Peripheral PD-1+NK cells could predict the 28-day mortality in sepsis patients.

Background: Unbalanced inflammatory response is a critical feature of sepsis, a life-threatening condition with significant global health burdens. Immune dysfunction, particularly that involving different immune cells in peripheral blood, plays a crucial pathophysiological role and shows early warning signs in sepsis. The objective is to explore the relationship between sepsis and immune subpopulations in peripheral blood, and to identify patients with a higher risk of 28-day mortality based on immunological subtypes with machine-learning (ML) model. Methods: Patients were enrolled according to the sepsis-3 criteria in this retrospective observational study, along with age- and sex-matched healthy controls (HCs). Data on clinical characteristics, laboratory tests, and lymphocyte immunophenotyping were collected. XGBoost and k-means clustering as ML approaches, were employed to analyze the immune profiles and stratify septic patients based on their immunological subtypes. Cox regression survival analysis was used to identify potential biomarkers and to assess their association with 28-day mortality. The accuracy of biomarkers for mortality was determined by the area under the receiver operating characteristic (ROC) curve (AUC) analysis. Results: The study enrolled 100 septic patients and 89 HCs, revealing distinct lymphocyte profiles between the two groups. The XGBoost model discriminated sepsis from HCs with an area under the receiver operating characteristic curve of 1.0 and 0.99 in the training and testing set, respectively. Within the model, the top three highest important contributions were the percentage of CD38+CD8+T cells, PD-1+NK cells, HLA-DR+CD8+T cells. Two clusters of peripheral immunophenotyping of septic patients by k-means clustering were conducted. Cluster 1 featured higher proportions of PD1+ NK cells, while cluster 2 featured higher proportions of naïve CD4+T cells. Furthermore, the level of PD-1+NK cells was significantly higher in the non-survivors than the survivors (15.1% vs 8.6%, P<0.01). Moreover, the levels of PD1+ NK cells combined with SOFA score showed good performance in predicting the 28-day mortality in sepsis (AUC=0.91,95%CI 0.82-0.99), which is superior to PD1+ NK cells only(AUC=0.69, sensitivity 0.74, specificity 0.64, cut-off value of 11.25%). In the multivariate Cox regression, high expression of PD1+ NK cells proportion was related to 28-day mortality (aHR=1.34, 95%CI 1.19 to 1.50; P<0.001). Conclusion: The study provides novel insights into the association between PD1+NK cell profiles and prognosis of sepsis. Peripheral immunophenotyping could potentially stratify the septic patients and identify those with a high risk of 28-day mortality.

Identification and experimental validation of cuproptosis regulatory program in a sepsis immune microenvironment through a combination of single-cell and bulk RNA sequencing.

Background: In spite of its high mortality rate and poor prognosis, the pathogenesis of sepsis is still incompletely understood. This study established a cuproptosis-based risk model to diagnose and predict the risk of sepsis. In addition, the cuproptosis-related genes were identified for targeted therapy. Methods: Single-cell sequencing analyses were used to characterize the cuproptosis activity score (CuAS) and intercellular communications in sepsis. Differential cuproptosis-related genes (CRGs) were identified in conjunction with single-cell and bulk RNA sequencing. LASSO and Cox regression analyses were employed to develop a risk model. Three external cohorts were conducted to assess the model's accuracy. Differences in immune infiltration, immune cell subtypes, pathway enrichment, and the expression of immunomodulators were further evaluated in distinct groups. Finally, various in-vitro experiments, such as flow cytometry, Western blot, and ELISA, were used to explore the role of LST1 in sepsis. Results: ScRNA-seq analysis demonstrated that CuAS was highly enriched in monocytes and was closely related to the poor prognosis of sepsis patients. Patients with higher CuAS exhibited prominent strength and numbers of cell-cell interactions. A total of five CRGs were identified based on the LASSO and Cox regression analyses, and a CRG-based risk model was established. The lower riskScore cohort exhibited enhanced immune cell infiltration, elevated immune scores, and increased expression of immune modulators, indicating the activation of an antibacterial response. Ultimately, in-vitro experiments demonstrated that LST1, a key gene in the risk model, was enhanced in the macrophage in response to LPS, which was closely related to the decrease of macrophage survival rate, the enhancement of apoptosis and oxidative stress injury, and the imbalance of the M1/M2 phenotype. Conclusions: This study constructed a cuproptosis-related risk model to accurately predict the prognosis of sepsis. We further characterized the cuproptosis-related gene LST1 to provide a theoretical framework for sepsis therapy.

Exosome-shuttled miR-150-5p from LPS-preconditioned mesenchymal stem cells down-regulate PI3K/Akt/mTOR pathway via Irs1 to enhance M2 macrophage polarization and confer protection against sepsis.

Rationale: Sepsis is a life-threatening organ dysfunction and lack of effective measures in the current. Exosomes from mesenchymal stem cells (MSCs) reported to alleviate inflammation during sepsis, and the preconditioning of MSCs could enhance their paracrine potential. Therefore, this study investigated whether exosomes secreted by lipopolysaccharide (LPS)-pretreated MSCs exert superior antiseptic effects, and explored the underlying molecular mechanisms. Methods: Exosomes were isolated and characterized from the supernatants of MSCs. The therapeutic efficacy of normal exosomes (Exo) and LPS-pretreated exosomes (LPS-Exo) were evaluated in terms of survival rates, inflammatory response, and organ damage in an LPS-induced sepsis model. Macrophages were stimulated with LPS and treated with Exo or LPS-Exo to confirm the results of the in vivo studies, and to explain the potential mechanisms. Results: LPS-Exo were shown to inhibit aberrant pro-inflammatory cytokines, prevent organ damages, and improve survival rates of the septic mice to a greater extent than Exo. In vitro, LPS-Exo significantly promoted the M2 polarization of macrophages exposed to inflammation. miRNA sequencing and qRT-PCR analysis identified the remarkable expression of miR-150-5p in LPS-Exo compared to that in Exo, and exosomal miR-150-5p was transferred into recipient macrophages and mediated macrophage polarization. Further investigation demonstrated that miR-150-5p targets Irs1 in recipient macrophages and subsequently modulates macrophage plasticity by down-regulating the PI3K/Akt/mTOR pathway. Conclusion: The current findings highly suggest that exosomes derived from LPS pre-conditioned MSCs represent a promising cell-free therapeutic method and highlight miR-150-5p as a novel molecular target for regulating immune hyperactivation during sepsis.

Deciphering the mechanism of cimifugin in mitigating LPS-induced neuroinflammation in BV-2 cells.

PURPOSE: Sepsis often triggers a systemic inflammatory response leading to multi-organ dysfunction, with complex and not fully understood pathogenesis. This study investigates the therapeutic effects of cimifugin on BV-2 cells under sepsis-induced stress conditions. METHODS: We utilized a BV-2 microglial cell model treated with lipopolysaccharide (LPS) to mimic sepsis. Assessments included cellular vitality, inflammatory cytokine quantification (6 interleukin [6IL]-1ß, interleukin 6 [IL-6], and tumor necrosis factor-α [TNF-α]) via enzyme-linked-immunosorbent serologic assay, and analysis of mRNA expression using real-time polymerase chain reaction. Oxidative stress and mitochondrial function were also evaluated to understand the cellular effects of cimifugin. RESULTS: Cimifugin significantly attenuated LPS-induced inflammatory responses, oxidative stress, and mitochondrial dysfunction. It enhanced cell viability and modulated the secretion and gene expression of inflammatory cytokines IL-1ß, IL-6, and TNF-α. Notably, cimifugin activated the deacetylase sirtuin 1-nuclear factor erythroid 2-related factor 2 pathway, contributing to its protective effects against mitochondrial damage. CONCLUSION: Cimifugin demonstrates the potential of being an effective treatment for sepsis--induced neuroinflammation, warranting further investigation.

Unraveling host-pathogen dynamics in a murine Model of septic peritonitis induced by vancomycin-resistant Enterococcus faecium.

Vancomycin-resistant Enterococcus faecium (E. faecium) infection is associated with higher mortality rates. Previous studies have emphasized the importance of innate immune cells and signalling pathways in clearing E. faecium, but a comprehensive analysis of host-pathogen interactions is lacking. Here, we investigated the interplay of host and E. faecium in a murine model of septic peritonitis. Following injection with a sublethal dose, we observed significantly increased murine sepsis score and histological score, decreased weight and bacterial burden, neutrophils and macrophages infiltration, and comprehensive activation of cytokine-mediated signalling pathway. In mice receiving a lethal dose, hypothermia significantly improved survival, reduced bacterial burden, cytokines, and CD86 expression of MHC-II+ recruited macrophages compared to the normothermia group. A mathematical model constructed by observational data from 80 animals, recapitulated the host-pathogen interplay, and further verified the benefits of hypothermia. These findings indicate that E. faecium triggers a severe activation of cytokine-mediated signalling pathway, and hypothermia can improve outcomes by reducing bacterial burden and inflammation.

Role of Kynurenine and Its Derivatives in the Neuroimmune System.

In recent years, there has been a growing realization of intricate interactions between the nervous and immune systems, characterized by shared humoral factors and receptors. This interplay forms the basis of the neuroimmune system, the understanding of which will provide insights into the pathogenesis of neurological diseases, in which the involvement of the immune system has been overlooked. Kynurenine and its derivatives derived from tryptophan have long been implicated in the pathogenesis of various neurological diseases. Recent studies have revealed their close association not only with neurological disorders but also with sepsis-related deaths. This review provides an overview of the biochemistry of kynurenine and its derivatives, followed by a discussion of their role via the modulation of the neuroimmune system in various diseases.

IL-8 Induces Neutrophil Extracellular Trap Formation in Severe Thermal Injury.

Neutrophil extracellular traps (NETs) have a dual role in the innate immune response to thermal injuries. NETs provide an early line of defence against infection. However, excessive NETosis can mediate the pathogenesis of immunothrombosis, disseminated intravascular coagulation (DIC) and multiple organ failure (MOF) in sepsis. Recent studies suggest that high interleukin-8 (IL-8) levels in intensive care unit (ICU) patients significantly contribute to excessive NET generation. This study aimed to determine whether IL-8 also mediates NET generation in patients with severe thermal injuries. IL-8 levels were measured in serum samples from thermally injured patients with ≥15% of the total body surface area (TBSA) and healthy controls (HC). Ex vivo NET generation was also investigated by treating isolated neutrophils with serum from thermal injured patients or normal serum with and without IL-8 and anti-IL-8 antibodies. IL-8 levels were significantly increased compared to HC on days 3 and 5 (p < 0.05) following thermal injury. IL-8 levels were also significantly increased at day 5 in septic versus non-septic patients (p < 0.001). IL-8 levels were also increased in patients who developed sepsis compared to HC at days 3, 5 and 7 (p < 0.001), day 10 (p < 0.05) and days 12 and 14 (p < 0.01). Serum containing either low, medium or high levels of IL-8 was shown to induce ex vivo NETosis in an IL-8-dependent manner. Furthermore, the inhibition of DNase activity in serum increased the NET-inducing activity of IL-8 in vitro by preventing NET degradation. IL-8 is a major contributor to NET formation in severe thermal injury and is increased in patients who develop sepsis. We confirmed that DNase is an important regulator of NET degradation but also a potential confounder within assays that measure serum-induced ex vivo NETosis.

Summary data-based Mendelian randomization and single-cell RNA sequencing analyses identify immune associations with low-level LGALS9 in sepsis.

Sepsis is one of the major challenges in intensive care units, characterized by the complexity of the host immune status. To gain a deeper understanding of the pathogenesis of sepsis, it is crucial to study the phenotypic changes in immune cells and their underlying molecular mechanisms. We conducted Summary data-based Mendelian randomization analysis by integrating genome-wide association studies data for sepsis with expression quantitative trait locus data, revealing a significant decrease in the expression levels of 17 biomarkers in sepsis patients. Furthermore, based on single-cell RNA sequencing data, we elucidated potential molecular mechanisms at single-cell resolution and identified that LGALS9 inhibition in sepsis patients leads to the activation and differentiation of monocyte and T-cell subtypes. These findings are expected to assist researchers in gaining a more in-depth understanding of the immune dysregulation in sepsis.

The potential immunological mechanisms of sepsis.

Sepsis is described as a life-threatening organ dysfunction and a heterogeneous syndrome that is a leading cause of morbidity and mortality in intensive care settings. Severe sepsis could incite an uncontrollable surge of inflammatory cytokines, and the host immune system's immunosuppression could respond to counter excessive inflammatory responses, characterized by the accumulated anti-inflammatory cytokines, impaired function of immune cells, over-proliferation of myeloid-derived suppressor cells and regulatory T cells, depletion of immune effector cells by different means of death, etc. In this review, we delve into the underlying pathological mechanisms of sepsis, emphasizing both the hyperinflammatory phase and the associated immunosuppression. We offer an in-depth exploration of the critical mechanisms underlying sepsis, spanning from individual immune cells to a holistic organ perspective, and further down to the epigenetic and metabolic reprogramming. Furthermore, we outline the strengths of artificial intelligence in analyzing extensive datasets pertaining to septic patients, showcasing how classifiers trained on various clinical data sources can identify distinct sepsis phenotypes and thus to guide personalized therapy strategies for the management of sepsis. Additionally, we provide a comprehensive summary of recent, reliable biomarkers for hyperinflammatory and immunosuppressive states, facilitating more precise and expedited diagnosis of sepsis.

Immune profiling of critically ill patients with acute kidney injury during the first week after various types of injuries: the REALAKI study.

BACKGROUND: Acute kidney injury (AKI) is common in hospitalized patients and results in significant morbidity and mortality. The objective of the study was to explore the systemic immune response of intensive care unit patients presenting with AKI, especially the association between immune profiles and persistent AKI during the first week after admission following various types of injuries (sepsis, trauma, surgery, and burns). METHODS: REALAKI is an ancillary analysis of the REAnimation Low Immune Status Marker (REALISM) cohort study, in which 359 critically ill patients were enrolled in three different intensive care units. Patients with end-stage renal disease were excluded from the REALAKI study. Clinical samples and data were collected three times after admission: at day 1 or 2 (D1-2), day 3 or 4 (D3-4) and day 5, 6 or 7 (D5-7). Immune profiles were compared between patients presenting with or without AKI. Patients with AKI at both D1-2 and D5-7 were defined as persistent AKI. A multivariable logistic regression model was performed to determine the independent association between AKI and patients' immunological parameters. RESULTS: Three hundred and fifty-nine patients were included in this analysis. Among them, 137 (38%) were trauma patients, 103 (29%) post-surgery patients, 95 (26%) sepsis patients, and 24 (7%) were burn patients. One hundred and thirty-nine (39%) patients presented with AKI at D1-2 and 61 (20%) at D5-7. Overall, 94% presented with persistent AKI at D5-7. Patients with AKI presented with increased pro and anti-inflammatory cytokines and altered innate and adaptive immune responses. The modifications observed in the immune profiles tended to be more pronounced with increasing KDIGO stages. In the logistic regression model, a statistically significant association was observed at D1-2 between AKI and CD10lowCD16low immature neutrophils (OR 3.03 [1.7-5.5]-p < 0.001). At D5-7, increased interleukin-10 (IL-10) levels and reduced ex vivo TNF-α production after LPS stimulation were significantly associated with the presence of AKI (OR 1.38 [1.12-1.71]-p = 0.001 and 0.51 [0.27-0.91]-p = 0.03, respectively). Patients who recovered from AKI between D1-2 and D5-7 compared to patients with persistent AKI at D5-7, tended to correct these alterations. CONCLUSION: Following various types of severe injuries, early AKI is associated with the initial inflammatory response. Presence of AKI at the end of the first week after injury is associated with injury-induced immunosuppression.

Multienzyme Active Nanozyme for Efficient Sepsis Therapy through Modulating Immune and Inflammation Inhibition.

Sepsis, a life-threatening condition caused by a dysregulated immune response to infection, leads to systemic inflammation, immune dysfunction, and multiorgan damage. Various oxidoreductases play a very important role in balancing oxidative stress and modulating the immune response, but they are stored inconveniently, environmentally unstable, and expensive. Herein, we develop multifunctional artificial enzymes, CeO2 and Au/CeO2 nanozymes, exhibiting five distinct enzyme-like activities, namely, superoxide dismutase, catalase, glutathione peroxidase, peroxidase, and oxidase. These artificial enzymes have been used for the biocatalytic treatment of sepsis via inhibiting inflammation and modulating immune responses. These nanozymes significantly reduce reactive oxygen species and proinflammatory cytokines, achieving multiorgan protection. Notably, CeO2 and Au/CeO2 nanozymes with enzyme-mimicking activities can be particularly effective in restoring immunosuppression and maintaining homeostasis. The redox nanozyme offers a promising dual-protective strategy against sepsis-induced inflammation and organ dysfunction, paving the way for biocatalytic-based immunotherapies for sepsis and related inflammatory diseases.

Integrated clustering of multiple immune marker trajectories reveals different immunotypes in severely injured patients.

BACKGROUND: The immune response of critically ill patients, such as those with sepsis, severe trauma, or major surgery, is heterogeneous and dynamic, but its characterization and impact on outcomes are poorly understood. Until now, the primary challenge in advancing our understanding of the disease has been to concurrently address both multiparametric and temporal aspects. METHODS: We used a clustering method to identify distinct groups of patients, based on various immune marker trajectories during the first week after admission to ICU. In 339 severely injured patients, we initially longitudinally clustered common biomarkers (both soluble and cellular parameters), whose variations are well-established during the immunosuppressive phase of sepsis. We then applied this multi-trajectory clustering using markers composed of whole blood immune-related mRNA. RESULTS: We found that both sets of markers revealed two immunotypes, one of which was associated with worse outcomes, such as increased risk of hospital-acquired infection and mortality, and prolonged hospital stays. This immunotype showed signs of both hyperinflammation and immunosuppression, which persisted over time. CONCLUSION: Our study suggest that the immune system of critically ill patients can be characterized by two distinct longitudinal immunotypes, one of which included patients with a persistently dysregulated and impaired immune response. This work confirms the relevance of such methodology to stratify patients and pave the way for further studies using markers indicative of potential immunomodulatory drug targets.

Integrated multi-omics and artificial intelligence to explore new neutrophils clusters and potential biomarkers in sepsis with experimental validation.

Background: Sepsis, causing serious organ and tissue damage and even death, has not been fully elucidated. Therefore, understanding the key mechanisms underlying sepsis-associated immune responses would lead to more potential therapeutic strategies. Methods: Single-cell RNA data of 4 sepsis patients and 2 healthy controls in the GSE167363 data set were studied. The pseudotemporal trajectory analyzed neutrophil clusters under sepsis. Using the hdWGCNA method, key gene modules of neutrophils were explored. Multiple machine learning methods were used to screen and validate hub genes for neutrophils. SCENIC was then used to explore transcription factors regulating hub genes. Finally, quantitative reverse transcription-polymerase chain reaction was to validate mRNA expression of hub genes in peripheral blood neutrophils of two mice sepsis models. Results: We discovered two novel neutrophil subtypes with a significant increase under sepsis. These two neutrophil subtypes were enriched in the late state during neutrophils differentiation. The hdWGCNA analysis of neutrophils unveiled that 3 distinct modules (Turquoise, brown, and blue modules) were closely correlated with two neutrophil subtypes. 8 machine learning methods revealed 8 hub genes with high accuracy and robustness (ALPL, ACTB, CD177, GAPDH, SLC25A37, S100A8, S100A9, and STXBP2). The SCENIC analysis revealed that APLP, CD177, GAPDH, S100A9, and STXBP2 were significant associated with various transcriptional factors. Finally, ALPL, CD177, S100A8, S100A9, and STXBP2 significantly up regulated in peripheral blood neutrophils of CLP and LPS-induced sepsis mice models. Conclusions: Our research discovered new clusters of neutrophils in sepsis. These five hub genes provide novel biomarkers targeting neutrophils for the treatment of sepsis.

Extracellular CIRP promotes Kupffer cell inflammatory polarization in sepsis.

Introduction: Sepsis is a life-threatening inflammatory condition caused by dysregulated host responses to infection. Extracellular cold-inducible RNA-binding protein (eCIRP) is a recently discovered damage-associated molecular pattern that causes inflammation and organ injury in sepsis. Kupffer cells can be activated and polarized to the inflammatory M1 phenotype, contributing to tissue damage by producing proinflammatory mediators. We hypothesized that eCIRP promotes Kupffer cell M1 polarization in sepsis. Methods: We stimulated Kupffer cells isolated from wild-type (WT) and TLR4-/- mice with recombinant mouse (rm) CIRP (i.e., eCIRP) and assessed supernatant IL-6 and TNFα levels by ELISA. The mRNA expression of iNOS and CD206 for M1 and M2 markers, respectively, was assessed by qPCR. We induced sepsis in WT and CIRP-/- mice by cecal ligation and puncture (CLP) and assessed iNOS and CD206 expression in Kupffer cells by flow cytometry. Results: eCIRP dose- and time-dependently increased IL-6 and TNFα release from WT Kupffer cells. In TLR4-/- Kupffer cells, their increase after eCIRP stimulation was prevented. eCIRP significantly increased iNOS gene expression, while it did not alter CD206 expression in WT Kupffer cells. In TLR4-/- Kupffer cells, however, iNOS expression was significantly decreased compared with WT Kupffer cells after eCIRP stimulation. iNOS expression in Kupffer cells was significantly increased at 20 h after CLP in WT mice. In contrast, Kupffer cell iNOS expression in CIRP-/- mice was significantly decreased compared with WT mice after CLP. CD206 expression in Kupffer cells was not different across all groups. Kupffer cell M1/M2 ratio was significantly increased in WT septic mice, while it was significantly decreased in CIRP-/- mice compared to WT mice after CLP. Conclusion: Our data have clearly shown that eCIRP induces Kupffer cell M1 polarization via TLR4 pathway in sepsis, resulting in overproduction of inflammatory cytokines. eCIRP could be a promising therapeutic target to attenuate inflammation by preventing Kupffer cell M1 polarization in sepsis.

Assessment of Modulating Effect of the Experimental Drug Astrabionorm on Synthesis of Proinflammatory Cytokine IL-1ß and Anti-Inflammatory Cytokine IL-10 in the Splenic Red Pulp Cells in a Mouse Model of Sepsis.

An indirect immunohistochemical method was used to study the production of proinflammatory (IL-1ß) and anti-inflammatory (IL-10) cytokines in the spleen cells of mature male C57BL/6 mice with an experimental model of sepsis and during treatment with a drug based on formic acid aldehyde (Astrabionorm). Clinical isolates of two strains of Pseudomonas aeruginosa were used. In the red pulp of the spleen, interleukin-positive cells represented by mononuclear forms were identified, as well as differences in the intensity of immunohistochemical staining of these cells for the studied interleukins in the two models used. A modulating role of the drug in the production of interleukins by the splenic red pulp cells during sepsis is assumed.

Glucans: A Therapeutic Alternative for Sepsis Treatment.

Sepsis treatment is a challenging condition due to its complexity, which involves host inflammatory responses to a severe and potentially fatal infection, associated with organ dysfunction. The aim of this study was to analyze the scientific literature on the immunomodulatory effects of glucans in a murine model of systemic infection induced by cecal ligation and puncture. This study comprises an integrative literature review based on systematic steps, with searches carried out in the PubMed, ScienceDirect, Scopus, Web of Science, and Embase databases. In most studies, the main type of glucan investigated was ß-glucan, at 50 mg/kg, and a reduction of inflammatory responses was identified, minimizing the occurrence of tissue damage leading to increased animal survival. Based on the data obtained and discussed in this review, glucans represent a promising biotechnological alternative to modulate the immune response and could potentially be used in the clinical management of septic individuals.

Neutrophil Depletion Changes the N-Glycosylation Pattern of IgG in Experimental Murine Sepsis.

Sepsis is a life-threatening condition with a rising disease burden worldwide. It is a multifactorial disease and is defined as a dysregulated host response to infection. Neutrophils have been shown to be involved in the pathogenesis of sepsis by exacerbating inflammation. However, the exact effector mechanism of action still remains a mystery. Changes in the glycosylation pattern of the immunoglobulin G (IgG) Fc region are described for several diseases including meningococcal sepsis. In this study, we investigated the possible contribution of neutrophils and neutrophil implication, potentially related to degranulation or neutrophil extracellular trap (NET) formation in changing the IgG Fc N-glycosylation pattern in a murine sepsis model. We have measured the serum level of cytokines/chemokines and immunoglobulins, the serum activity of neutrophil elastase (NE), and analyzed the IgG Fc glycosylation pattern by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) and Lectin enzyme-linked immunosorbent assay (ELISA). We observed an increased activity of NE- and neutrophil-associated cytokines such as keratinocyte chemoattractant (KC) with the development of sepsis. Regarding the IgG Fc N-glycosylation, we observed an increase in fucosylation and α1,3-galactosylation and a decrease for sialyation. Interestingly, these changes were not uniform for all IgG subclasses. After depletion of neutrophils, we saw a change in the exposure of fucose and α2,6-linked sialic acid during the time course of our experimental sepsis model. In conclusion, neutrophils can influence changes in the IgG glycosylation pattern in experimental sepsis.

Dynamic immune status analysis of peripheral blood mononuclear cells in patients with Klebsiella pneumoniae bloodstream infection sepsis using single-cell RNA sequencing.

Background: Klebsiella pneumoniae is a common Gram-negative bacterium. Blood infection caused by K. pneumoniae is one of the most common causes of human sepsis, which seriously threatens the life of patients. The immune status of peripheral blood mononuclear cells (PBMCs) based on single-cell RNA sequencing (scRNA-seq) in acute stage and recovery stage of sepsis caused by K. pneumoniae bloodstream infection has not been studied. Methods: A total of 13 subjects were included in this study, 3 healthy controls, 7 patients with K. pneumoniae bloodstream infection in the acute stage (4 patients died), and 3 patients in the recovery stage. Peripheral blood of all patients was collected and PBMCs were isolated for scRNA-seq analysis. We studied the changes of PBMCs components, signaling pathways, differential genes, and cytokines in acute and recovery stages. Results: During K. pneumoniae acute infection we observed a decrease in the proportion of T cells, most probably due to apoptosis and the function of T cell subtypes was disorder. The proportion of monocytes increased in acute stage. Although genes related to their phagocytosis function were upregulated, their antigen presentation capacity-associated genes were downregulated. The expression of IL-1ß, IL-18, IFNGR1 and IFNGR2 genes was also increased in monocytes. The proportion of DCs was depleted during the acute stage and did not recover during sepsis recovery. DCs antigen presentation was weakened during the acute stage but recovered fast during the recovery stage. pDCs response to MCP-1 chemokine was weakened, they recovered it quickly during the recovery stage. B cells showed apoptosis both in the acute stage and recovery stage. Their response to complement was weakened, but their antigen presentation function was enhanced. The proportion of NK cells stable during all disease's stages, and the expression of IFN-γ gene was upregulated. Conclusion: The proportion of PBMCs and their immune functions undergo variations throughout the course of the disease, spanning from the acute stage to recovery. These findings provide new insights into the mechanism of PBMCs immune function during K. pneumoniae bloodstream infection sepsis and recovery and sets the basis for further understanding and treatment.

Predicting Organ Dysfunction in Septic and Critically Ill Patients: A Prospective Cohort Study Using Rapid Ex Vivo Immune Profiling.

OBJECTIVES: While cytokine response patterns are pivotal in mediating immune responses, they are also often dysregulated in sepsis and critical illness. We hypothesized that these immunological deficits, quantifiable through ex vivo whole blood stimulation assays, may be indicative of subsequent organ dysfunction. DESIGN: In a prospective observational study, adult septic patients and critically ill but nonseptic controls were identified within 48 hours of critical illness onset. Using a rapid, ex vivo assay based on responses to lipopolysaccharide (LPS), anti-CD3/anti-CD28 antibodies, and phorbol 12-myristate 13-acetate with ionomycin, cytokine responses to immune stimulants were quantified. The primary outcome was the relationship between early cytokine production and subsequent organ dysfunction, as measured by the Sequential Organ Failure Assessment score on day 3 of illness (SOFAd3). SETTING: Patients were recruited in an academic medical center and data processing and analysis were done in an academic laboratory setting. PATIENTS: Ninety-six adult septic and critically ill nonseptic patients were enrolled. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Elevated levels of tumor necrosis factor and interleukin-6 post-endotoxin challenge were inversely correlated with SOFAd3. Interferon-gamma production per lymphocyte was inversely related to organ dysfunction at day 3 and differed between septic and nonseptic patients. Clustering analysis revealed two distinct immune phenotypes, represented by differential responses to 18 hours of LPS stimulation and 4 hours of anti-CD3/anti-CD28 stimulation. CONCLUSIONS: Our rapid immune profiling technique offers a promising tool for early prediction and management of organ dysfunction in critically ill patients. This information could be pivotal for early intervention and for preventing irreversible organ damage during the acute phase of critical illness.

invariant Natural Killer T Cells Modulate the Peritoneal Macrophage Response to Polymicrobial Sepsis.

INTRODUCTION: A dysregulated immune system is a major driver of the mortality and long-term morbidity from sepsis. With respect to macrophages, it has been shown that phenotypic changes are critical to effector function in response to acute infections, including intra-abdominal sepsis. Invariant natural killer T cells (iNKT cells) have emerged as potential central regulators of the immune response to a variety of infectious insults. Specifically, various iNKT cell:macrophage interactions have been noted across a spectrum of diseases, including acute events such as sepsis. However, the potential for iNKT cells to affect peritoneal macrophages during an abdominal septic event is as yet unknown. METHODS: Cecal ligation and puncture (CLP) was performed in both wild type (WT) and invariant natural killer T cell knockout (iNKT-/-) mice. 24 h following CLP or sham operation, peritoneal macrophages were collected for analysis. Analysis of macrophage phenotype and function was undertaken to include analysis of bactericidal activity and cytokine or superoxide production. RESULTS: Within iNKT-/- mice, a greater degree of intraperitoneal macrophages in response to the sepsis was noted. Compared to WT mice, within iNKT-/- mice, CLP did induce an increase in CD86+ and CD206+, but no difference in CD11b+. Unlike WT mice, intra-abdominal sepsis within iNKT-/- mice induced an increase in Ly6C-int (5.2% versus 14.9%; P < 0.05) and a decrease in Ly6C-high on peritoneal macrophages. Unlike phagocytosis, iNKT cells did not affect macrophage bactericidal activity. Although iNKT cells did not affect interleukin-6 production, iNKT cells did affect IL-10 production and both nitrite and superoxide production from peritoneal macrophages. CONCLUSIONS: The observations indicate that iNKT cells affect specific phenotypic and functional aspects of peritoneal macrophages during polymicrobial sepsis. Given that pharmacologic agents that affect iNKT cell functioning are currently in clinical trial, these findings may have the potential for translation to critically ill surgical patients with abdominal sepsis.