Increased prevalence of clonal hematopoiesis of indeterminate potential in hospitalized patients with COVID-19.

Clonal hematopoiesis of indeterminate potential (CHIP) leads to higher mortality, carries a cardiovascular risk and alters inflammation. All three aspects harbor overlaps with the clinical manifestation of COVID-19. This study aimed to identify the impact of CHIP on COVID-19 pathophysiology. 90 hospitalized patients were analyzed for CHIP. In addition, their disease course and outcome were evaluated. With a prevalence of 37.8%, the frequency of a CHIP-driver mutation was significantly higher than the prevalence expected based on median age (17%). CHIP increases the risk of hospitalization in the course of the disease but has no age-independent impact on the outcome within the group of hospitalized patients. Especially in younger patients (45 - 65 years), CHIP was associated with persistent lymphopenia. In older patients (> 65 years), on the other hand, CHIP-positive patients developed neutrophilia in the long run. To what extent increased values of cardiac biomarkers are caused by CHIP independent of age could not be elaborated solely based on this study. In conclusion, our results indicate an increased susceptibility to a severe course of COVID-19 requiring hospitalization associated with CHIP. Secondly, they link it to a differentially regulated cellular immune response under the pressure of SARS-CoV-2 infection. Hence, a patient's CHIP-status bears the potential to serve as biomarker for risk stratification and to early guide treatment of COVID-19 patients.

Extracellular Lactate Acts as a Metabolic Checkpoint and Shapes Monocyte Function Time Dependently.

Elevated blood lactate levels are frequently found in critically ill patients and thought to result from tissue hypoperfusion and cellular oxygen shortage. Considering the close relationship between immune cell function and intracellular metabolism, lactate is more than a glycolytic waste molecule but able to regulate the immune response. Our aim was to elucidate the temporal and mechanistic effect of extracellular lactate on monocytes. To this end, primary human monocytes and the human monocytic cell line MonoMac6 were stimulated with various toll-like-receptor agonists after priming with Na-L-lactate under constant pH conditions. As readout, cytokine production was measured, real-time assessment of intracellular energy pathways was performed, and intracellular metabolite concentrations were determined. Irrespective of the immunogenic stimulus, short-term Na-lactate-priming strongly reduced cytokine production capacity. Lactate and hexoses accumulated intracellularly and, together with a decreased glycolytic flux, indicate a lactate-triggered impairment of glycolysis. To counteract intracellular hyperglycemia, glucose is shunted into the branching polyol pathway, leading to sorbitol accumulation. In contrast, long-term priming with Na-L-lactate induced cellular adaption and abolished the suppressive effect. This lactate tolerance is characterized by a decreased cellular respiration due to a reduced complex-I activity. Our results indicate that exogenous lactate shapes monocyte function by altering the intracellular energy metabolism and acts as a metabolic checkpoint of monocyte activation.

Sevoflurane depletes macrophages from the melanoma microenvironment.

BACKGROUND: With more than 18 million annual new cases, cancer belongs to the major challenges of modern healthcare. Surgical resection of solid tumours under general anaesthesia is the prime therapy. Different aspects of anaesthesia are under discussion to independently influence the long-term outcome of cancer patients. Most recently, the commonly used volatile anaesthetics like sevoflurane have entered the spotlight, as retrospective studies suggest a detrimental outcome in certain cancer aetiologies with sparse mechanistic understanding. Our objective was to investigate this concept in a murine melanoma model, herein comparing the consequence of inhalative and injection anesthesia on tumour composition and growth. METHODS: We used a murine model of malignant melanoma in male, adult C57BL/6 mice (n = 92), induced by the subcutaneous injection of B16-F10 cells. We either exposed the melanoma cells to sevoflurane before implantation or subjected the animals to single or double anaesthesia with either volatile or injection drugs. After a maximum follow-up of 4 weeks, leucocytes within the tumour microenvironment (TME) were comprehensively analysed by flow cytometry with focus on tumor-associated macrophages (TAM). RESULTS: We found that exposure of melanoma cells to sevoflurane before implantation induced long-lasting transcriptome changes and aggravated tumour growth, without extensive changes of the TME. Contrastingly, both a single and double anaesthesia with sevoflurane led to a significant reduction of TAMs (injection vs. sevoflurane: 2,0 vs. 0.3% and 1.2 vs. 0.6%, respectively), whilst increasing PD-L1 expression on the remaining cells (mean fluorescent intensity injection vs. sevoflurane: 3,804 vs. 7,143 and 9,090 vs. 32,228, respectively). No changes in tumour growth were observed in these groups. CONCLUSION: In sharp contrast to the detrimental impact of sevoflurane on patients' outcome reported in retrospective clinical studies, we propose here that sevoflurane might actually exert a beneficial effect by decreasing TAMs within the TME, rendering the tumour again susceptible for cytotoxic T cells and immunotherapies. Further research is warranted to delineate, how these results translate into the clinic.

Sepsis Induces a Long-Lasting State of Trained Immunity in Bone Marrow Monocytes.

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.

Paternal sepsis induces alterations of the sperm methylome and dampens offspring immune responses-an animal study.

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.

Differential expression of TGF-ß superfamily members and role of Smad1/5/9-signalling in chondral versus endochondral chondrocyte differentiation.

Proteins of the transforming-growth-factor-ß (TGF-ß)-superfamily have a remarkable ability to induce cartilage and bone and the crosstalk of TGF-ß - and BMP-signalling pathways appears crucial during chondrocyte development. Aim was to assess the regulation of TGF-ß-superfamily members and of Smad2/3- and Smad1/5/9-signalling during endochondral in vitro chondrogenesis of mesenchymal stromal cells (MSC) relative to chondral redifferentiation of articular chondrocytes (AC) to adjust chondrocyte development of MSC towards a less hypertrophic phenotype. While MSC increased BMP4 and BMP7 and reduced TGFBR2 and TGFBR3-expression during chondrogenesis, an opposite regulation was observed during AC-redifferentiation. Antagonists CHRD and CHL2 rose significantly only in AC-cultures. AC showed higher initial BMP4, pSmad1/5/9 and SOX9 protein levels, a faster (re-)differentiation but a similar decline of pSmad2/3- and pSmad1/5/9-signalling versus MSC-cultures. BMP-4/7-stimulation of MSC-pellets enhanced SOX9 and accelerated ALP-induction but did not shift differentiation towards osteogenesis. Inhibition of BMP-signalling by dorsomorphin significantly reduced SOX9, raised RUNX2, maintained collagen-type-II and collagen-type-X lower and kept ALP-activity at levels reached at initiation of treatment. Conclusively, ALK1,2,3,6-signalling was essential for MSC-chondrogenesis and its prochondrogenic rather than prohypertrophic role may explain why inhibition of canonical BMP-signalling could not uncouple cartilage matrix production from hypertrophy as this was achieved with pulsed PTHrP-application.

Mapping the ligand-binding region of Borrelia hermsii fibronectin-binding protein.

Many pathogenic microorganisms express fibronectin-binding molecules that facilitate their adherence to the extracellular matrix and/or entry into mammalian cells. We have previously described a Borrelia recurrentis gene, cihC that encodes a 40-kDa surface receptor for both, fibronectin and the complement inhibitors C4bp and C1-Inh. We now provide evidence for the expression of a group of highly homologues surface proteins, termed FbpA, in three B. hermsii isolates and two tick-borne relapsing fever spirochetes, B. parkeri and B. turicatae. When expressed in Escherichia coli or B. burgdorferi, four out of five proteins were shown to selectively bind fibronectin, whereas none of five proteins were able to bind the human complement regulators, C4bp and C1-Inh. By applying deletion mutants of the B. hermsii fibronectin-binding proteins a putative high-affinity binding site for fibronectin was mapped to its central region. In addition, the fibronectin-binding proteins of B. hermsii were found to share sequence homology with BBK32 of the Lyme disease spirochete B. burgdorferi with similar function suggesting its involvement in persistence and/or virulence of relapsing fever spirochetes.