Ethanol Exposure Attenuates Immune Response in Sepsis via Sirtuin 2 Expression.

BACKGROUND: Sepsis and septic shock kill over 270,000 patients per year in the United States. Sepsis transitions from a hyper-inflammatory to a hypo-inflammatory phase. Alcohol dependence is a risk factor for mortality from sepsis. Ethanol (EtOH) exposure impairs pathogen clearance through mechanisms that are not fully understood. Sirtuin 2 (SIRT2) interferes with pathogen clearance in immune cells but its role in the effects of EtOH on sepsis is unknown. We studied the effect of EtOH exposure on hyper- and hypo-inflammation and the role of SIRT2 in mice. METHODS: We exposed C57Bl/6 (WT) mice to EtOH via drinking water and used intraperitoneal cecal slurry (CS)-induced sepsis to study: (i) 7-day survival, (ii) leukocyte adhesion (LA) in the mesenteric microcirculation during hyper- and hypo-inflammation, (iii) peritoneal cavity bacterial clearance, and (iv) SIRT2 expression in peritoneal macrophages. Using EtOH-exposed and lipopolysaccharide (LPS)-stimulated RAW 264.7 (RAW) cell macrophages for 4 hours or 24 hours, we studied: (i) tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), and SIRT2 expression, and (ii) the effect of the SIRT2 inhibitor AK-7 on inflammatory response at 24 hours. Lastly, we studied the effect of EtOH on sepsis in whole body Sirt2 knockout (SIRT2KO) mice during hyper- and hypo-inflammation, bacterial clearance, and 7-day survival. RESULTS: WT EtOH-sepsis mice showed: (i) Decreased survival, (ii) Muted LA in the microcirculation, (iii) Lower plasma TNF-α and IL-6 expression, (iv) Decreased bacterial clearance, and (v) Increased SIRT2 expression in peritoneal macrophages versus vehicle-sepsis. EtOH-exposed LPS-stimulated RAW cells showed: (i) Muted TNF-α, IL-6, and increased IL-10 expression at 4 hours, (ii) endotoxin tolerance at 24 hours, and (iii) reversal of endotoxin tolerance with the SIRT2 inhibitor AK-7. EtOH-exposed SIRT2KO-sepsis mice showed greater 7-day survival, LA, and bacterial clearance than WT EtOH-sepsis mice. CONCLUSION: EtOH exposure decreases survival and reduces the inflammatory response to sepsis via increased SIRT2 expression. SIRT2 is a potential therapeutic target in EtOH with sepsis.

SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure.

Alcohol abuse, reported by 1/8th critically ill patients, is an independent risk factor for death in sepsis. Sepsis kills over 270,000 patients/year in the US. We reported that the ethanol-exposure suppresses innate-immune response, pathogen clearance, and decreases survival in sepsis-mice via sirtuin 2 (SIRT2). SIRT2 is an NAD+-dependent histone-deacetylase with anti-inflammatory properties. We hypothesized that in ethanol-exposed macrophages, SIRT2 suppresses phagocytosis and pathogen clearance by regulating glycolysis. Immune cells use glycolysis to fuel increased metabolic and energy demand of phagocytosis. Using ethanol-exposed mouse bone marrow- and human blood monocyte-derived macrophages, we found that SIRT2 mutes glycolysis via deacetylating key glycolysis regulating enzyme phosphofructokinase-platelet isoform (PFKP), at mouse lysine 394 (mK394, human: hK395). Acetylation of PFKP at mK394 (hK395) is crucial for PFKP function as a glycolysis regulating enzyme. The PFKP also facilitates phosphorylation and activation of autophagy related protein 4B (Atg4B). Atg4B activates microtubule associated protein 1 light chain-3B (LC3). LC3 is a driver of a subset of phagocytosis, the LC3-associated phagocytosis (LAP), which is crucial for segregation and enhanced clearance of pathogens, in sepsis. We found that in ethanol-exposed cells, the SIRT2-PFKP interaction leads to decreased Atg4B-phosphorylation, decreased LC3 activation, repressed phagocytosis and LAP. Genetic deficiency or pharmacological inhibition of SIRT2 reverse PFKP-deacetylation, suppressed LC3-activation and phagocytosis including LAP, in ethanol-exposed macrophages to improve bacterial clearance and survival in ethanol with sepsis mice.

Sirtuins and Sepsis: Cross Talk between Redox and Epigenetic Pathways.

Sepsis and septic shock are the leading causes of death among hospitalized patients in the US. The immune response in sepsis transitions from a pro-inflammatory and pro-oxidant hyper-inflammation to an anti-inflammatory and cytoprotective hypo-inflammatory phase. While 1/3rd sepsis-related deaths occur during hyper-, a vast majority of sepsis-mortality occurs during the hypo-inflammation. Hyper-inflammation is cytotoxic for the immune cells and cannot be sustained. As a compensatory mechanism, the immune cells transition from cytotoxic hyper-inflammation to a cytoprotective hypo-inflammation with anti-inflammatory/immunosuppressive phase. However, the hypo-inflammation is associated with an inability to clear invading pathogens, leaving the host susceptible to secondary infections. Thus, the maladaptive immune response leads to a marked departure from homeostasis during sepsis-phases. The transition from hyper- to hypo-inflammation occurs via epigenetic programming. Sirtuins, a highly conserved family of histone deacetylators and guardians of homeostasis, are integral to the epigenetic programming in sepsis. Through their anti-inflammatory and anti-oxidant properties, the sirtuins modulate the immune response in sepsis. We review the role of sirtuins in orchestrating the interplay between the oxidative stress and epigenetic programming during sepsis.

Sirtuin 2 Dysregulates Autophagy in High-Fat-Exposed Immune-Tolerant Macrophages.

Obesity increases morbidity and resource utilization in sepsis patients. The immune response in sepsis transitions from an endotoxin-responsive hyper- to an endotoxin-tolerant hypo-inflammatory phase. The majority of sepsis mortality occurs during hypo-inflammation. We reported prolonged hypo-inflammation with increased sirtuin 2 (SIRT2) expression in obese-septic mice. The effect of direct exposure to high-fat/free fatty acid (FFA) and the role of SIRT2 in immune cells during the transition to hypo-inflammation is not well-understood. Autophagy, a degradation process of damaged protein/organelles, is dysregulated during sepsis. Here, we investigated the effect of direct FFA exposure and the role of SIRT2 expression on autophagy as macrophages transition from hyper-to hypo-inflammation. We found, FFA-exposed RAW 264.7 cells with lipopolysaccharide (LPS) stimulation undergo endotoxin-sensitive ("sensitive") hyper- followed by endotoxin tolerant ("tolerant") hypo-inflammatory phases; SIRT2 expression increases significantly in tolerant cells. Autophagy proteins LC3b-II, and beclin-1 increase in FFA-sensitive and decrease in tolerant cells; p62 expressions continue to accumulate in tolerant cells. We observed that SIRT2 directly deacetylates α-tubulin and impairs autophagy clearance. Importantly, we find SIRT2 inhibitor AK-7 treatment during endotoxin tolerant phase reverses autophagy dysregulation with improved autophagy clearance in FFA-tolerant cells. Thus, we report impaired autophagosome formation and autophagy clearance via increased SIRT2 expression in FFA-exposed tolerant macrophages.

Naturally acquired blocking human monoclonal antibodies to Plasmodium vivax reticulocyte binding protein 2b.

Plasmodium vivax preferentially invades reticulocytes and recognition of these cells is mediated by P. vivax Reticulocyte Binding Protein 2b (PvRBP2b) binding to human Transferrin receptor 1 (TfR1) and Transferrin (Tf). Longitudinal cohort studies in Papua New Guinea, Thailand and Brazil show that PvRBP2b antibodies are correlated with protection against P. vivax infection and disease. Here, we isolate and characterize anti-PvRBP2b human monoclonal antibodies from two individuals in Cambodia with natural P. vivax infection. These antibodies bind with high affinities and map to different regions of PvRBP2b. Several human antibodies block PvRBP2b binding to reticulocytes and inhibit complex formation with human TfR1-Tf. We describe different structural mechanisms for functional inhibition, including either steric hindrance with TfR1-Tf or the reticulocyte membrane. These results show that naturally acquired human antibodies against PvRBP2b can inhibit its function which is important for P. vivax invasion.

Immunoadjuvant effect of diethylcarbamazine in experimental filariasis.

Lymphatic filariasis caused by tissue dwelling nematodes is endemic in 73 countries and drugs have been administered to control or stop the infection. Resurgence of the infection after mass drug administration necessitates the study of several parasite antigens or adjuvants for vaccine developments. In this study, diethylcarbamazine (DEC) was evaluated for its efficacy as adjuvant against the filarial parasite; Brugia malayi microfilariae (mf) by combining with the Escherichia coli expressed recombinant BmShp-1 protein. Shp-1 is one of the sheath proteins expressed by adult female and microfilarial stage of the filarial parasite. Hence, immunoprophylactic efficacy of Shp-1 using DEC and alum adjuvants was compared in BALB/c mice model by an in situ micropore chamber method. Shp-1 antibody titre was high when the mice were immunized with Shp-1 along with DEC and they exhibited balanced Th1/Th2 profile. DEC also induced significantly high T-cell proliferation (P<0.001) when stimulated with Shp-1 compared to alum. Significantly high percentage protection against B. malayi microfilariae was observed in Shp-1+DEC immunized mice groups (P<0.05) and hence it is concluded that the need of repeated drug administration can be controlled when there is a possibility of developing protective immunity in the host against mf by vaccination.