In our model, there is clear evidence of the role of NO in vivo, with elevated plasma nitrate levels in the LPS- and FK565-treated groups and significant though incomplete reversibility with L-NAME. In vitro, there is clear dependence on NOSII, as demonstrated in NOSII–/– mice. Although incomplete reversibility of vascular hyporesponsiveness with L-NAME may be attributed to incomplete inhibition of NOSII, we do not rule out the possibility that other mediators (e.g., cyclooxygenase [COX]-II) may play a role in the vascular dysfunction.

LPS and FK565 differ in their ability to activate "professional" immune cells, such as dendrites and monocytes. Although LPS is a potent activator of these immune cells through TLR4, NOD1 agonists only weakly activate them and only at high concentrations (23). The lack of activation of professional immune cells is reflected in the different cytokine profiles and tissue injury markers seen in the LPS- versus FK565-treated animals. Thus, although 1 mg/kg of LPS was not enough to induce septic shock, there was clearly an increase in serum TNF at 90 minutes. This response was absent, or at least delayed, after administration of FK565. Furthermore, 6 mg/kg of LPS induced a significant TNF response at 1 hour and there was a corresponding fall in blood pressure, which had recovered by 2 hours. FK565 did not induce significant levels of TNF at 1 hour and there was also no fall in blood pressure at this time. Similarly, IL-1beta was not elevated in the serum of the FK565-treated rats, although it was elevated in the LPS 6-mg/kg group. TNF and IL-1beta are considered the mediators of septic shock, but our own model did not implicate these cytokines, which may provide important clues as to why anti-TNF or anti–IL-1beta therapies have had limited success in the treatment of sepsis.

The pattern of end-organ injury also differs between LPS (6 mg/kg) and FK565 (1 mg/kg). Although in both groups there was evidence of renal dysfunction and activation of the coagulation system, only in the LPS-treated animals was elevation in AST, ALT, CK, and amylase detected, indicating end-organ tissue damage. Also, there was no evidence of lung injury from histologic examination of the tissues in the FK565-treated rats, but LPS-treated rats had inflammation, with exudates and inflammatory cell infiltration further supported by MPO assays.

In the clinical setting, the host is exposed to both NOD1 ligands and LPS derived from gram-negative bacteria, as well as many other PAMPs. Furthermore, NOD1 ligands have been shown to synergize with other TLR agonists, including LPS, in their action on monocytic cells (35). In the current study, we identify an important new pathway, through NOD1, which mimics the cardiovascular and organ failure associated with septic shock. We do not elucidate the full signaling pathway for NOD1 in this study. LPS- or IL-1beta–induced NOSII may be mediated via Janus kinase (JAK)/signal transducers and activators of transcription (STAT), mitogen-activated protein kinases, and/or NF-{kappa}B. Although we showed previously that the effect of LPS on vessels is wholly TLR4 dependent (9), we cannot exclude the possibility that some effects of LPS are via NOD1, either directly or due to contaminants. However, the identification of a putative role for NOD1 in shock illustrates its potential as a novel therapeutic target.


   FOOTNOTES
 
Supported by the British Heart Foundation and Medical Research Council, UK. D.A.v.H. is supported by a Wellcome Trust Clinician Scientist grant.

* These authors contributed equally to this article and share first authorship. Professors Mitchell and Thiemermann are joint last authors. Back

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200608-1103OC on January 18, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form August 7, 2006; accepted in final form December 18, 2006


   REFERENCES
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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