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Background: Excessive alcohol intake is associated with adverse immune response-related effects, however, acute and chronic abuse differently modulate monocyte activation. In this study, we have evaluated the phenotypic and functional changes of monocytes in acutely intoxicated healthy volunteers (HV).
Methods: Twenty-two HV consumed individually adjusted amounts of alcoholic beverages until reaching a blood alcohol level of 1‰ after 4h (T4). Peripheral blood was withdrawn before and 2h (T2), 4h (T4), 6h (T6), 24h (T24), and 48h (T48) after starting the experiment and stained for CD14, CD16 and TLR4. CD14brightCD16-, CD14brightCD16+ and CD14dimCD16+ monocyte subsets and their TLR4 expression were analyzed by flow cytometry. Inflammasome activation via caspase-1 in CD14+ monocytes was measured upon an ex vivo in vitro LPS stimulation. Systemic IL-1β and adhesion capacity of isolated CD14+ monocytes upon LPS stimulation were evaluated.
Results: The percentage of CD14+ monocyte did not change following alcohol intoxication, whereas CD14brightCD16- monocyte subset significantly increased at T2 and T24, CD14brightCD16+ at T2, T4 and T6 and CD14dimCD16+ at T4 and T6. The relative fraction of TLR4 expressing CD14+ monocytes as well as the density of TLR4 surface presentation increased at T2 and decreased at T48 significantly. TLR4+CD14+ monocytes were significantly enhanced in all subsets at T2. TLR4 expression significantly decreased in CD14brightCD16- at T48, in CD14brightCD16+ at T24 and T48, increased in CD14dimCD16+ at T2. IL-1β release upon LPS stimulation decreased at T48, correlating with TLR4 receptor expression. Alcohol downregulated inflammasome activation following ex vivo in vitro stimulation with LPS between T2 and T48 vs. T0. The adhesion capacity of CD14+ monocytes decreased from T2 with significance at T4, T6 and T48. Following LPS administration, a significant reduction of adhesion was observed at T4 and T6.
Conclusions: Alcohol intoxication immediately redistributes monocyte subsets toward the pro-inflammatory phenotype with their subsequent differentiation into the anti-inflammatory phenotype. This is paralleled by a significant functional depression, suggesting an alcohol-induced time-dependent hyporesponsiveness of monocytes to pathogenic triggers.
Particulate autogenous tooth roots are used for alveolar bone augmentation surgery; however, dental plaque may provoke an inflammatory response that may counteract the desired graft consolidation process. Traditional mechanical cleaning of extracted teeth may be of support to lower a possible inflammatory response of the autograft. To test this assumption, extracted porcine teeth were left either uncleaned or underwent mechanical cleaning with a toothbrush and toothpaste before being fragmented and subjected to acid lysis, termed as unclean acid dentine lysate (ucADL) and clean acid dentine lysate (cADL), respectively. The inflammatory responses of murine macrophage RAW 264.7 cells being exposed to the respective acid dentine lysates were evaluated at the level of inflammatory gene expression and IL6 immunoassays. We report here that acid lysates obtained from uncleaned teeth provoked a robust increase in IL1β, IL6, and COX2 in RAW 264.7 cells. The mechanical removal of dental plaque significantly reduced the inflammatory response. Consistently, Limulus tests revealed that tooth cleaning lowers the presence of endotoxins in dentine lysates. To further prove the involvement of endotoxins, a toll-like receptor 4 (TLR4) inhibitor TAK242 was introduced. TAK242 abolished the inflammatory response provoked by acid lysates obtained from uncleaned teeth in RAW 264.7 cells. Moreover, nuclear translocation and phosphorylation of the TLR4 downstream NFκB-p65 were attenuated at the presence of cleaned versus uncleaned dentine lysates. Taken together, our data support the importance of dental plaque removal of teeth being extracted for alveolar bone augmentation surgery.
We report here that RUFY4, a newly characterized member of the ‘RUN and FYVE domain-containing’ family of proteins previously associated with autophagy enhancement, is highly expressed in alveolar macrophages (AM). We show that RUFY4 interacts with mitochondria upon stimulation by microbial-associated molecular patterns of AM and dendritic cells. RUFY4 interaction with mitochondria and other organelles is dependent on a previously uncharacterized OmpH domain located immediately upstream of its C-terminal FYVE domain. Further, we demonstrate that rufy4 messenger RNA can be translated from an alternative translation initiation codon, giving rise to a N-terminally truncated form of the molecule lacking most of its RUN domain and with enhanced potential for its interaction with mitochondria. Our observations point towards a role of RUFY4 in selective mitochondria clearance in activated phagocytes.