Comparison of Single and Repeated Dosing of Anti-Inflammatory Human Umbilical Cord Mesenchymal Stromal Cells in a Mouse Model of Polymicrobial Sepsis

Summary Mesenchymal stromal cells (MSCs) ameliorate pre-clinical sepsis and sepsis-associated acute kidney injury (SA-AKI) but clinical trials of single-dose MSCs have not indicated robust efficacy. This study investigated immunomodulatory effects of a novel MSC product (CD362-selected human umbilical cord-derived MSCs [hUC-MSCs]) in mouse endotoxemia and polymicrobial sepsis models. Initially, mice received intra-peritoneal (i.p.) lipopolysaccharide (LPS) followed by single i.p. doses of hUC-MSCs or vehicle. Next, mice underwent cecal ligation and puncture (CLP) followed by intravenous (i.v.) doses of hUC-MSCs at 4 h or 4 and 28 h. Analyses included serum/plasma assays of biochemical indices, inflammatory mediators and the AKI biomarker NGAL; multi-color flow cytometry of peritoneal macrophages (LPS) and intra-renal immune cell subpopulations (CLP) and histology/immunohistochemistry of kidney (CLP). At 72 h post-LPS injections, hUC-MSCs reduced serum inflammatory mediators and peritoneal macrophage M1/M2 ratio. Repeated, but not single, hUC-MSC doses administered at 48 h post-CLP resulted in lower serum concentrations of inflammatory mediators, lower plasma NGAL and reversal of sepsis-associated depletion of intra-renal T cell and myeloid cell subpopulations. Hierarchical clustering analysis of all 48-h serum/plasma analytes demonstrated partial co-clustering of repeated-dose hUC-MSC CLP animals with a Sham group but did not reveal a distinct signature of response to therapy. It was concluded that repeated doses of CD362-selected hUC-MSCs are required to modulate systemic and local immune/inflammatory events in polymicrobial sepsis and SA-AKI. Inter-individual variability and lack of effect of single dose MSC administration in the CLP model are consistent with observations to date from early-phase clinical trials. Graphical Abstract

Platelet Transfusions Can Increase or Decrease the Inflammatory Response and Mortality in a Murine Model of Neonatal Polymicrobial Sepsis

Thrombocytopenia affects 18-35% of all neonates in the Neonatal Intensive Care Unit and ~70% of those born extremely prematurely, with sepsis being a frequent cause. Platelet transfusions (PTx) are frequently given to septic preterm neonates at higher platelet count (PC) thresholds than those used in adults, in an attempt to reduce their bleeding risk. However, in the largest randomized trial of neonatal PTx thresholds, infants transfused at a higher PC threshold had a significantly higher mortality and/or major bleeding compared to infants transfused at a lower threshold. We hypothesized that the deleterious effects of PTx would be related to a potential "developmental mismatch" resulting from adult platelets being transfused into a neonate. Among other developmental differences, adult platelets (human and murine) exhibit significantly higher surface P-selectin expression following activation than neonatal platelets. P-selectin is essential for the interaction of platelets with immune cells. Thus, we hypothesized that adult platelets transfused into septic neonates would be consumed faster than endogenous neonatal platelets (due to higher potential for immune interaction), and would increase inflammation and mortality. To test these hypotheses, we used a published murine model of neonatal sepsis, consisting of injecting cecal slurry (CS) into C57BL/6 pups. CS batches were prepared by isolating the cecal content of adult C57BL/6 mice, which was weighted, aliquoted and frozen until use. Three different CS batches were prepared and injected IP into post-natal day 10 pups at a dose of 1.1 (CS1) or 1.0 mg/g (CS2 and 3). Two hours after infection, pups were transfused with washed platelets from adult GFP mice (5x10 7 platelets/g) or Tyrode's buffer (control). Weights, PCs and GFP platelet % were measured before, 4h and 24h post-infection. Blood was collected via terminal bleed at 24h, and plasma separated for quantification of 31 cytokines by multiplex. Despite identical preparation, CS batches varied greatly in their 24h mortality (11% vs 73% vs. 30% for CS1, 2 and 3, respectively). Moreover, PTx had different effects on the mortality of pups infected with different CS batches, increasing the 24h mortality of pups infected with CS1 (30% in transfused vs 11% in non-transfused, RR 2.70, 95% CI 1.02-7.15) but decreasing the mortality of pups infected with CS2 (46% vs. 73%) or CS3 (9% vs. 30%), with a combined RR of 0.52; 95% CI 0.30-0.91. Bacterial counts differed between CS batches, but did not correlate with mortality. Comparison of the microbiome composition using deep sequencing revealed an increased presence of pathogenic bacterial species (Legionella, Sutterella, and Helicobacter species) in CS2 and 3 compared to CS1, and a relative abundance of beneficial bacterial (Actinobacteria and Proteobacteria) in CS1. Different CS batches also elicited different cytokine responses, with significant differences noted in G-CSF, IL-1α, IL-1β, IL-3, IL-7, IL-12p70, and IL-15 levels (p<0.05). For all of these cytokines, except G-CSF, levels were lower in mice infected with CS1 compared to CS2 or 3. Next, we investigated the effects of PTx on the plasma cytokine profile of mice infected with CS1 or CS2/3 (combined), compared to their infected, non-transfused littermates. For nearly all cytokines, PTx increased the response after infection with CS1, but decreased it after infection with CS2/3, with a significant difference in mean global cytokine effect (p<0.0001). For individual cytokines, however, these differences only reached statistical significance for LIX (CXCL5, p=0.04) and approached significance for IL15 and IL17 (p=0.06). Finally, we developed a mathematical model to compare the consumption of endogenous neonatal platelets (GFP-) to that of transfused adult platelets (GFP+) in pups infected with CS1 vs. CS2. In both, the calculated percent consumption was higher for adult platelets than for neonatal platelets (54.8% vs. 32.6% for CS1 and 56.5% vs. 40.4% for CS2). In conclusion, our findings support the hypothesis that adult transfused platelets are consumed faster than endogenous platelets in early neonatal sepsis, and demonstrate that platelet transfusions can either enhance or attenuate the neonatal inflammatory response and the mortality in a model of murine polymicrobial sepsis, depending on the bacterial composition of the inoculum and/or the severity of the sepsis. Disclosures Stowell: Grifols: Speakers Bureau; Argenx: Speakers Bureau; Alexion: Consultancy.

Therapeutic role of Filarial HSP70 in murine models of polymicrobial sepsis and H1N1 Influenza

Nematodes characteristically modulate effector immune responses by synthesizing and releasing both anti-inflammatory as well as proinflammatory molecules in infected hosts. Pre-clinical studies suggest that immuno-modulatory molecules and synthetic small molecules that mimic parasite products could have therapeutic value to ameliorate tissue damage found in inflammatory diseases. We report here identification of a glycoprotein from filarial parasite, a homologue of mammalian Heat Shock Protein 70 with immunostimulatory attributes. The purified native glycoprotein designated as FHSP70 and its recombinant protein moiety, WFL were found to be TLR2 and TLR4 agonists in vitro in human myeloid cells and induce systemic inflammatory cytokines in vivo. Cecal ligation and puncture (CLP) performed in mice which leads to onset of poly microbial sepsis and mortality could be treated by therapeutic administration of a single dose of FHSP70, along with antibiotics, suggesting its potential as a immunotherapeutic adjuvant for clinical management of Sepsis. Intra-nasal administration of WFL to mice followed by challenge with virulent human Influenza-A virus resulted in decreased viral growth as well as improved survival. The protective effect was demonstrable by both prophylactic as well as therapeutic intranasal administration of WFL. Further, therapeutic administration of WFL by intraperitoneal route 5 days post viral challenge also resulted in significant decrease in viral load in the respiratory tract.One sentence SummarySystemic administration of a Filarial HSP70 acts as an adjuvant therapy, through immuno-modulation, for improved survival against murine Polymicrobial Sepsis and Viral Infection while its intra nasal administration protects mice prophylactically as well as therapeutically against H1N1 Influenza viral challenge.

Lactate promotes macrophage HMGB1 lactylation, acetylation, and exosomal release in polymicrobial sepsis

High circulating levels of lactate and high mobility group box-1 (HMGB1) are associated with the severity and mortality of sepsis. However, it is unclear whether lactate could promote HMGB1 release during sepsis. The present study demonstrated a novel role of lactate in HMGB1 lactylation and acetylation in macrophages during polymicrobial sepsis. We found that macrophages can uptake extracellular lactate via monocarboxylate transporters (MCTs) to promote HMGB1 lactylation via a p300/CBP-dependent mechanism. We also observed that lactate stimulates HMGB1 acetylation by Hippo/YAP-mediated suppression of deacetylase SIRT1 and β-arrestin2-mediated recruitment of acetylases p300/CBP to the nucleus via G protein-coupled receptor 81 (GPR81). The lactylated/acetylated HMGB1 is released from macrophages via exosome secretion which increases endothelium permeability. In vivo reduction of lactate production and/or inhibition of GPR81-mediated signaling decreases circulating exosomal HMGB1 levels and improves survival outcome in polymicrobial sepsis. Our results provide the basis for targeting lactate/lactate-associated signaling to combat sepsis.