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The COVID-19 pandemic has exerted great pressure on national health systems, which have aimed to ensure comprehensive healthcare at all times. Healthcare professionals working with COVID-19 patients are on the frontline and thereby confronted with enormous demands. Although early reports exist on the psychological impact of the pandemic on frontline medical staff working in Asia, little is known about its impact on healthcare professionals in other countries and across various work sectors. The present cross-sectional, online survey sought to investigate common work stressors among healthcare professionals, their psychological stress as well as coping resources during the pandemic. A sample of 575 healthcare professionals (57% male) in three different sectors (hospital, prehospital emergency care, and outpatient service) reported their experiences concerning work and private stressors, psychological stress, and coping strategies between April 17, 2020 and June 5, 2020. To capture pandemic-specific answers, most of the items were adapted or newly developed. Exploratory factor analyses (EFA) were conducted to detect underlying latent factors relating to COVID-specific work stressors. In a next step, the effects of these latent stressors across various work sectors on psychological stress (perceived stress, fatigue, and mood) were examined by means of structural equation models (SEM). To add lived experience to the findings, responses to open-ended questions about healthcare professionals' stressors, effective crisis measures and prevention, and individual coping strategies were coded inductively, and emergent themes were identified. The EFA revealed that the examined work stressors can be grouped into four latent factors: \"fear of transmission\", \"interference of workload with private life\", \"uncertainty/lack of knowledge\", and \"concerns about the team\". The SEM results showed that \"interference of workload with private life\" represented the pivotal predictor of psychological stress. \"Concerns about the team\" had stress-reducing effects. The latent stressors had an equal effect on psychological stress across work sectors. On average, psychological stress levels were moderate, yet differed significantly between sectors (all p < .001); the outpatient group experienced reduced calmness and more stress than the other two sectors, while the prehospital group reported lower fatigue than the other two sectors. The prehospital group reported significantly higher concerns about the team than the hospital group (p < .001). In their reports, healthcare professionals highlighted regulations such as social distancing and the use of compulsory masks, training, experience and knowledge exchange, and social support as effective coping strategies during the pandemic. The hospital group mainly mentioned organizational measures such as visiting bans as effective crisis measures, whereas the prehospital sector most frequently named governmental measures such as contact restrictions. The study demonstrated the need for sector-specific crisis measures to effectively address the specific work stressors faced by the outpatient sector in particular. The results on pandemic-specific work stressors reveal that healthcare professionals might benefit from coping strategies that facilitate the utilization of social support. At the workplace, team commitment and knowledge exchange might buffer against adverse psychological stress responses. Schedules during pandemics should give healthcare workers the opportunity to interact with families and friends in ways that facilitate social support outside work. Future studies should investigate cross-sector stressors using a longitudinal design to identify both sector- and time-specific measures. Ultimately, an international comparison of stressors and measures in different sectors of healthcare systems is desirable.

Sepsis is defined as a life-threatening condition, resulting from a dysregulated and harmful response of the hosts' immune system to infection. Apart from this, the (over-)compensating mechanisms counterbalancing the inflammatory response have been proven to render the host susceptible to further infections and increase delayed mortality. Our study aimed to unravel the heterogeneity of immune response in early sepsis and to explain the biology behind it. A systematic search of public repositories yielded 949 microarray samples from patients with sepsis of different infectious origin and early after clinical manifestation. These were merged into a meta-expression set, and after applying sequential conservative bioinformatics filtering, an in-deep analysis of transcriptional heterogeneity, as well as a comparison to samples of healthy controls was performed. We can identify two distinct clusters of patients (cluster 1: 655 subjects, cluster 2: 294 subjects) according to their global blood transcriptome. While both clusters exhibit only moderate differences in direct comparison, a comparison of both clusters individually to healthy controls yielded strong expression changes of genes involved in immune responses. Both comparisons found similar regulated genes, with a stronger dysregulation occurring in the larger patient cluster and implicating a loss of monocyte and T cell function, co-occurring with an activation of neutrophil granulocytes. We propose a consistent-but in its extent varying-presence of immunosuppression, occurring as early in sepsis as its clinical manifestation and irrespective of the infectious origin. While certain cell types possess contradictory activation states, our finding underlines the urgent need for an early host-directed therapy of sepsis side-by-side with antibiotics.

Innate immune memory describes the functional reprogramming of innate immune cells after pathogen contact, leading to either a boosted (trained immunity) or a diminished (immune tolerance) response to a secondary stimulus. Immune tolerance or \"sepsis-induced immunosuppression\" is a typical hallmark of patients after sepsis survival, characterized by hypo-responsiveness of the host's immune system. This condition renders the host vulnerable for a persisting infection or the occurrence of secondary, often opportunistic infections, along with an increased mortality rate. The mechanisms involved in the maintenance of this long-lasting condition are not examined yet. Polymicrobial abdominal sepsis was induced in 12 week old male C57BL/6 mice by cecal ligation and puncture. Mice were euthanized 3 months after insult. Immune cell composition of the spleen and whole blood, as well as stem and progenitor cells of the bone marrow, were assessed by flow cytometry. Whole blood and bone marrow monocytes were stimulated with LPS and supernatant levels of TNF and IL-6 detected by ELISA. Furthermore, naïve bone marrow monocytes were analyzed for metabolic (Seahorse technology) and transcriptomic (RNA sequencing) changes. Flow cytometric analysis revealed an increase of inflammatory monocytes and regulatory T cells in the spleen, whereby immune composition of whole blood kept unchanged. Granulocyte-monocyte progenitor cells are increased in sepsis survivors. Systemic cytokine response was unchanged after LPS challenge. In contrast, cytokine response of post-septic naïve bone marrow monocytes was increased. Metabolic analysis revealed enhanced glycolytic activity, whereas mitochondrial indices were not affected. In addition, RNA sequencing analysis of global gene expression in monocytes revealed a sustained signature of 367 differentially expressed genes. We here demonstrate that sepsis via functional reprogramming of naïve bone marrow monocytes induces a cellular state of trained immunity, which might be counteracted depending on the compartmental localization of the cell. These findings shed new light on the complex aftermath of sepsis and open up a new pathophysiological framework in need for further research.

Soluble Delta-like ligand 1 (sDLL1) has demonstrated promising results as an early biomarker of bacterial sepsis, but its role in viral infections remains unclear. This study investigated the association between sDLL1 levels and clinical outcomes in patients hospitalized for COVID-19. In this secondary analysis of a single-center prospective observational trial, we measured plasma sDLL1 levels in 46 patients admitted with PCR-confirmed SARS-CoV2 infection between December 2020 and April 2021. Patients were divided into a high-sDLL1 group, upper quartile of patients with the highest measured sDLL1 levels, and low-sDLL1 group, lower three quartiles of patients. Clinical outcomes, including secondary infections, organ dysfunction, and mortality, were compared between groups. Patients in the high-sDLL1 group ( n  = 11, 24%) showed higher rates of secondary infections (63% vs. 20%, OR 7, CI 1.6 to 31, p  = 0.01) with higher odds of pulmonary secondary infections (46% vs. 11%, OR 6.5, CI 1.3 to 31, p  = 0.03). Organ dysfunction was more prevalent in the high-sDLL1-group, indicated by a higher maximal Sequential Organ-Failure Assessment (SOFA) score (median (IQR) 11 (8.5–14) vs. 3 (0.5-8), p  < 0.01) as well as higher rates of vasopressor support (64% vs. 26%, OR 5.1, CI 1.2 to 21, p  = 0.03) and renal replacement therapy (36% vs. 9%, OR 6.1, CI 1.1 to 3.6, p  < 0.05). The high-sDLL1 group also showed increased 90-day mortality (45% vs. 11%, OR 6.5, CI 1.3 to 31, p  = 0.03). These findings suggest that high levels of sDLL1 are associated with adverse outcomes in viral sepsis, warranting further investigation in larger, prospective studies.

Postoperative immunosuppression has been recognized as an important driver of surgery-related morbidity and mortality. It is characterized by lymphocyte depression and impaired monocyte capability to present foreign antigens to T-cells via Major Histocompatibility Complex, Class II (MHC-II) molecules. In patients with postoperative abdominal sepsis, we previously detected a persisting differential binding of the CCCTC-Binding Factor (CTCF), a superordinate regulator of transcription, inside the MHC-II region with specific impact on human leucocyte antigen (HLA) gene expression. In this prospective exploratory study, we investigated to which extent major surgery affects the MHC-II region of circulating CD14+-monocytes.BACKGROUNDPostoperative immunosuppression has been recognized as an important driver of surgery-related morbidity and mortality. It is characterized by lymphocyte depression and impaired monocyte capability to present foreign antigens to T-cells via Major Histocompatibility Complex, Class II (MHC-II) molecules. In patients with postoperative abdominal sepsis, we previously detected a persisting differential binding of the CCCTC-Binding Factor (CTCF), a superordinate regulator of transcription, inside the MHC-II region with specific impact on human leucocyte antigen (HLA) gene expression. In this prospective exploratory study, we investigated to which extent major surgery affects the MHC-II region of circulating CD14+-monocytes.In non-immunocompromised patients undergoing elective major abdominal surgery, a postoperative loss of monocyte HLA-DR surface receptor density was accompanied by a decline in the transcription levels of the classical MHC-II genes HLA-DRA, HLA-DRB1, HLA-DPA1 and HLA-DPB1. The surgical event decreased the expression of the transcriptional MHC-II regulators CIITA and CTCF and led to a lower CTCF enrichment at an intergenic sequence within the HLA-DR subregion. During the observation period, we found a slow and only incomplete restoration of monocyte HLA-DR surface receptor density as well as a partial recovery of CIITA, HLA-DRA and HLA-DRB1 expression. In contrast, transcription of HLA-DPA1, HLA-DPB1, CTCF and binding of CTCF within the MHC-II remained altered.RESULTSIn non-immunocompromised patients undergoing elective major abdominal surgery, a postoperative loss of monocyte HLA-DR surface receptor density was accompanied by a decline in the transcription levels of the classical MHC-II genes HLA-DRA, HLA-DRB1, HLA-DPA1 and HLA-DPB1. The surgical event decreased the expression of the transcriptional MHC-II regulators CIITA and CTCF and led to a lower CTCF enrichment at an intergenic sequence within the HLA-DR subregion. During the observation period, we found a slow and only incomplete restoration of monocyte HLA-DR surface receptor density as well as a partial recovery of CIITA, HLA-DRA and HLA-DRB1 expression. In contrast, transcription of HLA-DPA1, HLA-DPB1, CTCF and binding of CTCF within the MHC-II remained altered.In circulating monocytes, major surgery does not globally affect MHC-II transcription but rather induces specific changes in the expression of selected HLA genes, followed by differential recovery patterns and accompanied by a prolonged reduction of CTCF expression and binding within the MHC-II region. Our results hint toward a long-lasting impact of a major surgical intervention on monocyte functionality, possibly mediated by epigenetic changes that endure the life span of the individual cell.CONCLUSIONIn circulating monocytes, major surgery does not globally affect MHC-II transcription but rather induces specific changes in the expression of selected HLA genes, followed by differential recovery patterns and accompanied by a prolonged reduction of CTCF expression and binding within the MHC-II region. Our results hint toward a long-lasting impact of a major surgical intervention on monocyte functionality, possibly mediated by epigenetic changes that endure the life span of the individual cell.

The highly conserved Notch signaling pathway essentially participates in immunity through regulation of developmental processes and immune cell activity. In the adaptive immune system, the impact of the Notch cascade in T and B differentiation is well studied. In contrast, the function, and regulation of Notch signaling in the myeloid lineage during infection is poorly understood. Here we show that TLR signaling, triggered through LPS stimulation or infection with various Gram-negative and -positive bacteria, stimulates Notch receptor ligand Delta-like 1 (DLL1) expression and Notch signaling in human blood-derived monocytes. TLR activation induces DLL1 indirectly, through stimulated cytokine expression and autocrine cytokine receptor-mediated signal transducer and activator of transcription 3 (STAT3). Furthermore, we reveal a positive feedback loop between Notch signaling and Janus kinase (JAK)/STAT3 pathway during infection that involves Notch-boosted IL-6. Inhibition of Notch signaling by γ-secretase inhibitor DAPT impairs TLR4-stimulated accumulation of NF-κB subunits p65 in the nucleus and subsequently reduces LPS- and infection-mediated IL-6 production. The reduced IL-6 release correlates with a diminished STAT3 phosphorylation and reduced expression of STAT3-dependent target gene programmed death-ligand 1 (PD-L1). Corroborating recombinant soluble DLL1 and Notch activator oxaliplatin stimulate STAT3 phosphorylation and expression of immune-suppressive PD-L1. Therefore we propose a bidirectional interaction between Notch signaling and STAT3 that stabilizes activation of the transcription factor and supports STAT3-dependent remodeling of myeloid cells toward an immuno-suppressive phenotype. In summary, the study provides new insights into the complex network of Notch regulation in myeloid cells during infection. Moreover, it points to a participation of Notch in stabilizing TLR-mediated STAT3 activation and STAT3-mediated modulation of myeloid functional phenotype through induction of immune-suppressive PD-L1.

Elucidating the mechanisms contributing to the dysregulated host response to infection as part of the syndrome is a current challenge in sepsis research. Peripheral blood mononuclear cells are widely used in immunological studies. Density gradient centrifugation, a common method, is of limited use for blood drawn from patients with sepsis. A significant number of low-density granulocytes co-purify contributing to low purity of isolated peripheral blood mononuclear cells. Whole blood anticoagulated with lithium heparin was drawn from patients with sepsis (n=14) and healthy volunteers (n=11). Immediately after drawing, the plasma fraction was removed and PBMC were isolated from the cellular fraction by density gradient centrifugation. Samples derived from patients with sepsis were subsequently incubated with cluster of differentiation 15 MicroBeads and granulocytes were depleted using magnetic-activated cell sorting. Core cellular functions as antigen presentation and cytokine secretion were analyzed in cells isolated from healthy volunteers (n=3) before and after depletion to confirm consistent functionality. We report here that depleting CD15 + cells after density gradient centrifugation is a feasible way to get rid of the low-density granulocyte contamination. Afterwards, the purity of isolated, functionally intact peripheral blood mononuclear cells is comparable to healthy volunteers. Information on the isolation purity and identification of the containing cell types are necessary for good comparability between different studies. Depletion of CD15 + cells after density gradient centrifugation is an easy but highly efficient way to gain a higher quality and more reliability in studies using peripheral blood mononuclear cells from septic patients without affecting the functionality of the cells.

Critically ill patients, especially following trauma or extensive surgery, experience a systemic immune response, consisting of a pro-inflammatory as well as a counterbalancing anti-inflammatory response. Pro-inflammation is necessary for the initiation of homeostatic control and wound healing of the organism. However, when the counterbalancing mechanisms dominate, a condition of secondary immunodeficiency occurs, which renders the patient susceptible for opportunistic or secondary infections. However, the incidence of this condition is yet illusive. For a period of 3 months (May to July 2017), 110 consecutive patients admitted to the surgical ICU of the Heidelberg University Hospital, a tertiary university hospital, were enrolled in the study. Monocyte HLA-DR (mHLA-DR), a long-known surrogate of monocyte function, was assessed quantitatively once on admission utilizing a novel point-of-care flow cytometer with single-use cartridges (Accelix system). Patients were followed up for further 28 days and data on ICU stay, antibiotic therapy, microbiological findings, and mechanical ventilation were recorded. Statistical analysis was performed to evaluate the incidence of immunosuppression-defined by different thresholds-as well as its consequence in terms of outcome and clinical course. Depending on the HLA-DR threshold applied for stratification (≤8,000/≤5,000/≤2,000 molecules/cell), a large group of patients (85.5/68.2/40.0%) already presented with a robust decrease of HLA-DR on admission, independent of the cause for critical illness. Analyzed for survival, neither threshold was able to stratify patients with a higher mortality. However, both thresholds of 2,000 and 5,000 were able to discriminate patients with longer ICU stay, ventilation time and duration of antibiotic therapy, as well as higher count of microbiological findings. Moreover, a mHLA-DR value ≤2,000 molecules/cell was associated with higher incidence of overall antibiotic therapy. Single assessment of mHLA-DR using a novel point-of-care flow cytometer is able to stratify patients according to their risk of a complicated course. Therefore, this device overcomes the technical boundaries for measuring cellular biomarkers and paves the way for future studies involving personalized immunotherapy to patients with a high immunological risk profile independent of their background. German Clinical Trials Register; ID: DRKS00012348.

Integrity of epithelial and endothelial cell barriers is of critical importance for health, barrier disruption is a hallmark of numerous diseases, of which many are driven by carbonyl stressors such as methylglyoxal (MG). Carnosine and anserine exert some MG-quenching activity, but the impact of these and of other histidine containing dipeptides on cell barrier integrity has not been explored in detail. In human proximal tubular (HK-2) and umbilical vein endothelial (HUVEC) cells, exposure to 200 µM MG decreased transepithelial resistance (TER), i.e. increased ionic permeability and permeability for 4-, 10- and 70-kDa dextran, membrane zonula occludens (ZO-1) abundance was reduced, methylglyoxal 5-hydro-5-methylimidazolones (MG-H1) formation was increased. Carnosine, balenine (ß-ala-1methyl-histidine) and anserine (ß-ala-3-methyl-histidine) ameliorated MG-induced reduction of TER in both cell types. Incubation with histidine, 1-/3-methylhistidine, but not with ß-alanine alone, restored TER, although to a lower extent than the corresponding dipeptides. Carnosine and anserine normalized transport and membrane ZO-1 abundance. Aminoguanidine, a well-described MG-quencher, did not mitigate MG-induced loss of TER. Our results show that the effects of the dipeptides on epithelial and endothelial resistance and junction function depend on the methylation status of histidine and are not exclusively explained by their quenching activity.

Background Sepsis represents the utmost severe consequence of infection, involving a dysregulated and self-damaging immune response of the host. While different environmental exposures like chronic stress or malnutrition have been well described to reprogram the germline and subsequently offspring attributes, the intergenerational impact of sepsis as a tremendous immunological stressor has not been examined yet. Methods Polymicrobial sepsis in 12-week-old male C57BL/6 mice was induced by cecal ligation and puncture (CLP), followed by a mating of the male survivors (or appropriate sham control animals) 6 weeks later with healthy females. Alveolar macrophages of offspring animals were isolated and stimulated with either LPS or Zymosan, and supernatant levels of TNF-α were quantified by ELISA. Furthermore, systemic cytokine response to intraperitoneally injected LPS was assessed after 24 h. Also, morphology, motility, and global DNA methylation of the sepsis survivors’ sperm was examined. Results Comparative reduced reduction bisulfite sequencing (RRBS) of sperm revealed changes of DNA methylation ( n  = 381), most pronounced in the intergenic genome as well as within introns of developmentally relevant genes. Offspring of sepsis fathers exhibited a slight decrease in body weight, with a more pronounced weight difference in male animals (CLP vs. sham). Male descendants of sepsis fathers, but not female descendants, exhibited lower plasma concentrations of IL-6, TNF-alpha, and IL-10 24 h after injection of LPS. In line, only alveolar macrophages of male descendants of sepsis fathers produced less TNF-alpha upon Zymosan stimulation compared to sham descendants, while LPS responses kept unchanged. Conclusion We can prove that male—but surprisingly not female—descendants of post-sepsis fathers show a dampened systemic as well as pulmonary immune response. Based on this observation of an immune hypo-responsivity, we propose that male descendants of sepsis fathers are at risk to develop fungal and bacterial infections and might benefit from therapeutic immune modulation.