Platelets have a wide range of functions including critical roles in hemostasis, thrombosis, and immunity. We hypothesized that during acute inflammation, such as in life-threatening sepsis, there are fundamental changes in the sites of platelet production and phenotypes of resultant platelets. Here, we showed during sepsis that the spleen is a major site of megakaryopoiesis and platelet production. Sepsis provoked an adrenergic-dependent mobilization of megakaryocyteerythrocyte progenitors (MEPs) from the bone marrow to the spleen where interleukin-3 (IL-3) induced their differentiation into megakaryocytes.In the spleen, immune-skewed megakaryocytes produced a CD40 ligand-high platelet population with potent immunomodulatory functions. Transfusions of post-sepsis platelets enriched from splenic production enhanced immune responses and reduced overall mortality in sepsis-challenged animals. These findings identify a spleen-derived protective platelet population that may be broadly immunomodulatory in acute inflammatory states such as sepsis.

Highlights Comparison of FDA PIs to EMA SmPCs showed little harmonization. Comparison of CDC VISs to EMA PLs showed even less harmonization. No pattern was observed in the number of labeling elements harmonized over time.

Sepsis, the leading cause of mortality in hospitals, currently lacks effective early diagnostics. A new cellular host response test, the IntelliSep test, may provide an indicator of the immune dysregulation characterizing sepsis. The objective of this study was to examine the correlation between the measurements performed using this test and biological markers and processes associated with sepsis. Phorbol myristate acetate (PMA), an agonist of neutrophils known to induce neutrophil extracellular trap (NET) formation, was added to whole blood of healthy volunteers at concentrations of 0, 200, and 400 nM and then evaluated using the IntelliSep test. Separately, plasma from a cohort of subjects was segregated into Control and Diseased populations and tested for levels of NET components (citrullinated histone (cit-H3) DNA and neutrophil elastase (NE) DNA) using customized ELISA assays and correlated with ISI scores from the same patient samples. Significant increases in IntelliSep Index (ISI) scores were observed with increasing concentrations of PMA in healthy blood (0 and 200: p < 10−10; 0 and 400: p < 10−10). Linear correlation was observed between the ISI and quantities of NE DNA and Cit-H3 DNA in patient samples. Together these experiments demonstrate that the IntelliSep test is associated with the biological processes of leukocyte activation and NETosis and may indicate changes consistent with sepsis.

Microbes, toxins, therapeutics and cells are often instilled into lungs of mice to model diseases and test experimental interventions. Consistent pulmonary delivery is critical for experimental power and reproducibility, but we observed variation in outcomes between handlers using different anesthetic approaches for intranasal dosing into mice. We therefore used a radiotracer to quantify lung delivery after intranasal dosing under inhalational (isoflurane) versus injectable (ketamine/xylazine) anesthesia in C57BL/6 mice. We found that ketamine/xylazine anesthesia resulted in delivery of a greater proportion (52±9%) of an intranasal dose to lungs relative to isoflurane anesthesia (30±15%). This difference in pulmonary dose delivery altered key outcomes in models of viral and bacterial pneumonia, with mice anesthetized with ketamine/xylazine for intranasal infection with influenza A virus or Pseudomonas aeruginosa developing more robust lung inflammation responses relative to control animals randomized to isoflurane anesthesia. Pulmonary dosing efficiency through oropharyngeal aspiration was not affected by anesthetic method and resulted in delivery of 63±8% of dose to lungs, and a non-surgical intratracheal dosing approach further increased lung delivery to 92±6% of dose. Use of either of these more precise dosing methods yielded greater experimental power in the bacterial pneumonia model relative to intranasal infection. Both anesthetic approach and dosing route can impact pulmonary dosing efficiency. These factors affect experimental power and so should be considered when planning and reporting studies involving delivery of fluids to lungs of mice.

Microbes, toxins, therapeutics and cells are often instilled into lungs of mice to model diseases and test experimental interventions. Consistent pulmonary delivery is critical for experimental power and reproducibility, but we observed variation in outcomes between handlers using different anesthetic approaches for intranasal dosing into mice. We therefore used a radiotracer to quantify lung delivery after intranasal dosing under inhalational (isoflurane) versus injectable (ketamine/xylazine) anesthesia in C57BL/6 mice. We found that ketamine/xylazine anesthesia resulted in delivery of a greater proportion (52±9%) of an intranasal dose to lungs relative to isoflurane anesthesia (30±15%). This difference in pulmonary dose delivery altered key outcomes in a model of viral pneumonia, with mice anesthetized with ketamine/xylazine for intranasal infection with influenza A virus developing worse lung pathology and more consistently losing body weight relative to control animals randomized to isoflurane anesthesia. Pulmonary dosing efficiency through oropharyngeal aspiration was not affected by anesthetic method and resulted in delivery of 63±8% of dose to lungs, and a non-surgical intratracheal dosing approach further increased lung delivery to 92±6% of dose. We conclude that anesthetic approach and dosing route can impact pulmonary dosing efficiency. These factors should be considered when planning and reporting studies involving delivery of fluids to lungs of mice.