Proteolytic Cleavage of AMPKα and Intracellular MMP9 Expression Are Both Required for TLR4-Mediated mTORC1 Activation and HIF-1α Expression in Leukocytes.

LPS-induced TLR4 activation alters cellular bioenergetics and triggers proteolytic cleavage of AMPKα and HIF-1α expression in leukocytes. In human leukocytes, and more specifically neutrophils, AMPKα cleavage yields 55- and 35-kDa protein fragments. In this study, we address the mechanism by which AMPKα is cleaved and its relevance to human health. Our data indicate that AMPKα cleavage is linked to MMP9 expression and that both are required for mammalian target of rapamycin complex-1 and S6K1 activation and HIF-1α expression in LPS-stimulated human and mice leukocytes. Three key observations support this conclusion. First, no changes in AMPKα and TLR4 signaling intermediates (mammalian target of rapamycin complex-1/S6 kinase 1/HIF-1α) were detected in LPS-stimulated MMP9-deficient mice leukocytes. Second, rMMP9 cleaved human AMPKα ex vivo, producing degradation products similar in size to those detected following LPS stimulation. Third, MMP9 inhibitors prevented AMPKα degradation and HIF-1α expression in LPS-activated human leukocytes, whereas AMPK activators blocked MMP9 and HIF-1α expression. Significantly, AMPKα degradation, MMP9, and TLR4 signaling intermediates were all detected in leukocytes from patients with type 2 diabetes mellitus and patients following cardiopulmonary bypass surgery. Plasma from these two patient cohorts induced AMPKα cleavage and TLR4 signaling intermediates in healthy donor leukocytes and either a TLR4 inhibitor or polymyxin prevented these outcomes. Detection of AMPKα degradation, MMP9 expression, and TLR4 signaling intermediates described in this study in leukocytes, the most readily available human cells for clinical investigation, may provide a powerful tool for further exploring the role of TLR4 signaling in human diseases and lead to identification of new, context-specific therapeutic modalities for precision medicine.

Cellular metabolic regulators: novel indicators of low-grade inflammation in humans.

OBJECTIVE: The Toll-like receptor 4 (TLR4) ligand endotoxin triggers robust systemic inflammatory responses in humans at doses equal to or greater than 1 ng/kg. In this study, we tested the hypothesis that evidence of TLR4-induced responses would be detectable in leukocytes challenged with endotoxin doses that are below the threshold needed to trigger a characteristic systemic inflammatory phenotype in humans. METHODS: Subjects were challenged with endotoxin at 1, 0.5, or 0.1 ng/kg (n = 5 per dose). Systemic responses were monitored for 24 hours. Blood samples, collected at designated intervals, were used to determine plasma cytokines levels, total and differential leukocyte counts, expression of leukocyte cell surface receptors, and changes in the leukocyte transcriptome. Western blotting was used to determine changes in leukocyte protein expression. RESULTS: We found that in vivo endotoxin at doses below 1.0 ng/kg triggers weak and variable responses in humans. In marked contrast, we show that endotoxin at a concentration as low as 0.1 ng/kg triggers a transient decline in cellular ATP levels in leukocytes. This is associated with the appearance of a unique protein expression signature in leukocytes. The protein expression signature includes 3 prominent features: (i) AMP-activated protein kinase subunit α (AMPKα) degradation, (ii) increased hypoxia inducible factor-1 (HIF-1) α expression, and (iii) autophagy, collectively indicative of a regulated metabolic response. An indistinguishable response phenotype was observed in human leukocytes treated with endotoxin in vitro. CONCLUSIONS: These data demonstrate for the first time in humans that a TLR4 ligand concentration that is below the threshold needed to trigger clinically evident systemic inflammatory manifestations initiates a transient decline in ATP levels, AMPKα degradation, HIF-1α expression, and autophagy in leukocytes. This establishes that low-grade TLR4 activation exerts control over leukocyte metabolism in the absence of systemic inflammatory indicators.

A novel model of common Toll-like receptor 4- and injury-induced transcriptional themes in human leukocytes.

INTRODUCTION: An endotoxin challenge, sepsis, and injury/trauma, trigger significant changes in human peripheral blood leukocytes (PBL) gene expression. In this study, we have sought to test the hypothesis that the Toll-like receptor 4 (TLR4) induced transcription patterns elicited in humans exposed to in vivo endotoxin would parallel gene expression patterns observed in trauma patients with initial non-infectious injury. In addition, we sought to identify functional modules that are commonly affected by these two insults of differing magnitude and duration. METHODS: PBL were obtained from seven adult human subject experimental groups. The groups included a group of healthy, hospitalized volunteers (n = 15), that comprised four study groups of subjects challenged with intravenous endotoxin, without or with cortisol, and two serial samplings of trauma patients (n = 5). The PBL were analyzed for gene expression using a 8,793 probe microarray platform (Gene Chip® Focus, Affymetrix). The expression of a subset of genes was determined using qPCR. RESULTS: We describe sequential selection criteria of gene expression data that identifies 445 genes that are significantly differentially expressed (both P ≤ 0.05 and > 1.2 fold-change) in PBL derived from human subjects during the peak of systemic inflammatory responses induced by in vivo endotoxin, as well as in PBL obtained from trauma patients at 1 to 12 days after admission. We identified two functional modules that are commonly represented by this analysis. The first module includes more than 50 suppressed genes that encode ribosomal proteins or translation regulators. The second module includes up-regulated genes encoding key enzymes associated with glycolysis. Finally, we show that several circadian clock genes are also suppressed in PBL of surgical ICU patients. CONCLUSIONS: We identified a group of > 400 genes that exhibit similar expression trends in PBL derived from either endotoxin-challenged subjects or trauma patients. The suppressed translational and circadian clock modules, and the upregulated glycolytic module, constitute a robust and long lasting PBL gene expression signature that may provide a tool for monitoring systemic inflammation and injury.

Polymorphisms of heat shock protein-70 (HSPA1B and HSPA1L loci) do not influence infection or outcome risk in critically ill surgical patients.

Heat shock proteins (HSP) are induced in various stress conditions and have many cytoprotective effects, including formation of protein complexes for antigen presentation, stabilizing intracellular proteins, and facilitating protein folding. The HSP-70 gene exhibits polymorphisms at the HSPA1B and HSPA1L loci that reportedly influence cytokine levels and clinical outcomes in critically ill patients. These HSP variations also have been linked to TNF-beta polymorphisms associated with poor outcomes. This study further evaluated outcomes and risk of infection of HSP polymorphisms in critically ill patients. Seventy-six consecutive surgical intensive care unit uninfected patients with established systemic inflammatory response features were prospectively enrolled. Genomic DNA was isolated from whole blood samples and specific fragments, including the relevant polymorphic sites, were amplified by PCR, and restriction digestions were performed. Genotypes were determined by electrophoresis and all were confirmed by direct sequencing. Plasma cytokine levels for TNF-alpha were assayed in a subset of patients by enzyme-linked immunoabsorbent assay. None of the HSP alleles bore a significant relationship to nosocomial infection rates, organ specific dysfunctions, or mortality. No linkage of HSP genotype to common TNF-alpha or TNF-beta genotypes could be demonstrated, although the HSPA1L CT polymorphism was associated with higher levels of TNF-alpha compared with the TT genotype. These data suggest that polymorphisms of the HSPA1L or HSPA1B loci do not influence infection or other highly morbid outcomes in surgical intensive care unit patients.

Insulin-Dependent Regulation of mTORC2-Akt-FoxO Suppresses TLR4 Signaling in Human Leukocytes: Relevance to Type 2 Diabetes.

Leukocyte signaling in patients with systemic insulin resistance is largely unexplored. We recently discovered the presence of multiple Toll-like receptor 4 (TLR4) signaling intermediates in leukocytes from patients with type 2 diabetes or acute insulin resistance associated with cardiopulmonary bypass surgery. We extend this work to show that in addition to matrix metalloproteinase 9, hypoxia-inducible factor 1α, and cleaved AMPKα, patient leukocytes also express IRS-1 phosphorylated on Ser(312), Akt phosphorylated on Thr(308), and elevated TLR4 expression. Similar signaling intermediates were detected in leukocytes and neutrophils treated with lipopolysaccharide (LPS), a ligand of TLR4, in vitro. In contrast, insulin, but not LPS, induced mammalian target of rapamycin complex 2 (mTORC2)-dependent phosphorylation of Akt on Ser(473) and FoxO1/O3a on Thr(24/32) in leukocytes and neutrophils. Insulin suppressed LPS-induced responses in a dose- and time-dependent manner. AS1842856, a FoxO1 inhibitor, also suppressed TLR4 signaling. We propose that insulin is a homeostatic regulator of leukocyte responses to LPS/TLR4 and that the signaling intermediates expressed in leukocytes of patients with type 2 diabetes indicate TLR4 signaling dominance and deficient insulin signaling. The data suggest that insulin suppresses LPS/TLR4 signals in leukocytes through the mTORC2-Akt-FoxO signaling axis. Better understanding of leukocyte signaling in patients with type 2 diabetes may shed new light on disease causation and progression.

Physiologic variability at the verge of systemic inflammation: multiscale entropy of heart rate variability is affected by very low doses of endotoxin.

INTRODUCTION: Human injury or infection induces systemic inflammation with characteristic neuroendocrine responses. Fluctuations in autonomic function during inflammation are reflected by beat-to-beat variation in heart rate, termed heart rate variability (HRV). In the present study, we determine threshold doses of endotoxin needed to induce observable changes in markers of systemic inflammation, investigate whether metrics of HRV exhibit a differing threshold dose from other inflammatory markers, and investigate the size of data sets required for meaningful use of multiscale entropy (MSE) analysis of HRV. METHODS: Healthy human volunteers (n = 25) were randomized to receive placebo (normal saline) or endotoxin/lipopolysaccharide (LPS): 0.1, 0.25, 0.5, 1.0, or 2.0 ng/kg administered intravenously. Vital signs were recorded every 30 min for 6 h and then at 9, 12, and 24 h after LPS. Blood samples were drawn at specific time points for cytokine measurements. Heart rate variability analysis was performed using electrocardiogram epochs of 5 min. Multiscale entropy for HRV was calculated for all dose groups to scale factor 40. RESULTS: The lowest significant threshold dose was noted in core temperature at 0.25 ng/kg. Endogenous tumor necrosis factor α and interleukin 6 were significantly responsive at the next dosage level (0.5 ng/kg) along with elevations in circulating leukocytes and heart rate. Responses were exaggerated at higher doses (1 and 2 ng/kg). Time domain and frequency domain HRV metrics similarly suggested a threshold dose, differing from placebo at 1.0 and 2.0 ng/kg, below which no clear pattern in response was evident. By applying repeated-measures analysis of variance across scale factors, a significant decrease in MSE was seen at 1.0 and 2.0 ng/kg by 2 h after exposure to LPS. Although not statistically significant below 1.0 ng/kg, MSE unexpectedly decreased across all groups in an orderly dose-response pattern not seen in the other outcomes. CONCLUSIONS: By using repeated-measures analysis of variance across scale factors, MSE can detect autonomic change after LPS challenge in a group of 25 subjects using electrocardiogram epochs of only 5 min and entropy analysis to scale factor of only 40, potentially facilitating MSE's wider use as a research tool or bedside monitor. Traditional markers of inflammation generally exhibit threshold dose behavior. In contrast, MSE's apparent continuous dose-response pattern, although not statistically verifiable in this study, suggests a potential subclinical harbinger of infectious or other insult. The possible derangement of autonomic complexity prior to or independent of the cytokine surge cannot be ruled out. Future investigation should focus on confirmation of overt inflammation following observed decreases in MSE in a clinical setting.

Continuous enteral and parenteral feeding each reduces heart rate variability but differentially influences monocyte gene expression in humans.

Enteral (EN) or parenteral (PN) nutrition is used to support critically ill patients until oral feeding resumes. Enteral nutrition is assumed preferable to PN, but the differential influence on immune function is not well defined. Autonomic nervous activity is known to influence innate immune responses, and we hypothesized that EN and PN could influence both autonomic signaling and gene activation in peripheral blood monocytes (PBMs). Ten subjects (aged 18-36 years) received continuous EN or PN for 72 h. Peripheral blood monocytes were isolated from whole blood before and after continuous feeding and were analyzed for gene expression using a microarray platform. Gene expression after feeding was compared from baseline and between groups. To measure autonomic outflow, subjects also underwent heart rate variability (HRV) monitoring during feeding. Time and frequency domain HRV data were compared between groups and five orally fed subjects for changes from baseline and changes over time. During continuous EN and PN, subjects exhibited declines in both time and frequency domain HRV parameters compared with baseline and with PO subjects, indicating a loss of vagal/parasympathetic tone. However, PN feeding had a much greater influence on PBM gene expression compared with baseline than EN, including genes important to innate immunity. Continuous EN and PN are both associated with decreasing vagal tone over time, yet contribute differently to PBM gene expression, in humans. These preliminary findings support assumptions that PN imposes a systemic inflammatory risk but also imply that continuous feeding, independent of route, may impart additional risk through different mechanisms.

Relationship of basal heart rate variability to in vivo cytokine responses after endotoxin exposure.

Autonomic inputs from the sympathetic and parasympathetic nervous systems, as measured by heart rate variability (HRV), have been reported to correlate to the severity injury and responses to infectious challenge among critically ill patients. In addition, parasympathetic/vagal activity has been shown experimentally to exert anti-inflammatory effects via attenuation of splanchnic tissue TNF-alpha production. We sought to define the influence of gender on HRV responses to in vivo endotoxin challenge in healthy humans and to determine if baseline HRV parameters correlated with endotoxin-mediated circulating cytokine responses. Young (<30 years of age), healthy subjects (n = 30) received endotoxin (2 ng/kg), and HRV and blood samples were obtained serially thereafter. Plasma cytokines were measured by enzyme-linked immunosorbent assay, and HRV parameters were determined by analysis of serial 5-min epochs of heart rate monitoring. In addition, calculation of multiscale entropy deriving from cardiac monitoring data was performed. The influence of factors such as gender, body mass index, and resting heart rate on HRV after endotoxin exposure was assessed. We found that gender, body mass index, or resting heart rate did not significantly alter the HRV response after endotoxin exposure. Using entropy analysis, we observed that females had significantly higher entropy values at 24 h after endotoxin exposure. Using a serially sampling protocol for cytokine determination, we found a significant correlation of several baseline HRV parameters (percentage of interval differences of successive interbeat intervals more than 50 ms, r = 0.42, P < 0.05; high-frequency variability, r = 0.4, P < 0.05; and low-frequency/high-frequency ratio, r = -0.43, P < 0.05) on TNF-alpha release after endotoxin exposure.

A single nucleotide polymorphism in the Mdm2 promoter and risk of sepsis.

BACKGROUND: The Mdm2-SNP309(T/G) polymorphism has been shown to upregulate transcription of Mdm2 and subsequently attenuate the p53 pathway. Its role in regulating the human response to acute illness has not been reported. METHODS: Patients from the surgical intensive care unit were prospectively enrolled. SNP309 genotype was determined, and a genotype-based comparison of clinical outcomes was performed. RESULTS: Of the 85 enrolled patients, 41 had wild type (T/T) and 44 had mutant (32 T/G and 12 G/G) genotypes. The mutant-genotype group tended to have a longer LOS in both the surgical intensive care unit (P = .40) and the hospital (P = .08), but these trends did not reach significance. No observable genotype-based differences were noted in any other measured parameters. CONCLUSIONS: The Mdm2-SNP309(G) allele may be associated with longer LOS. However, it does not appear to influence any other clinical characteristics, nor can it be used to predict clinical outcome.