IL-12p40 deletion aggravates lipopolysaccharide-induced cardiac dysfunction in mice.

Background: Cardiac dysfunction is one of the most common complications of sepsis and is associated with the adverse outcomes and high mortality of sepsis patients. IL-12p40, the common subunit of IL-12 and IL-23, has been shown to be involved in a variety of inflammation-related diseases, such as psoriasis and inflammatory bowel disease. However, the role of IL-12p40 in lipopolysaccharide (LPS)-induced cardiac dysfunction remains obscure. This study aimed to explore the role of IL-12p40 in LPS-induced cardiac dysfunction and its potential mechanisms. Methods: In this study, mice were treated with LPS and the cardiac expression of IL-12p40 was determined. Then, IL-12p40-/- mice were used to detect the role and mechanisms of IL-12p40 in LPS-induced cardiac injury. In addition, monocytes were adoptively transferred to IL-12p40-/- mice to explore their effects on LPS-induced cardiac dysfunction. Results: The results showed that cardiac IL-12p40 expression was significantly increased after treated with LPS. In addition, IL-12p40 deletion significantly aggravated LPS-induced cardiac dysfunction, evidenced by the increased serum levels of cardiomyocyte injury markers and heart injury scores, as well as by the deteriorated cardiac function. Moreover, IL-12p40 deletion increased LPS-induced monocyte accumulation and cardiac expression of inflammatory cytokines, as well as enhanced the activation of the NF-κB and MAPK pathways. Furthermore, adoptive transfer WT mouse monocytes to IL-12p40-/- mice alleviated LPS-induced cardiac dysfunction and decreased the phosphorylation of p65. Conclusion: IL-12p40 deletion significantly aggravated LPS-induced cardiac injury and cardiac dysfunction in mice by regulating the NF-κB and MAPK signaling pathways, and this process was related to monocytes. Therefore, IL-12p40 show a protective role in SIC, and IL-12p40 deficiency or anti-IL-12p40 monoclonal antibodies may be detrimental to patients with SIC.

Single-cell transcriptomic analysis of the immune cell landscape in the aged mouse brain after ischemic stroke.

BACKGROUND: Ischemic stroke is a medical emergency that primarily affects the elderly. A complex immune response in the post-stroke brain constitutes a key component of stroke pathophysiology. This study aimed to determine how stroke affects immune cell populations in the aged brain based on molecular profiles of individual cells. METHODS: Single-cell RNA sequencing and a new transient ischemic stroke mouse model with late reperfusion were used. RESULTS: We generated, for the first time, a composite picture of immune cell populations in the stroke aged brain at single-cell resolution. We discovered at least 6 microglial subsets in the stroke aged brain, including a potentially stroke-specific subtype. Moreover, we identified major cell subpopulations formed by infiltrated myeloid cells after stroke, and revealed their unique molecular profiles. CONCLUSIONS: This study provided the first scRNA-seq data set for immune cells in the stroke aged brain, and offered novel insights into post-stroke immune cell heterogeneity.

A Nomogram Model for Predicting Type-2 Myocardial Infarction Induced by Acute Upper Gastrointestinal Bleeding.

OBJECTIVE: To examine the independent risk factors of type-2 myocardial infarction (T2MI) elicited by acute upper gastrointestinal bleeding (AUGIB), and to establish a nomogram model for the prediction of AUGIB-induced T2MI. METHODS: A nomogram model was established on the basis of a retrospective study that involved 533 patients who suffered from AUGIB in the Department of Critical Care Medicine (CCM) or Emergency Intensive Care Unit (EICU) of Renmin Hospital of Wuhan University, Wuhan, China, from January 2017 to December 2020. The predictive accuracy and discriminative power of the nomogram were initially evaluated by internal validation, which involved drawing the receiver operating characteristic (ROC) curve, calculating the area under the curve (AUC), plotting the calibration curve derived from 1000 resampled bootstrap data sets, and computing the root mean square error (RMSE). The predictive ability of the nomogram was further validated through the prospective and multicenter study conducted by the investigators, which enrolled 240 AUGIB patients [including 88 cases from Renmin Hospital of Wuhan University, 73 cases from Qilu Hospital of Shandong University (Qingdao), and 79 cases from Northern Jiangsu People's Hospital)], who were admitted to the Department of CCM or EICU, from February 2021 to July 2021. RESULTS: Among the 533 patients in the training cohort, 78 (14.6%) patients were assigned to the T2MI group and 455 (85.4%) patients were assigned to the non-T2MI group. The multivariate analysis revealed that age >65, hemorrhagic shock, cerebral stroke, heart failure, chronic kidney disease, increased blood urea nitrogen, decreased hematocrit, and elevated D-Dimer were independent risk factors for AUGIB-induced T2MI. All these factors were incorporated into the nomogram model. The AUC for the nomogram for predicting T2MI was 0.829 (95% CI, 0.783-0.875) in the internal validation cohort and 0.848 (95% CI, 0.794-0.902) in the external validation cohort. The calibration curve for the risk of T2MI exhibited good consistency between the prediction by the nomogram and the actual clinical observation in both the internal validation (RMSE=0.016) and external validation (RMSE=0.020). CONCLUSION: The nomogram was proven to be a useful tool for the risk stratification of T2MI in AUGIB patients, and is helpful for the early identification of AUGIB patients who are prone to T2MI for early intervention, especially in emergency departments and intensive care units.

Neutrophil-to-lymphocyte ratio is a powerful predictor of adult patients with acute respiratory distress syndrome who might benefit from corticosteroid therapy.

BACKGROUND: There is no convincing pharmacological treatment for patients withacute respiratory distress syndrome (ARDS). The efficacy of corticosteroids in ARDS patients remains controversial. Neutrophil-to-Lymphocyte Ratio (NLR) has displayed as a good biomarker for inflammation and immune status, and thus a prognostic marker in some critical patients of ARDS. In this study, we hypothesized that NLR could also serve as an indicator for the efficacy of corticosteroid therapy in ARDS patients. METHODS: Subjects included in this retrospective cohort study with ARDS patients who were admitted to an academic hospital in Wuhan, China, from May 1st, 2020 to April 20th, 2021. Multivariable logisitic regression model was used to evaluate risk factors of 30-day in-hospital mortality and ventilator-free days. Multi-Cox regression model was used to assess the efficacy of corticosteroid treatment in terms of NLR cutoff value. RESULTS: Among the 357 patients in our study, 89 (24.9%) had NLR≥14.35 and 268 (75.1%) had NLR<14.35. Among them, 53 patients with NLR≥14.35 (58.9%) received corticosteroids and 99 patients with NLR<14.35 (37.1%) received corticosteroids. Post-adjustment analysis (by APACHE II score and age) revealed that corticosteroid treatment was associated with a decreased risk of 30-day mortality in the NLR≥14.35 group but with an increased risk of death in the NLR<14.35 group. Use of corticosteroid in NLR≥14.35 group significantly increased ventilator-free days (7.0 vs. 13.0, P<0.001). CONCLUSION: NLR may be used to help identify ARDS patients who may benefit from corticosteroid treatment. Large-sized randomized controlled trials are warranted to determine the optimal cutoff value of NLR.

Development and Evaluation of a Novel Mouse Model of Asphyxial Cardiac Arrest Revealed Severely Impaired Lymphopoiesis After Resuscitation.

Background Animal disease models represent the cornerstone in basic cardiac arrest (CA) research. However, current experimental models of CA and resuscitation in mice are limited. In this study, we aimed to develop a mouse model of asphyxial CA followed by cardiopulmonary resuscitation (CPR), and to characterize the immune response after asphyxial CA/CPR. Methods and Results CA was induced in mice by switching from an O2/N2 mixture to 100% N2 gas for mechanical ventilation under anesthesia. Real-time measurements of blood pressure, brain tissue oxygen, cerebral blood flow, and ECG confirmed asphyxia and ensuing CA. After a defined CA period, mice were resuscitated with intravenous epinephrine administration and chest compression. We subjected young adult and aged mice to this model, and found that after CA/CPR, mice from both groups exhibited significant neurologic deficits compared with sham mice. Analysis of post-CA brain confirmed neuroinflammation. Detailed characterization of the post-CA immune response in the peripheral organs of both young adult and aged mice revealed that at the subacute phase following asphyxial CA/CPR, the immune system was markedly suppressed as manifested by drastic atrophy of the spleen and thymus, and profound lymphopenia. Finally, our data showed that post-CA systemic lymphopenia was accompanied with impaired T and B lymphopoiesis in the thymus and bone marrow, respectively. Conclusions In this study, we established a novel validated asphyxial CA model in mice. Using this new model, we further demonstrated that asphyxial CA/CPR markedly affects both the nervous and immune systems, and notably impairs lymphopoiesis of T and B cells.

Cardiac arrest and resuscitation activates the hypothalamic-pituitary-adrenal axis and results in severe immunosuppression.

In patients who are successfully resuscitated after initial cardiac arrest (CA), mortality and morbidity rates are high, due to ischemia/reperfusion injury to the whole body including the nervous and immune systems. How the interactions between these two critical systems contribute to post-CA outcome remains largely unknown. Using a mouse model of CA and cardiopulmonary resuscitation (CA/CPR), we demonstrate that CA/CPR induced neuroinflammation in the brain, in particular, a marked increase in pro-inflammatory cytokines, which subsequently activated the hypothalamic-pituitary-adrenal (HPA) axis. Importantly, this activation was associated with a severe immunosuppression phenotype after CA. The phenotype was characterized by a striking reduction in size of lymphoid organs accompanied by a massive loss of immune cells and reduced immune function of splenic lymphocytes. The mechanistic link between post-CA immunosuppression and the HPA axis was substantiated, as we discovered that glucocorticoid treatment, which mimics effects of the activated HPA axis, exacerbated post-CA immunosuppression, while RU486 treatment, which suppresses its effects, significantly mitigated lymphopenia and lymphoid organ atrophy and improved CA outcome. Taken together, targeting the HPA axis could be a viable immunomodulatory therapeutic to preserve immune homeostasis after CA/CPR and thus improve prognosis of post-resuscitation CA patients.

MCC950, a selective NLPR3 inflammasome inhibitor, improves neurologic function and survival after cardiac arrest and resuscitation.

BACKGROUND: Cardiac arrest (CA) is associated with high morbidity and mortality, even after spontaneous circulation is re-established. This dire situation is partly due to post-CA syndrome for which no specific and effective intervention is available. One key component of post-CA syndrome is sterile inflammation, which affects various organs including the brain. A major effector of sterile inflammation is activated NLRP3 inflammasome, which leads to increased release of interleukin (IL)-1ß. However, how NLRP3 inflammasome impacts neuroinflammation and neurologic outcome after CA is largely undefined. METHODS: Mice were subjected to a potassium-based murine CA and cardiopulmonary resuscitation (CPR) model. MCC950 was used to suppress activation of NLRP3 inflammasome after CA/CPR. Levels of protein and mRNA were examined by Western blotting and quantitative PCR, respectively. Immunologic changes were assessed by measuring cytokine expression and immune cell compositions. CA outcomes, including neurologic deficits, bacterial load in the lung, and survival rate, were evaluated. RESULTS: Using our CA/CPR model, we found that NLRP3 inflammasome was activated in the post-CA brain, and that pro-inflammatory cytokine levels, including IL-1ß, were increased. After treatment with MCC950, a potent and selective NLRP3 inflammasome inhibitor, mice exhibited improved functional recovery and survival rate during the 14-day observational period after CA/CPR. In line with these findings, IL-1ß mRNA levels in the post-CA brain were significantly suppressed after MCC950 treatment. Interestingly, we also found that in MCC950- vs. vehicle-treated CA mice, immune homeostasis in the spleen was better preserved and bacterial load in the lung was significantly reduced. CONCLUSIONS: Our data demonstrate that activation of NLRP3 inflammasome could be a key event shaping the post-CA immuno- and neuro-pathology, and identify this pathway as a unique and promising therapeutic target to improve outcomes after CA/CPR.

Small ubiquitin-like modifier 2 (SUMO2) is critical for memory processes in mice.

Small ubiquitin-like modifier (SUMO1-3) conjugation (SUMOylation), a posttranslational modification, modulates almost all major cellular processes. Mounting evidence indicates that SUMOylation plays a crucial role in maintaining and regulating neural function, and importantly its dysfunction is implicated in cognitive impairment in humans. We have previously shown that simultaneously silencing SUMO1-3 expression in neurons negatively affects cognitive function. However, the roles of the individual SUMOs in modulating cognition and the mechanisms that link SUMOylation to cognitive processes remain unknown. To address these questions, in this study, we have focused on SUMO2 and generated a new conditional Sumo2 knockout mouse line. We found that conditional deletion of Sumo2 predominantly in forebrain neurons resulted in marked impairments in various cognitive tests, including episodic and fear memory. Our data further suggest that these abnormalities are attributable neither to constitutive changes in gene expression nor to alterations in neuronal morphology, but they involve impairment in dynamic SUMOylation processes associated with synaptic plasticity. Finally, we provide evidence that dysfunction on hippocampal-based cognitive tasks was associated with a significant deficit in the maintenance of hippocampal long-term potentiation in Sumo2 knockout mice. Collectively, these data demonstrate that protein conjugation by SUMO2 is critically involved in cognitive processes.

PERK (Protein Kinase RNA-Like ER Kinase) Branch of the Unfolded Protein Response Confers Neuroprotection in Ischemic Stroke by Suppressing Protein Synthesis.

Background and Purpose- Ischemic stroke impairs endoplasmic reticulum (ER) function, causes ER stress, and activates the unfolded protein response. The unfolded protein response consists of 3 branches controlled by ER stress sensor proteins, which include PERK (protein kinase RNA-like ER kinase). Activated PERK phosphorylates eIF2α (eukaryotic initiation factor 2 alpha), resulting in inhibition of global protein synthesis. Here, we aimed to clarify the role of the PERK unfolded protein response branch in stroke. Methods- Neuron-specific and tamoxifen-inducible PERK conditional knockout (cKO) mice were generated by cross-breeding Camk2a-CreERT2 with Perkf/f mice. Transient middle cerebral artery occlusion was used to induce stroke. Short- and long-term stroke outcomes were evaluated. Protein synthesis in the brain was assessed using a surface-sensing-of-translation approach. Results- After tamoxifen-induced deletion of Perk in forebrain neurons was confirmed in PERK-cKO mice, PERK-cKO and control mice were subjected to transient middle cerebral artery occlusion and 3 days or 3 weeks recovery. PERK-cKO mice had larger infarcts and worse neurological outcomes compared with control mice, suggesting that PERK-induced eIF2α phosphorylation and subsequent suppression of translation protects neurons from ischemic stress. Indeed, better stroke outcomes were observed in PERK-cKO mice that received postischemic treatment with salubrinal, which can restore the ischemia-induced increase in phosphorylated eIF2α in these mice. Finally, our data showed that post-treatment with salubrinal improved functional recovery after stroke. Conclusions- Here, we presented the first evidence that postischemic suppression of translation induced by PERK activation promotes recovery of neurological function after stroke. This confirms and further extends our previous observations that recovery of ER function impaired by ischemic stress critically contributes to stroke outcome. Therefore, future research should include strategies to improve stroke outcome by targeting unfolded protein response branches to restore protein homeostasis in neurons.

Mitochondrial Autophagy and NLRP3 Inflammasome in Pulmonary Tissues from Severe Combined Immunodeficient Mice after Cardiac Arrest and Cardiopulmonary Resuscitation.

BACKGROUND: The incidence of cancer, diabetes, and autoimmune diseases has been increasing. Furthermore, there are more and more patients with solid organ transplants. The survival rate of these immunocompromised individuals is extremely low when they are severely hit-on. In this study, we established cardiac arrest cardiopulmonary resuscitation (CPR) model in severe combined immunodeficient (SCID) mice, analyzed the expression and activation of mitochondrial autophagy and NLRP3 inflammasome/caspase-1, and explored mitochondrial repair and inflammatory injury in immunodeficiency individual during systemic ischemia-reperfusion injury. METHODS: A potassium chloride-induced cardiac arrest model was established in C57BL/6 and nonobese diabetic/SCID (NOD/SCID) mice. One hundred male C57BL/6 mice and 100 male NOD/SCID mice were randomly divided into five groups (control, 2 h post-CPR, 12 h post-CPR, 24 h post-CPR, and 48 h post-CPR). A temporal dynamic view of alveolar epithelial cells, macrophages, and neutrophils from bronchoalveolar lavage fluid (BALF) was obtained using Giemsa staining. Spatial characterization of phenotypic analysis of macrophages in the lung interstitial tissue was analyzed by flow cytometry. The morphological changes of mitochondria 48 h after CPR were studied by transmission electron microscopy and quantified according to the Flameng grading system. Western blotting analysis was used to detect the expression and activation of the markers of mitochondrial autophagy, NLRP3 inflammasome, and caspase-1. RESULTS: (1) In NOD/SCID mice, macrophages were disintegrated in BALF, and many alveolar epithelial cells were shed at 48 h after resuscitation. Compared with C57BL/6 mice, the ratio of macrophages/total cells peaked at 12 h and was significantly higher in NOD/SCID mice (31.17 ± 4.13 vs. 49.69 ± 2.43, t = 14.46, P = 0.001). After 24 h, the results showed a downward trend. Furthermore, a large number of macrophages were disintegrated in the BALF. (2) Mitochondrial autophagy was present in both C57BL/6 and NOD/SCID mice after CPR, but it began late in the NOD/SCID mice. Compared with C57BL/6 mice, phos-ULK1 (Ser327) expression was significantly lower at 2 h and 12 h after CPR (2 h after CPR: 1.88 ± 0.36 vs. 1.12 ± 0.11, t = -1.36, P < 0.01 and 12 h after CPR: 1.52 ± 0.16 vs. 1.05 ± 0.12, t = -0.33, P < 0.01), whereas phos-ULK1 (Ser757) expression was significantly higher at 2 h and 12 h after CPR in NOD/SCID mice (2 h after CPR: 1.28 ± 0.12 vs. 1.69 ± 0.14, t = 1.7, P < 0.01 and 12 h after CPR: 1.33 ± 0.10 vs. 1.94 ± 0.13, t = 2.75, P < 0.01). (3) Furthermore, NLRP3 inflammasome/caspase-1 activation in the pulmonary tissues occurred early and for only a short time in C57BL/6 mice, but this phenomenon was sustained in NOD/SCID mice. The expression of the NLRP3 inflammasome increased modestly in the C57 mice, but the increase was higher in the NOD/SCID mice than in the C57BL/6 mice, especially at 12, 24, 48 h after CPR (48 h after CPR: 1.46 ± 0.13 vs. 2.97 ± 0.19, t = 5.34, P = 0.001). The expression of caspase-1-20 generally followed the same pattern as the NLRP3 inflammasome. CONCLUSIONS: There is a regulatory relationship between the NLRP3 inflammasome and mitochondrial autophagy after CPR in the healthy mice. This regulatory relationship was disturbed in the NOD/SCID mice because the signals for mitochondrial autophagy occurred late, and NLRP3 inflammasome- and caspase-1-dependent cell injury was sustained.