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The clinical and immunological performance of 28 days survival model of cecal ligation and puncture in humanized mice.

ABSTRACT: Sepsis triggers a coordinated and thorough immune system response with long-term unfavorable sequelae after the initial insult. Long-term recovery from sepsis has garnered increasing attention recently, but a lack of suitable animal models impairs progress in this area. Our study, therefore, aimed to address the performance of the immune system in a survivable model of sepsis (cecal ligation and sepsis; CLP) for up to 28 d after the initial injury in humanized mice. Our model mimics human sepsis with weight loss and post-sepsis hypothermia. Within the first 7 d of sepsis, the M1 inflammatory cell subtype predominated, as evidenced by increased CD16 expression, but at 28 d, a mixed population of M1 and M2 inflammatory cells emerged, as evidenced by increased secretion of transforming growth factor TGF? and CD206 expression. This change was accompanied by normalized production of interleukin (IL)-6, tumor necrosis factor TNF? and IL-10 at 28 d. Furthermore, the ability of MO to become regulatory DC or the frequency of endogenous DC were severely affected at 28 days. Thus, sepsis results in profound and persistent changes in the function of myeloid cells up to 28 days after CLP demonstrating the persistence of the new acquired immunological features long after resolution of the sepsis.

SUBMITTER: Laudanski K

PROVIDER: S-EPMC5513410 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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The clinical and immunological performance of 28 days survival model of cecal ligation and puncture in humanized mice.

Laudanski Krzysztof K Lapko Natalia N Zawadka Mateusz M Zhou Benjamin X BX Danet-Desnoyers Gwenn G Worthen George S GS

PloS one 20170717 7

Sepsis triggers a coordinated and thorough immune system response with long-term unfavorable sequelae after the initial insult. Long-term recovery from sepsis has garnered increasing attention recently, but a lack of suitable animal models impairs progress in this area. Our study, therefore, aimed to address the performance of the immune system in a survivable model of sepsis (cecal ligation and sepsis; CLP) for up to 28 d after the initial injury in humanized mice. Our model mimics human sepsis ...[more]

PMID: 28715505

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Project description:BACKGROUND:The paradigm shift from crystalloid to plasma resuscitation of traumatic hemorrhagic shock has improved patient outcomes due in part to plasma-mediated reversal of catecholamine and inflammation-induced endothelial injury, decreasing vascular permeability and attenuating organ injury. Since sepsis induces a similar endothelial injury as seen in hemorrhage, we hypothesized that plasma resuscitation would increase 48-h survival in a rat sepsis model. METHODS:Adult male Sprague-Dawley rats (375-425?g) were subjected to 35% cecal ligation and puncture (CLP) (t?=?0?h). Twenty-two hours post-CLP and prior to resuscitation (t?=?22?h), animals were randomized to resuscitation with normal saline (NS, 10?cc/kg/h) or pooled rat fresh frozen plasma (FFP, 3.33?cc/kg/h). Resuscitation under general anesthesia proceeded for the next 6 h (t?=?22?h to t?=?28?h); lactate was checked every 2?h, and fluid volumes were titrated based on lactate clearance. Blood samples were obtained before (t?=?22?h) and after resuscitation (t?=?28?h), and at death or study conclusion. Lung specimens were obtained for calculation of wet-to-dry weight ratio. Fisher exact test was used to analyze the primary outcome of 48-h survival. ANOVA with repeated measures was used to analyze the effect of FFP versus NS resuscitation on blood gas, electrolytes, blood urea nitrogen (BUN), creatinine, interleukin (IL)-6, IL-10, catecholamines, and syndecan-1 (marker for endothelial injury). A two-tailed alpha level of <0.05 was used for all statistical tests. RESULTS:Thirty-three animals were studied: 14 FFP, 14 NS, and 5 sham. Post-CLP but preresuscitation (t?=?22?h) variables between FFP and NS animals were similar and significantly deranged compared with sham animals. FFP significantly increased 48-h survival compared to NS (n?=?8 [57%] vs n?=?2 [14%]), attenuated the post-resuscitation (t?=?28?h) levels of epinephrine (mean 2.2 vs 7.0?ng/mL), norepinephrine, (3.8 vs 8.9?ng/mL), IL-6 (3.8 vs 18.7?ng/mL), and syndecan-1 (21.8 vs 31.0?ng/mL) (all P?<?0.05), improved the post-resuscitation PO2 to FiO2 ratio (353 vs 151), and reduced the pulmonary wet-to-dry weight ratio (5.28 vs 5.94) (all P?<?0.05). CONCLUSION:Compared to crystalloid, plasma resuscitation increased 48-h survival in a rat sepsis model, improved pulmonary function and decreased pulmonary edema, and attenuated markers for inflammation, endothelial injury, and catecholamines.

Project description:Sepsis is the leading cause of death in intensive care units. MicroRNA-34a (miR-34a) is involved in sepsis progression, while its underlying mechanisms on sepsis-induced lung injury remain obscure. Oxidative stress, pyroptosis, and inhibition of autophagy can result in organ injury. MiR-34a has been reported to regulate oxidative stress and autophagy via inhibiting silent information regulator T1 (SIRT1) and autophagy gene 4B (ATG4B) signaling. This study aimed at identifying the function of miR-34a in oxidative stress, inflammation, pyroptosis, and autophagy in sepsis-induced lung injury. Male 8-week-old C57BL/6 mice were subjected to cecal ligation and puncture and treated with miR-34a antagomir/agomir. Survival (n = 10), histopathological changes (n = 6), and lung wet-to-dry ratio (n = 6) were recorded and assayed. Other detection (n = 6) was performed to investigate the level of oxidative stress, inflammation, pyroptosis, and autophagy in lung tissues. Results showed that miR-34a down-regulation ameliorated lung injury in septic mice as reflected by decreased lung injury scores (decrease from 3.00 ± 0.32 to 2.00 ± 0.32) and wet-to-dry ratio (0.36-fold decrease). MiR-34a down-regulation also decreased reactive oxygen species accumulation (0.36-fold decrease), and promoted superoxide dismutase activity and the expression of SIRT1 (1.24-fold increase), heme oxygenase-1 and nuclear factor erythroid 2 like 2 to inhibit oxidative stress in septic mice. Moreover, miR-34a down-regulation suppressed inflammatory response and pyroptosis in septic mice, as evidenced by decreased level of pro-inflammatory factors including tumor necrosis factor ?, interleukin-6 (IL-6), IL-1?, and IL-18, activity of caspase-1 (0.51-fold decrease) and expression of nucleotide-binding domain and leucine-rich repeat protein-3 (0.48-fold decrease), apoptosis-associated speck-like protein containing a CARD, cleaved-caspase-1, and cleaved-gasdermin D (0.36-fold decrease), and increased level of anti-inflammatory factors IL-10. MiR-34a down-regulation also enhanced autophagy in septic mice as evidenced by more autolysosomes and elevated expressions of ATG4B (0.90-fold increase), beclin1, ATG9, and LC3 II/I. Among these experiments, miR-34a up-regulation showed opposite effects on oxidative stress, inflammatory response, pyroptosis, and autophagy in septic mice. Additionally, miR-34a could bind to the 3'-untranslated region of SIRT1 and ATG4B. In conclusion, our findings demonstrated that miR-34a was implicated in oxidative stress, inflammation, pyroptosis, and autophagy in the development of sepsis. MiR-34a inhibition had a potential to alleviate sepsis-induced lung injury.

Project description:BackgroundIndividual T cell responses vary significantly based on the microenvironment present at the time of immune response and on prior induced T cell memory. While the cecal ligation and puncture (CLP) model is the most commonly used murine sepsis model, the contribution of diverse T cell responses has not been explored. We defined T cell subset responses to CLP using single-cell RNA sequencing and examined the effects of prior induced T cell memory (Immune Education) on these responses. We hypothesized that Immune Education prior to CLP would alter T cell responses at the single cell level at a single, early post-CLP time point.MethodsSplenic T cells were isolated from C57BL/6 mice. Four cohorts were studied: Control, Immune-Educated, CLP, and Immune-Educated CLP. At age 8 weeks, Immune-Educated and Immune-Educated CLP mice received anti-CD3ϵ antibody; Control and CLP mice were administered an isotype control. CLP (two punctures with a 22-gauge needle) was performed at 12-13 weeks of life. Mice were sacrificed at baseline or 24-hours post-CLP. Unsupervised clustering of the transcriptome library identified six distinct T cell subsets: quiescent naïve CD4+, primed naïve CD4+, memory CD4+, naïve CD8+, activated CD8+, and CD8+ cytotoxic T cell subsets. T cell subset specific gene set enrichment analysis and Hurdle analysis for differentially expressed genes (DEGs) were performed.ResultsT cell responses to CLP were not uniform - subsets of activated and suppressed T cells were identified. Immune Education augmented specific T cell subsets and led to genomic signatures favoring T cell survival in unoperated and CLP mice. Additionally, the combination of Immune Education and CLP effected the expression of genes related to T cell activity in ways that differed from CLP alone. Validating our finding that IL7R pathway markers were upregulated in Immune-Educated CLP mice, we found that Immune Education increased T cell surface IL7R expression in post-CLP mice.ConclusionImmune Education enhanced the expression of genes associated with T cell survival in unoperated and CLP mice. Induction of memory T cell compartments via Immune Education combined with CLP may increase the model's concordance to human sepsis.

Project description:Sepsis is one of the most prevalent diseases in the world. The development of cardiac dysfunction in sepsis results in an increase of mortality. It is known that Bruton's tyrosine kinase (BTK) plays a role in toll-like receptor signaling and NLRP3 inflammasome activation, two key components in the pathophysiology of sepsis and sepsis-associated cardiac dysfunction. In this study we investigated whether pharmacological inhibition of BTK (ibrutinib 30 mg/kg and acalabrutinib 3 mg/kg) attenuates sepsis associated cardiac dysfunction in mice. 10-week old male C57BL/6 mice underwent CLP or sham surgery. One hour after surgery mice received either vehicle (5% DMSO + 30% cyclodextrin i.v.), ibrutinib (30 mg/kg i.v.), or acalabrutinib (3 mg/kg i.v.). Mice also received antibiotics and an analgesic at 6 and 18 h. After 24 h, cardiac function was assessed by echocardiography in vivo. Cardiac tissue underwent western blot analysis to determine the activation of BTK, NLRP3 inflammasome and NF-κB pathway. Serum analysis of 33 cytokines was conducted by a multiplex assay. When compared to sham-operated animals, mice subjected to CLP demonstrated a significant reduction in ejection fraction (EF), fractional shortening (FS), and fractional area change (FAC). The cardiac tissue from CLP mice showed significant increases of BTK, NF-κB, and NLRP3 inflammasome activation. CLP animals resulted in a significant increase of serum cytokines and chemokines (TNF-α, IL-6, IFN-γ, KC, eotaxin-1, eotaxin-2, IL-10, IL-4, CXCL10, and CXCL11). Delayed administration of ibrutinib and acalabrutinib attenuated the decline of EF, FS, and FAC caused by CLP and also reduced the activation of BTK, NF-κB, and NLRP3 inflammasome. Both ibrutinib and acalabrutinib significantly suppressed the release of cytokines and chemokines. Our study revealed that delayed intravenous administration of ibrutinib or acalabrutinib attenuated the cardiac dysfunction associated with sepsis by inhibiting BTK, reducing NF-κB activation and the activation of the inflammasome. Cytokines associated with sepsis were significantly reduced by both BTK inhibitors. Acalabrutinib is found to be more potent than ibrutinib and could potentially prove to be a novel therapeutic in sepsis. Thus, the FDA-approved BTK inhibitors ibrutinib and acalabrutinib may be repurposed for the use in sepsis.

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The clinical and immunological performance of 28 days survival model of cecal ligation and puncture in humanized mice.

Publications

The clinical and immunological performance of 28 days survival model of cecal ligation and puncture in humanized mice.

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