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Sepsis and shock states impose mitochondrial stress, and in response, adaptive mechanisms such as fission, fusion and mitophagy are induced to eliminate damaged portions of or entire dysfunctional mitochondria. The mechanisms underlying these events are being elucidated; yet a direct link between loss of mitochondrial membrane potential ΔΨm and the initiation of fission, fusion and mitophagy remains to be well characterized. The direct association between the magnitude of the ΔΨm and the capacity for mitochondria to buffer Ca 2+ renders Ca 2+ uniquely suited as the signal engaging these mechanisms in circumstances of mitochondrial stress that lower the ΔΨm. Herein, we show that the calcium/calmodulin-dependent protein kinase (CaMK) IV mediates an adaptive slowing in oxidative respiration that minimizes oxidative stress in the kidneys of mice subjected to either cecal ligation and puncture (CLP) sepsis or endotoxemia. CaMKIV shifts the balance towards mitochondrial fission and away from fusion by 1) directly phosphorylating an activating Serine616 on the fission protein DRP1 and 2) reducing the expression of the fusion proteins Mfn1/2 and OPA-1. CaMKIV, through its function as a direct PINK1 kinase and regulator of Parkin expression, also enables mitophagy. These data support that CaMKIV serves as a keystone linking mitochondrial stress with the adaptive mechanisms of mitochondrial fission, fusion and mitophagy that mitigate oxidative stress in the kidneys of mice responding to sepsis., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Clinical and biologic phenotypes of sepsis are proposed in human studies, yet it is unknown whether prognostic or drug response phenotypes are present in animal models of sepsis. Using a biotelemetry-enhanced, murine cecal ligation and puncture (CLP) model, we determined phenotypes of polymicrobial sepsis prior to physiologic deterioration, and the association between phenotypes and outcome in a randomized trial of prompt or delayed antibiotics and fluids., Methods: We performed a secondary analysis of male C57BL/6J mice in two observational cohorts and two randomized, laboratory animal experimental trials. In cohort 1, mice (n = 118) underwent biotelemetry-enhanced CLP, and we applied latent class mixed models to determine optimal number of phenotypes using clinical data collected between injury and physiologic deterioration. In cohort 2 (N = 73 mice), inflammatory cytokines measured at 24 h after deterioration were explored by phenotype. In a subset of 46 mice enrolled in two trials from cohort 1, we tested the association of phenotypes with the response to immediate (0 h) vs. delayed (2 to 4 h) antibiotics or fluids initiated after physiologic deterioration., Results: Latent class mixture modeling derived a two-class model in cohort 1. Class 2 (N = 97) demonstrated a shorter time to deterioration (mean SD 7.3 (0.9) vs. 9.7 (3.2) h, p < 0.001) and lower heart rate at 7 h after injury (mean (SD) 564 (55) vs. 626 (35) beats per minute, p < 0.001). Overall mortality was similar between phenotypes (p = 0.75). In cohort 2 used for biomarker measurement, class 2 mice had greater plasma concentrations of IL6 and IL10 at 24 h after CLP (p = 0.05). In pilot randomized trials, the effects of sepsis treatment (immediate vs. delayed antibiotics) differed by phenotype (p = 0.03), with immediate treatment associated with greater survival in class 2 mice only. Similar differential treatment effect by class was observed in the trial of immediate vs. delayed fluids (p = 0.02)., Conclusions: We identified two sepsis phenotypes in a murine cecal ligation and puncture model, one of which is characterized by faster deterioration and more severe inflammation. Response to treatment in a randomized trial of immediate versus delayed antibiotics and fluids differed on the basis of phenotype.

Objectives: The physiology of nearly all mammalian organisms are entrained by light and exhibit circadian rhythm. The data derived from animal studies show that light influences immunity, and these neurophysiologic pathways are maximally entrained by the blue spectrum. Here, we hypothesize that bright blue light reduces acute kidney injury by comparison with either bright red or standard, white fluorescent light in mice subjected to sepsis. To further translational relevance, we performed a pilot clinical trial of blue light therapy in human subjects with appendicitis., Design: Laboratory animal research, pilot human feasibility trial., Setting: University basic science laboratory and tertiary care hospital., Subjects: Male C57BL/6J mice, adult (> 17 yr) patients with acute appendicitis., Interventions: Mice underwent cecal ligation and puncture and were randomly assigned to a 24-hour photoperiod of bright blue, bright red, or ambient white fluorescent light. Subjects with appendicitis were randomized to receive postoperatively standard care or standard care plus high-illuminance blue light., Measurements and Main Results: Exposure to bright blue light enhanced bacterial clearance from the peritoneum, reduced bacteremia and systemic inflammation, and attenuated the degree of acute kidney injury. The mechanism involved an elevation in cholinergic tone that augmented tissue expression of the nuclear orphan receptor REV-ERBα and occurred independent of alterations in melatonin or corticosterone concentrations. Clinically, exposure to blue light after appendectomy was feasible and reduced serum interleukin-6 and interleukin-10 concentrations., Conclusions: Modifying the spectrum of light may offer therapeutic utility in sepsis.

Objectives: Sepsis, the acute organ dysfunction caused by a dysregulated host response to infection, poses a serious public health burden. Current management includes early detection, initiation of antibiotics and fluids, and source control as necessary. Although observational data suggest that delays of even a few hours in the initiation of antibiotics or IV fluids is associated with survival, these findings are controversial. There are no randomized data in humans, and prior animal studies studied time from experimental manipulation, not from the onset of clinical features of sepsis. Using a recently developed murine cecal ligation and puncture model that precisely monitors physiologic deterioration, we hypothesize that incremental hourly delays in the first dose of antibiotics, in the first bolus of fluid resuscitation, or a combination of the two at a clinically relevant point of physiologic deterioration during polymicrobial sepsis will shorten survival., Design: Randomized laboratory animal experimental trial., Setting: University basic science laboratory., Subjects: Male C57BL/6J, female C57BL/6J, aged (40-50 wk old) male C57BL/6J, and BALB/C mice., Interventions: Mice (n = 200) underwent biotelemetry-enhanced cecal ligation and puncture and were randomized after meeting validated criteria for acute physiologic deterioration. Treatment groups consisted of a single dose of imipenem/cilastatin, a single bolus of 30 mL/kg fluid resuscitation, or a combination of the two. Mice were allocated to receive treatment at the time of meeting deterioration criteria, after a 2-hour delay or after a 4-hour delay., Measurements and Main Results: Hourly delays in the initiation of antibiotic therapy led to progressively shortened survival in our model (p < 0.001). The addition of fluid resuscitation was unable to rescue animals, which received treatment 4 hours after meeting enrollment criteria. Systemic inflammation was increased, and host physiology was increasingly deranged with hourly delays to antibiotics., Conclusions: We conclude that antibiotic therapy is highly time sensitive, and efforts should be made to deliver this critical therapy as early as possible in sepsis, perhaps extending into the first point of medical contact outside the hospital.

During sepsis and shock states, mitochondrial dysfunction occurs. Consequently, adaptive mechanisms, such as fission, fusion, and mitophagy, are induced to eliminate damaged portions or entire dysfunctional mitochondria. The regulatory PINK1/Parkin and DJ-1 pathways are strongly induced by mitochondrial depolarization, although a direct link between loss of mitochondrial membrane potential (Δ Ψ ) and mitophagy has not been identified. Mitochondria also buffer Ca 2+ , and their buffering capacity is dependent on Δ Ψ Here, we characterize a role for calcium/calmodulin-dependent protein kinase (CaMK) I in the regulation of these mechanisms. Loss of Δ Ψ with either pharmacologic depolarization or LPS leads to Ca 2+ -dependent mitochondrial recruitment and activation of CaMKI that precedes the colocalization of PINK1/Parkin and DJ-1. CaMKI is required and serves as both a PINK1 and Parkin kinase. The mechanisms operate in both immune and nonimmune cells and are induced in in vivo models of endotoxemia, sepsis, and hemorrhagic shock. These data support the idea that CaMKI links mitochondrial stress with the PINK1/Parkin and DJ-1 mechanisms of mitophagy.-Zhang, X., Yuan, D., Sun, Q., Xu, L., Lee, E., Lewis, A. J., Zuckerbraun, B. S., Rosengart, M. R. Calcium/calmodulin-dependent protein kinase regulates the PINK1/Parkin and DJ-1 pathways of mitophagy during sepsis., (© FASEB.)

Animal models of sepsis exhibit considerable variability in the temporal development of the physiologic response, which reduces the power of studies, particularly if interventions are tested at arbitrary time points. We developed a biotelemetry-based model of cecal ligation and puncture (CLP) that standardizes the testing of time-sensitive therapies to specific criteria of physiologic deterioration. In this study we seek to further define the variability in physiologic response to CLP sepsis and conduct a cost analysis detailing the potential for reducing animal usage. We have further characterized the variability in physiologic response after CLP in mice and determined peaks in the temporal distribution of points of physiologic decline. Testing therapies at physiologic thresholds reduces the variability found in historical fixed time-based models. Though initial cost is higher with biotelemetry, this is eventually offset by the significantly reduced number of mice needed to conduct physiologically relevant sepsis experiments.

Objectives: Murine models of critical illness are commonly used to test new therapeutic interventions. However, these interventions are often administered at fixed time intervals after the insult, perhaps ignoring the inherent variability in magnitude and temporality of the host response. We propose to use wireless biotelemetry monitoring to define and validate criteria for acute deterioration and generate a physiology-based murine cecal ligation and puncture model that is more similar to the conduct of human trials of sepsis., Design: Laboratory and animal research., Setting: University basic science laboratory., Subjects: Male C57BL/6 mice., Interventions: Mice underwent cecal ligation and puncture, and an HD-X11 wireless telemetry monitor (Data Sciences International) was implanted that enabled continuous, real-time measurement of heart rate, core temperature, and mobility. We performed a population-based analysis to determine threshold criteria that met face validity for acute physiologic deterioration. We assessed construct validity by temporally matching mice that met these acute physiologic deterioration thresholds with mice that had not yet met deterioration threshold. We analyzed matched blood samples for blood gas, inflammatory cytokine concentration, cystatin C, and alanine aminotransferase., Measurements and Main Results: We observed that a 10% reduction in both heart rate and temperature sustained for greater than or equal to 10 minutes defined acute physiologic deterioration. There was significant variability in the time to reach acute deterioration threshold across mice, ranging from 339 to 529 minutes after cecal ligation and puncture. We found adequate construct validity, as mice that met criteria for acute deterioration had significantly worse shock, systemic inflammation (elevated tumor necrosis factor-α, p = 0.003; interleukin-6, p = 0.01; interleukin-10, p = 0.005), and acute kidney injury when compared with mice that had not yet met acute deterioration criteria., Conclusions: We defined a murine threshold for acute physiologic deterioration after cecal ligation and puncture that has adequate face and construct validity. This model may enable a more physiology-based model for evaluation of novel therapeutics in critical illness.

Background: Calcium plays an essential role in nearly all cellular processes. As such, cellular and systemic calcium concentrations are tightly regulated. During sepsis, derangements in such tight regulation frequently occur, and treating hypocalcemia with parenteral calcium administration remains the current practice guideline., Objective: We investigated whether calcium administration worsens mortality and organ dysfunction using an experimental murine model of sepsis and explored the mechanistic role of the family of calcium/calmodulin-dependent protein kinases in mediating these physiological effects. To highlight the biological relevance of these observations, we conducted a translational study of the association between calcium administration, organ dysfunction, and mortality among a cohort of critically ill septic ICU patients., Design: Prospective, randomized controlled experimental murine study and observational clinical cohort analysis., Setting: University research laboratory and eight ICUs at a tertiary care center., Patients: A cohort of 870 septic ICU patients., Subjects: C57Bl/6 and CaMKK mice., Interventions: Mice underwent cecal ligation and puncture polymicrobial sepsis and were administered with calcium chloride (0.25 or 0.25 mg/kg) or normal saline., Measurements and Main Results: Administering calcium chloride to septic C57Bl/6 mice heightened systemic inflammation and vascular leak, exacerbated hepatic and renal dysfunction, and increased mortality. These events were significantly attenuated in CaMKK mice. In a risk-adjusted analysis of septic patients, calcium administration was associated with an increased risk of death, odds ratio 1.92 (95% CI, 1.00-3.68; p = 0.049), a significant increase in the risk of renal dysfunction, odds ratio 4.74 (95% CI, 2.48-9.08; p < 0.001), and a significant reduction in ventilator-free days, mean decrease 3.29 days (0.50-6.08 days; p = 0.02)., Conclusions: Derangements in calcium homeostasis occur during sepsis that is sensitive to calcium administration. This altered calcium signaling, transduced by the calmodulin-dependent protein kinase kinase cascade, mediates heightened inflammation and vascular leak that culminates in elevated organ dysfunction and mortality. In the clinical management of septic patients, calcium supplementation provides no benefit and may impose harm.

Dysregulated Ca(2+) handling is prevalent during sepsis and postulated to perpetuate the aberrant inflammation underlying subsequent organ dysfunction and death. The signal transduction cascades mediating these processes are unknown. Here, we identify that CaMKIα mediates the Mϕ response to LPS in vitro and the inflammation and organ dysfunction of sepsis in vivo. We show that LPS induced active pThr(177)-CaMKIα in RAW 264.7 cells and murine peritoneal Mϕ, which if inhibited biochemically with STO609 (CaMKK inhibitor) or by RNAi, reduces LPS-induced production of IL-10. Transfection of constitutively active CaMKIα (CaMKI293), but not a kinase-deficient mutant (CaMKI293(K49A)), induces IL-10 release. This production of IL-10 is mediated by CaMKIα-dependent regulation of p38 MAPK activation. CaMKIα activity also mediates the cellular release of HMGB1 by colocalizing with and regulating the packaging of HMGB1 into secretory lysosomes. During endotoxemia, mice receiving in vivo CaMKIα(RNAi) display reduced systemic concentrations of IL-10 and HMGB1 in comparison with mice receiving NT(RNAi). These data support the biological relevance of CaMKIα-dependent IL-10 production and HMGB1 secretion. In a CLP model of sepsis, CaMKIα(RNAi) mice display reduced systemic concentrations of IL-10, IL-6, TNF-α, and HMGB1 in comparison with NT(RNAi) mice, which correlate with reductions in the development of renal dysfunction. These data support that CaMKIα signaling is integral to the Mϕ responding to LPS and may also be operant in vivo in regulating the inflammation and organ dysfunction consequent to sepsis.

The wavelength of light is a critical determinant of light's capacity to entrain adaptive biological mechanisms, such as enhanced immune surveillance, that precede and prepare us for the active circadian day, a time when the risk of encountering pathogen is highest. Light rich in the shorter wavelength visible blue spectrum maximally entrains these circadian rhythms. We hypothesized that exposure to blue light during sepsis will augment immunity and improve outcome. Using a clinically relevant Klebsiella pneumoniae acute lower respiratory tract infection model, we show that blue spectrum light shifts autonomic tone toward parasympathetic predominance and enhances immune competence, as characterized by accelerated pathogen clearance that is accompanied by reduced alveolar neutrophil influx, inflammation, and improved survival. Blue light functioned through an optic‐cholinergic pathway and expansion of splenic Ccr2+ monocytes to increase control of the infection and improve survival. The "keystone" mediating these effects is the circadian clock protein Rev‐Erbα, and biochemical activation with Rev‐Erbα agonist SR9009 enhanced mononuclear cell phagocytosis in vitro and recapitulated the enhanced pathogen elimination in vivo observed with blue light. These findings underscore the potential therapeutic value of blue light and modulating Rev‐Erbα to enhance host immunity against infection. [ABSTRACT FROM AUTHOR]

Objective: To determine that 1) an age-dependent loss of inducible autophagy underlies the failure to recover from AKI in older, adult animals during endotoxemia, and 2) pharmacologic induction of autophagy, even after established endotoxemia, is of therapeutic utility in facilitating renal recovery in aged mice. Design: Murine model of endotoxemia and cecal ligation and puncture (CLP) induced acute kidney injury (AKI). Setting: Academic research laboratory. Subjects: C57Bl/6 mice of 8 (young) and 45 (adult) weeks of age. Intervention: Lipopolysaccharide (1.5 mg/kg), Temsirolimus (5 mg/kg), AICAR (100 mg/kg). Measurements and Main Results: Herein we report that diminished autophagy underlies the failure to recover renal function in older adult mice utilizing a murine model of LPS-induced AKI. The administration of the mTOR inhibitor temsirolimus, even after established endotoxemia, induced autophagy and protected against the development of AKI. Conclusions: These novel results demonstrate a role for autophagy in the context of LPS-induced AKI and support further investigation into like interventions that have potential to alter the natural history of disease. [ABSTRACT FROM AUTHOR]

In patients with sepsis, diagnosis of blood stream infection (BSI) is a key concern to the therapist. Direct verification of pathogens in the blood stream executed by blood cultures (BC) still is regarded as the gold standard up to date. The quickest possible initiation of an appropriate antimicrobial therapy is a cornerstone of an effective therapy. Moreover, in this view BC can also serve to identify antimicrobial agents to target the pathogen. However, when employing BC the time needed until microbiological results are available ranges from 24 up to 72 h. Moreover, infections caused by multiple pathogens often remain undetected and concurrent antibiotic therapy may lower the overall sensitivity. Alternative pathogen characterization can be performed by polymerase chain reaction (PCR) based amplification methods. Results using PCR can be obtained within 6–8 h. Therefore, the time delay until an appropriate therapy can be reduced enormously. Moreover, these methods have the potential to enhance the sensitivity in the diagnosis of blood stream infections. Therefore, PCR based methods might be a valuable adjunct to present procedures of diagnosing bacteraemia. [Copyright &y& Elsevier]