The aim of this study was to assess the frequency of the two most common polymorphisms C677T and A 1298C of the methylenetetrahydrofolate reductase (MTHFR) gene, as well as the coexistence of both these genetic variants in healthy subjects from part of the Algerian population (Aures Region). A total of 94 apparently healthy subjects were enrolled in the study group. The frequency of the both investigated genotypes of the both MTHFR gene polymorphisms (C677T and A1298C) was determined by using the Real-Time Polymerase Chain Reaction-Fluorescence Resonance Energy Transfer (Real-Time PCR-FRET) technic. The frequencies of C and T alleles of C677T polymorphism were 127 (67.55%), 61 (32.45%), and for CC, CT, and TT genotypes were 44 (46.48%), 39 (41.8%) and 11 (11.70%) respectively. Regarding the frequencies at position 1298, for A and C alleles were 147 (78.19%), 41 (21.81%), and for AA, AC, and CC genotypes were 60 (63.82%), 27 (28.72%) and 7 (7.44%) respectively. Also, our results indicated that no significant differences in the percentage distributions of the C677T (P=0.518) and A1298C (P=0.514) polymorphisms between males and females carriers. As noted in the findings, the most frequent coexistence of genotypes were 677CT/1298AA (29.78%), 677CC/1298AA (22.34%) and 677CC/1298AC (17.02%%). The coexistence of 677TT/1298AA (11.70%), 677CT/1298 AC (11.70%) and 677CC/1298 CC (7.44%) genotypes was observed less frequently and for 677TT/1298AC, 677CT/1298CC, 677TT/1298CC genotypes, it has been no observed in the studied population. The frequencies of MTHFR 677 C and T alleles were 0.66 and 0.31, whereas those of MTHFR1298 A and C alleles were 0.77 and 0.21, respectively. The allelic distributions of the C677T polymorphism remain intermediate in the Aures region (Northeast of Algeria); that support the idea of a north-south gradient. For the A1298C SNP, our funding appears to be lower compared across populations. In addition, the frequency and coexistence of genotypes of the C677T and A 1298C MTHFR gene polymorphisms in the region studied are similar to other ethnic group populations.

OBJECTIVE:

   Cirrhosis downregulates phagocyte oxidant production via their antibacterial superoxide-generating system, NADPH oxidase (NOX2) and increases patients' susceptibility to infection and mortality rate. To explore novel biochemical parameters that explain susceptibility to infections, we investigated the expression of NOX2 and partners in neutrophils of patients with severe alcoholic cirrhosis and have provided a novel approach to restore superoxide production capacity in patients' neutrophils and blood.

DESIGN:

   Neutrophils were isolated from patients with decompensated alcoholic cirrhosis. NOX2 activity was assessed after stimulation of purified neutrophils or whole blood with the bacterial-derived peptide fMet-Leu-Phe. The expression of NOX2 and partners was studied by western blot analysis, flow cytometry and reverse transcription-PCR.

RESULTS:

   The impaired superoxide production by patients' neutrophils was associated with a severe deficient expression of the NADPH oxidase catalytic core flavocytochrome-b558 (gp91 ^phox /NOX2 and p22 ^phox ), its cytosolic partner p47 ^phox but not p67 ^phox . NOX2 expression decreased rapidly by protein degradation involving elastase released during degranulation of healthy neutrophils stimulated with fMet-Leu-Phe, or highly present in patients' plasma. Interestingly, the deficient superoxide production was reversed by treatment of patients' neutrophils and whole blood with toll-like receptor 7/8 (TLR7/8) agonists. This treatment stimulated a rapid NOX2 transcription and translation through a process involving mammalian target of rapamycin (mTOR) whose expression was also deficient in patients' neutrophils. NOX2 expression was also increased by the TLR4 agonist lipopolysaccharide but with only a modest improvement of reactive oxygen species production.

CONCLUSION:

   Impairment of neutrophil oxidants production in alcoholic cirrhosis is associated with NOX2 degradation and deficient mTOR-dependent translational machinery. The NOX2 depletion can be reversed via TRL7/8 activation and might be used to restore antimicrobial responses of immunocompromised patients.

   Polymorphonuclear neutrophils (PN) play a key role in host defense mechanisms against microbial infections. This innate function is dependent on two major PN responses ; a massive production of superoxide anion (02-°) by the NADPH oxidase system (a phenomenon termed respiratory burst) leading to the formation of hydrogen peroxide (H2O2), and the release of granular enzymes (degranulation) among which Myeloperoxidase (MPO) which uses H2O2 to kill bacteria. In some pathologies such as decompensated alcoholic cirrhosis, patients exhibit an increased susceptibility for mostly lethal infections suggesting deficiencies of defensive responses of PN.
   In this study, we compared the MPO release from PN of these cirrhotic patients and healthy volunteers in response to stimulation by the bacterial peptide fMet-Leu-Phe(fMLP), and the activation state of major signaling effectors potentially involved(AKT, p44/42-MAP kinases (ERK1/2) et p38-MAP-kinases). In addition, the, respective contribution of the three signaling effectors in MPO degranulation was explored using healthy PN and pharmacological antagonists. Our results show that fMLP-induced myeloperoxidase release was strongly impaired in patients’ neutrophils whereas the intracellular myeloperoxidase stock was unaltered. The fMLP-induced activation of signaling effectors measured by their phosphorylated forms was also strongly deficient despite a normal expression of the three effectors and the fMLP receptor, fPR. However, based on the pharmacological inhibition of the signaling effectors in healthy neutrophils, AKT and p38-MAPK, but not ERK1/2, up-regulated fMLP-induced myeloperoxidase exocytosis. Interestingly, patients’ neutrophils also exhibited a defective bactericidal capacity that was reversed ex-vivo by the Toll-like receptor 7/8agonist CL097. This agent potentiated both the degree and duration of the AKT/p38-
MAPK signaling axis induced by fMLP, thus enhancing myeloperoxidase release.

   In conclusion, this study provides first evidences that neutrophils from patients with decompensated alcoholic cirrhosis exhibit a deficient AKT/p38-MAPK signaling, myeloperoxidase release and bactericidal activity. These deficiencies which may increase patients’ susceptibility to bacterial infections, can be reversed via TLR7/8 activation, and thus raises therapeutical perspectives to boost host-defense mechanisms of immuno-depressed patients.
   Key words: Neutrophil, Exocytosis, Myeloperoxidase, Hepatitis, Phagocytes, Signaling, Reactive oxygen species.

BACKGROUND & AIMS:

Myeloperoxidase exocytosis and production of hydrogen peroxide via the neutrophil superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase contribute to efficient elimination of bacteria. Cirrhosis impairs immune functions and increases susceptibility to bacterial infection. We recently showed that neutrophils from patients with decompensated alcoholic cirrhosis exhibit a severe impairment of formylpeptide receptor (fPR)-mediated intracellular signaling and superoxide production. Here, we performed ex vivo studies with these patients' neutrophils to further investigate myeloperoxidase release, bactericidal capacity and signaling events following fPR stimulation by the formylpeptide formyl-met-leu-phe (fMLP).

METHODS:

   Myeloperoxidase release was studied by measuring extracellular myeloperoxidase activity. Activation of signaling effectors was studied by Western blot and their respective contribution to myeloperoxidase release studied using pharmacological antagonists.

RESULTS:

   fMLP-induced myeloperoxidase release was strongly impaired in patients' neutrophils whereas the intracellular myeloperoxidase stock was unaltered. The fMLP-induced phosphorylation of major signaling effectors, AKT, ERK1/2 and p38-MAP-Kinases, was also strongly deficient despite a similar expression of signaling effectors or fPR. However, based on effector inhibition in healthy neutrophils, AKT and p38-MAPK but not ERK1/2 upregulated fMLP-induced myeloperoxidase exocytosis. Interestingly, patients' neutrophils exhibited a defective bactericidal capacity that was reversed ex vivo by the TLR7/8 agonist CL097, through potentiation of the fMLP-induced AKT/p38-MAPK signaling axis and myeloperoxidase release.

CONCLUSIONS:

   We provide first evidence that neutrophils from patients with decompensated alcoholic cirrhosis exhibit a deficient AKT/p38-MAPK signaling, myeloperoxidase release and bactericidal activity, which can be reversed via TLR7/8 activation. These defects, together with the previously described severe deficient superoxide production, may increase cirrhotic patients' susceptibility to bacterial infections.