Derangement in gut barrier function occurs in many clinical settings. Endotoxin translocation has been evidenced in some cases and more frequently than in systemic bacterial translocation. Still, endotoxin is only representative of Gram-negative bacteria, microbial translocation may occur more regularly than previously reported after endotoxin measurement. The aim of this study was: to develop a tool allowing us to measure PGN, representative of both Gram-negative and Gram-positive; to address its presence within the bloodstream of patients in a clinical situation known to favor translocation of microbial products; to perform a survey during and after surgery to assess the frequency of the translocation of microbial products; and to attempt linking PGN translocation with the inflammatory process.
AAS is thought to be associated with endotoxin and bacterial translocation through the gut barrier, following manipulation of the gut by the surgeon and aortic clamping. However, evidence to prove this link was difficult to gather, probably because bacteria translocated into the bloodstream are rapidly killed and do not give rise to positive hemocultures [30
]. Several studies, including ours, aimed to address this question by measuring circulating endotoxin [8
]. However, this approach is hindered by the presence of many interfering or blocking molecules (soluble CD14, LPS-binding protein, lipoproteins) [15
]. Thus the determination of LPS levels in the plasma of patients after surgery was not reliable enough, and did not allow for the demonstration that translocation was taking place systematically.
In the present study, we set up a new method for detecting bacterial NOD2 agonist in plasma, using a cell line transfected with NOD2, a general sensor of PGN through its minimal motif MDP [20
], and a reporter gene under the control of the NF-κB transcription factor. Measurement of PGN is relevant in many aspects. First, it is the major component of Gram-positive bacteria and is also found in Gram-negative bacteria. Thus, when LPS detection addresses only Gram-negative bacteria, PGN detection addresses both types. Second, we showed that our system efficiently detected anaerobic bacterial PGN, expected to be more representative of the intestinal flora. Finally, the specificity of our test was confirmed by the use of a frameshift mutant of NOD2, which cannot be activated by bacterial PGN or its fragment [20
]. This system showed that translocation of pathogen-associated molecular patterns (PAMPs) occurred in abdominal aortic surgery with a frequency higher than that measured so far, and that the assay of NOD2 agonist in plasma is a useful and sensitive tool for early detection of a bacterial product in the bloodstream. As the exact nature of circulating PGN is unknown, we used the terms 'NOD2 agonist' to name the circulating PAMP found in patients' plasma. The circulating bacterial material is probably closer to PGN than MDP. Indeed, in the HEK239T test, similarly to biochemically purified PGN, plasma samples led to a positive signal when added after the transfection step, in contrast to MDP, which had to be added in the presence of the transfection reagent in order to be detected. This is the first time that the NOD2 agonist was measured and found in AAS patients. Its detection is more reliable than that of LPS. Our results concur with those of a study of hemorrhagic shock in rat, showing that 30% had detectable amounts of LPS, but 73% were positive for circulating NOD2 agonist [30
On the other hand, CAS patients (our negative control) did not show peaks for circulating NOD2 agonist or endotoxin, and had a relatively lower inflammatory response after the surgery, even if in some rare cases NOD2 agonist was detected in their plasma. Of course the CAS group underwent a shorter and less severe insult (shorter duration of surgery, less blood loss, rare blood transfusion), which may account for the lower inflammatory response on POD1 and POD2. Nevertheless, CAS patients were relevant as a control group for AAS patients. The groups were comparable in terms of weight, sex ratio, and pathology prior to surgery (atherosclerosis, diabetes, smoking habits). They were also undergoing vascular surgery, but without intestinal manipulation for CAS patients. We concluded that bacterial translocation is indeed tightly linked to reclination of the gut during surgery, in agreement with our previous observation [9
]. Indeed, laparatomy and handing are sufficient to induce degradation in the intestinal brush border membrane [36
Regarding medications, especially for statins, the CAS patients were higher consumers than AAS patients, although the difference was not statistically significant. Statins are known to have pleiotropic effects such as a reduction in inflammatory response, stabilization of atheroscleortic plaques, and improvement in vascular endothelial function, as well as a lipid lowering effect [37
]. In our study, statin use had no significant effect on the levels of NOD2 agonist and inflammatory markers in either group. These observations suggest that differences in inflammatory response or circulating NOD2 agonist between the two surgery groups during the observational period were not related to statin use.
As expected, levels of inflammatory markers were higher in AAS than in CAS patients. In AAS patients, all endogenous markers of inflammation increased after circulating NOD2 agonist appeared. From these results, we assumed that bacterial translocation, which occurs before aortic clamping following abdomen incision and gut manipulation, may also contribute to a systemic inflammatory response in AAS patients. As illustrated by some correlations between NOD2 agonist and inflammatory markers (cortisol, IL-10), the presence of circulating NOD2 agonist contributes to the inflammatory response that was associated with a higher number of postoperative complications in AAS patients. Indeed, levels of IL-10 and cortisol are known to correlate with disease severity [8
]. Furthermore, it is worth mentioning that PGN and PGN-derived structures are known to prime cells and enhance a further response to LPS [42
], to synergize with cytokines [43
], TREM-1 ligand [44
], and other PAMPs [45
] to favor inflammation, and to induce shock and organ dysfunction in vivo