In this study, we have shown that peritoneal macrophages, obtained from patients with cirrhosis and AF, and the presence of bactDNA are primed to synthesise significantly higher amounts of NO than macrophages obtained from patients without bactDNA, and this is associated with marked activation of the cytokines implicated in the type 1 immune response.
Bacterial infections are common complications in patients with advanced cirrhosis, and SBP is the most frequent and clinically relevant.1
The classical pathogenic theory of SBP suggests that bacteria of intestinal origin move across the intestinal wall,5
reaching mesenteric lymph nodes and other organs. Bacteria can then obtain access to AF, and a SBP episode may eventually develop if the local bactericidal mechanisms are insufficient to mount an adequate response.16,17
We have recently described the presence of bactDNA in patients with cirrhosis and culture negative non-neutrocytic AF, a fact that we interpret as molecular evidence of BT.6
It is likely that bacteria reaching AF may activate the cellular component of the innate immune system, modifying its inflammatory response and NO synthesising ability, a situation similar to what has been described in in vitro models.9,18
Thus the aim of this study was to assess the macrophage immune response according to the presence or absence of bactDNA in patients with cirrhosis.
BactDNA is characterised by the presence of short repeated sequences of unmethylated CpG dinucleotides. Different experimental studies have shown the immunomodulatory role of these fragments, capable of inducing a similar immune response to that produced by a complete microorganism in vitro,19–21
thus becoming potent activators of cells of the innate immune system (namely, NK, macrophages, and dendritic cells) by binding toll-like receptor 9.7
BactDNA mediated macrophage activation leads to TNF-α, IL-6, and IL-12 synthesis.20,22
These last two cytokines activate B and T cells23,24
and modulate IFN-γ production by contributing to NK cell activation in vivo and in vitro.8,25,26
As IFN-γ receptors are also expressed on the macrophage surface, the presence of increased levels of IFN-γ further enhances TNF-α production and upregulates the transcription of the iNOS gene27–32
leading to an increase in macrophage NO synthesising capability.
Peritoneal macrophages from cirrhotic patients with ascites and a previous episode of SBP have been shown to be activated, with NO overproduction,33
a situation similar to that described here, as suggested by the fact that NOx levels were higher in patients with molecular evidence of BT (group II) compared with patients in group I (table 2). Also, the existence of a direct and significant correlation between iNOS and NOx levels strongly suggests that NOx detected in the supernatant of cultures of peritoneal macrophages is iNOS derived. One of the best described functions of iNOS is its role in the macrophage mediated response to infectious agents.11,12
Furthermore, iNOS produces physiological concentrations of NO in the nanomolar range.34
Micromolar concentrations observed in our study are consistent with these data, considering overactivation of macrophages in response to bacterial DNA.
As described previously, the increase in NO synthesis is not an isolated phenomenon but a consequence of a complex sequence of events in which several cytokines play a significant role. In fact, NO has been shown to downregulate cytokine release from macrophages.35
Supporting this, the reported differences in the ability of macrophages to synthesise NO is associated with a marked inflammatory scenario. We observed significant basal differences in all four cytokines evaluated (table 2) between both groups of patients, which indicates two clearly differentiated populations according to the absence (group I) or presence (group II) of bactDNA.
We observed a positive correlation between IL-2, IL-12, and IFN-γ and iNOS values, and the cytokine dependent induction of iNOS has been reported previously.36
Elevated production of IFN-γ by CpG DNA activated NK cells is mediated by IL-1237
and, in fact, we observed a significant correlation between basal levels of these two cytokines, which clearly suggests activation of a type I immune response in these patients. Supporting this assumption, TNF-α, which is directly synthesised by macrophages and also enhanced by IFN-γ, was significantly higher in patients in group II compared with group I.
In summary, peritoneal macrophages from patients with cirrhosis and ascites and the presence of bactDNA are primed for higher cytokine and NO synthesis. Our data suggest identification of a new subset of patients, with a marked pre-activation of the cellular component of the immune system, probably in response to the immunogenicity of CpG motifs present in bactDNA.