Extracellular Lactate Acts as a Metabolic Checkpoint and Shapes Monocyte Function Time Dependently

Elevated blood lactate levels are frequently found in critically ill patients and thought to result from tissue hypoperfusion and cellular oxygen shortage. Considering the close relationship between immune cell function and intracellular metabolism, lactate is more than a glycolytic waste molecule but able to regulate the immune response. Our aim was to elucidate the temporal and mechanistic effect of extracellular lactate on monocytes. To this end, primary human monocytes and the human monocytic cell line MonoMac6 were stimulated with various toll-like-receptor agonists after priming with Na-L-lactate under constant pH conditions. As readout, cytokine production was measured, real-time assessment of intracellular energy pathways was performed, and intracellular metabolite concentrations were determined. Irrespective of the immunogenic stimulus, short-term Na-lactate-priming strongly reduced cytokine production capacity. Lactate and hexoses accumulated intracellularly and, together with a decreased glycolytic flux, indicate a lactate-triggered impairment of glycolysis. To counteract intracellular hyperglycemia, glucose is shunted into the branching polyol pathway, leading to sorbitol accumulation. In contrast, long-term priming with Na-L-lactate induced cellular adaption and abolished the suppressive effect. This lactate tolerance is characterized by a decreased cellular respiration due to a reduced complex-I activity. Our results indicate that exogenous lactate shapes monocyte function by altering the intracellular energy metabolism and acts as a metabolic checkpoint of monocyte activation.

Individuals co-exposed to sand fly saliva and filarial parasites exhibit altered monocyte function

Background In Mali, cutaneous leishmaniasis (CL) and filariasis are co-endemic. Previous studies in animal models of infection have shown that sand fly saliva enhance infectivity of Leishmania parasites in naïve hosts while saliva-specific adaptive immune responses may protect against cutaneous and visceral leishmaniasis. In contrast, the human immune response to Phlebotomus duboscqi (Pd) saliva, the principal sand fly vector in Mali, was found to be dichotomously polarized with some individuals having a Th1-dominated response and others having a Th2-biased response. We hypothesized that co-infection with filarial parasites may be an underlying factor that modulates the immune response to Pd saliva in endemic regions. Methodology/Principal findings To understand which cell types may be responsible for polarizing human responses to sand fly saliva, we investigated the effect of salivary glands (SG) of Pd on human monocytes. To this end, elutriated monocytes were cultured in vitro, alone, or with SG, microfilariae antigen (MF ag) of Brugia malayi, or LPS, a positive control. The mRNA expression of genes involved in inflammatory or regulatory responses was then measured as were cytokines and chemokines associated with these responses. Monocytes of individuals who were not exposed to sand fly bites (mainly North American controls) significantly upregulated the production of IL-6 and CCL4; cytokines that enhance leishmania parasite establishment, in response to SG from Pd or other vector species. This selective inflammatory response was lost in individuals that were exposed to sand fly bites which was not changed by co-infection with filarial parasites. Furthermore, infection with filarial parasites resulted in upregulation of CCL22, a type-2 associated chemokine, both at the mRNA levels and by its observed effect on the frequency of recruited monocytes. Conclusions/Significance Together, our data suggest that SG or recombinant salivary proteins from Pd alter human monocyte function by upregulating selective inflammatory cytokines.

MO132IMPACT OF MONOCYTE CHEMOATTRACTANTS ON IN-HOSPITAL MORTALITY IN RELATION TO KIDNEY FUNCTION IN PATIENTS WITH COVID-19

Background and Aims Patients with chronic kidney disease (CKD) are at higher risk of severe complications and mortality due to Covid-19 than patients with other known risk factors. The association between CKD and mortality persist in analyses adjusted for covariates known to associate with worse Covid-19 outcomes, suggesting that CKD confers a risk beyond that associated to comorbid conditions. The mechanisms underlying the increased susceptibility to severe Covid-19 in CKD remains unclear but morphologic and functional differences in monocytes have been associated with prolonged hospitalization in other cohorts of patients. The monocyte is capable to contribute to the pathophysiology through different mechanisms. There is however insufficient information on factors that orchestrate different aspects of monocyte function and how these factors relate to outcomes. Increased knowledge into which features of monocyte function that contribute to risks in CKD patients with Covid-19 is important to guide treatment strategies. The aim of the present study was to examine the concentrations of monocyte chemoattractant markers MCP-1 (Monocyte Chemoattractant Protein-1; CCL2) and MIP-1α (Macrophage Inflammatory Protein 1-α; CCL3) in patients with Covid-19 and normal or impaired kidney function and to compare that to CKD patients matched for sex and eGFR, and sex matched healthy subjects. We analyzed the impact of these monocyte chemoattractant markers on in-hospital and 30 days mortality by logistic and multiple regression analyses. We related this to established risk factors for morbidity and mortality in Covid-19 patients, e.g. CRP and IL-6. Method We prospectively included 110 patients with Covid-19 (mean age 59 yr., mean eGFR 75 ml/min/1.73m2) admitted to Danderyd University Hospital, Stockholm, Sweden, during the first pandemic wave in April to May 2020 and 33 sex and eGFR-matched patients (mean age 51 yr., eGFR 52 ml/min/1.73m2) with CKD and 35 sex matched healthy subjects (mean age 47 yr., eGFR 101 ml/min/1.73m2).We used Luminex assays to analyze MCP-1, MIP-1α and IL-6 and routine laboratory tests to determine white blood cell count (WBC) and CRP. Results Patients with Covid-19 had significantly lower concentrations of MIP-1α (p<0.001), and higher IL-6 (p<0.001) and CRP (p<0.001) than patients with CKD and healthy subjects (Kruskal-Wallis), there were no differences in MCP-1 between groups. We found significant negative correlations between MCP-1 (p<0.05), MIP-1α (p<0.05) and IL-6 (p<0.05) with eGFR in patients with Covid-19 (Spearman´s rank correlation). Logistic regression analysis (Odds ratio, OR, 95% Confidence Intervals (CI)) and Cox proportional hazard models (Hazard ratio, HR), both adjusted for age, showed significant associations between in-hospital mortality and WBC, CRP, IL-6, MCP-1 and MIP-1α (Table, Figure). Similar findings were observed also for 30 days mortality. Conclusion We demonstrate that factors related to monocyte recruitment and activation, MCP-1 and MIP-1α, are associated with in-hospital and 30-days mortality in CKD patients with Covid-19. In this patient group general inflammatory markers as IL-6 and CRP are also associated with risk of mortality. These data contribute to an increased understanding of the impact of monocyte activation in Covid-19 and may be of value when treatment strategies are evaluated.

Sustained Immunoparalysis in Endotoxin-Tolerized Monocytic Cells

Sepsis is associated with a strong inflammatory reaction triggering a complex and prolonged immune response. Septic patients have been shown to develop sustained immunosuppression due to a reduced responsiveness of leukocytes to pathogens. Changes in cellular metabolism of leukocytes have been linked to this phenomenon and contribute to the ongoing immunological derangement. However, the underlying mechanisms of these phenomena are incompletely understood. In cell culture models, we mimicked LPS tolerance conditions to provide evidence that epigenetic modifications account for monocyte metabolic changes which cause immune paralysis in restimulated septic monocytes. In detail, we observed differential methylation of CpG sites related to metabolic activity in human PBMCs 18 h after septic challenge. The examination of changes in immune function and metabolic pathways was performed in LPS-tolerized monocytic THP-1 cells. Passaged THP-1 cells, inheriting initial LPS challenge, presented with dysregulation of cytokine expression and oxygen consumption for up to 7 days after the initial LPS treatment. Proinflammatory cytokine concentrations of TNFα and IL1β were significantly suppressed following a second LPS challenge (p<0.001) on day 7 after first LPS stimulation. However, the analysis of cellular metabolism did not reveal any noteworthy alterations between tolerant and nontolerant THP-1 monocytes. No quantitative differences in ATP and NADH synthesis or participating enzymes of energy metabolism occurred. Our data demonstrate that the function and epigenetic modifications of septic and tolerized monocytes can be examined in vitro with the help of our LPS model. Changes in CpG site methylation and monocyte function point to a correlation between epigenetic modification in metabolic pathways and reduced monocyte function under postseptic conditions.