Rationale: Acute respiratory distress syndrome (ARDS) remains a major cause of respiratory failure in critically ill patients. Mesenchymal stromal cells (MSCs) are a promising candidate for a cell-based therapy. However, the mechanisms of MSCs' effects in ARDS are not well understood. In this study, we focused on the paracrine effect of MSCs on macrophage polarization and the role of extracellular vesicle (EV)-mediated mitochondrial transfer.Objectives: To determine the effects of human MSCs on macrophage function in the ARDS environment and to elucidate the mechanisms of these effects.Methods: Human monocyte-derived macrophages (MDMs) were studied in noncontact coculture with human MSCs when stimulated with LPS or bronchoalveolar lavage fluid (BALF) from patients with ARDS. Murine alveolar macrophages (AMs) were cultured ex vivo with/without human MSC-derived EVs before adoptive transfer to LPS-injured mice.Measurements and Main Results: MSCs suppressed cytokine production, increased M2 macrophage marker expression, and augmented phagocytic capacity of human MDMs stimulated with LPS or ARDS BALF. These effects were partially mediated by CD44-expressing EVs. Adoptive transfer of AMs pretreated with MSCderived EVs reduced inflammation and lung injury in LPS-injured mice. Inhibition of oxidative phosphorylation in MDMs prevented the modulatory effects of MSCs. Generating dysfunctional mitochondria in MSCs using rhodamine 6G pretreatment also abrogated these effects.Conclusions: In the ARDS environment, MSCs promote an antiinflammatory and highly phagocytic macrophage phenotype through EV-mediated mitochondrial transfer. MSC-induced changes in macrophage phenotype critically depend on enhancement of macrophage oxidative phosphorylation. AMs treated with MSCderived EVs ameliorate lung injury in vivo.

Mesenchymal stromal cells (MSC) have been reported to improve bacterial clearance in preclinical models of Acute Respiratory Distress Syndrome (ARDS) and sepsis. The mechanism of this effect is not fully elucidated yet. The primary objective of this study was to investigate the hypothesis that the antimicrobial effect of MSC in vivo depends on their modulation of macrophage phagocytic activity which occurs through mitochondrial transfer. We established that selective depletion of alveolar macrophages (AM) with intranasal (IN) administration of liposomal clodronate resulted in complete abrogation of MSC antimicrobial effect in the in vivo model of Escherichia coli pneumonia. Furthermore, we showed that MSC administration was associated with enhanced AM phagocytosis in vivo. We showed that direct coculture of MSC with monocyte‐derived macrophages enhanced their phagocytic capacity. By fluorescent imaging and flow cytometry we demonstrated extensive mitochondrial transfer from MSC to macrophages which occurred at least partially through tunneling nanotubes (TNT)‐like structures. We also detected that lung macrophages readily acquire MSC mitochondria in vivo, and macrophages which are positive for MSC mitochondria display more pronounced phagocytic activity. Finally, partial inhibition of mitochondrial transfer through blockage of TNT formation by MSC resulted in failure to improve macrophage bioenergetics and complete abrogation of the MSC effect on macrophage phagocytosis in vitro and the antimicrobial effect of MSC in vivo. Collectively, this work for the first time demonstrates that mitochondrial transfer from MSC to innate immune cells leads to enhancement in phagocytic activity and reveals an important novel mechanism for the antimicrobial effect of MSC in ARDS. Stem Cells 2016;34:2210–2223

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SummaryMatrix metalloproteinases (MMPs) are a family of proteolytic enzymes that perform multiple roles in the normal immune response to infection. MMPs facilitate leucocyte recruitment, cytokine and chemokine processing, defensin activation and matrix remodelling. However, excess MMP activity following infection may lead to immunopathology that causes host morbidity or mortality and favours pathogen dissemination or persistence. Here, we review the normal functions of MMPs in immunity and then discuss viral and bacterial infections where excess MMP activity has been implicated in pathology, specifically examining HIV, HTLV-1, hepatitis B, endotoxin shock, Helicobacter pylori and Mycobacterium tuberculosis . Tissue destruction may be exacerbated further by bacterial-derived enzymes which activate the host proMMPs. Finally, the potential for therapeutic targeting of excess MMP activity in infection is considered.

Rationale: Simvastatin inhibits inflammatory responses in vitro and in murine models of lung inflammation in vivo. As simvastatin modulates a number of the underlying processes described in acute lung injury (ALI), it may be a potential therapeutic option. Objectives: To investigate in vivo if simvastatin modulates mechanisms important in the development of ALI in a model of acute lung inflammation induced by inhalation of lipopolysaccharide (LPS) in healthy human volunteers. Methods: Thirty healthy subjects were enrolled in a double-blind, placebo-controlled study. Subjects were randomized to receive 40 mg or 80 mg of simvastatin or placebo (n 5 10/group) for 4 days before inhalation of 50 mg LPS. Measurements were performed in bronchoalveolar lavage fluid (BALF) obtained at 6 hours and plasma obtained at 24 hours after LPS challenge. Nuclear translocation of nuclear factor-kB (NF-kB) was measured in monocyte-derived macrophages. Measurements and Main Results: Pretreatment with simvastatin reduced LPS-induced BALF neutrophilia, myeloperoxidase, tumor necrosis factor-a, matrix metalloproteinases 7, 8, and 9, and C-reactive protein (CRP) as well as plasma CRP (all P , 0.05 vs. placebo). There was no significant difference between simvastatin 40 mg and 80 mg. BALF from subjects post-LPS inhalation induced a threefold up-regulation in nuclear NF-kB in monocyte-derived macrophages (P , 0.001); pretreatment with simvastatin reduced this by 35% (P , 0.001). Conclusions: Simvastatin has antiinflammatory effects in the pulmonary and systemic compartment in humans exposed to inhaled LPS. Clinical trial registered with www.controlled-trials.com (ISRCTN21056528).

This article has an online data supplement.

Treatment with simvastatin appears to be safe and may be associated with an improvement in organ dysfunction in ALI. These clinical effects may be mediated by a reduction in pulmonary and systemic inflammation. Clinical trial registered with www.controlled-trials.com (ISRCTN70127774).

Background In acute respiratory distress syndrome (ARDS) unrelated to COVID-19, two phenotypes, based on the severity of systemic inflammation (hyperinflammatory and hypoinflammatory), have been described. The hyperinflammatory phenotype is known to be associated with increased multiorgan failure and mortality. In this study, we aimed to identify these phenotypes in COVID-19-related ARDS. Methods In this prospective observational study done at two UK intensive care units, we recruited patients with ARDS due to COVID-19. Demographic, clinical, and laboratory data were collected at baseline. Plasma samples were analysed for interleukin-6 (IL-6) and soluble tumour necrosis factor receptor superfamily member 1A (TNFR1) using a novel point-of-care assay. A parsimonious regression classifier model was used to calculate the probability for the hyperinflammatory phenotype in COVID-19 using IL-6, soluble TNFR1, and bicarbonate levels. Data from this cohort was compared with patients with ARDS due to causes other than COVID-19 recruited to a previous UK multicentre, randomised controlled trial of simvastatin (HARP-2). Findings Between March 17 and April 25, 2020, 39 patients were recruited to the study. Median ratio of partial pressure of arterial oxygen to fractional concentration of oxygen in inspired air (PaO 2 /FiO 2 ) was 18 kpa (IQR 15–21) and acute physiology and chronic health evaluation II score was 12 (10–16). 17 (44%) of 39 patients had died by day 28 of the study. Compared with survivors, patients who died were older and had lower PaO 2 /FiO 2 . The median probability for the hyperinflammatory phenotype was 0·03 (IQR 0·01–0·2). Depending on the probability cutoff used to assign class, the prevalence of the hyperinflammatory phenotype was between four (10%) and eight (21%) of 39, which is lower than the proportion of patients with the hyperinflammatory phenotype in HARP-2 (186 [35%] of 539). Using the Youden index cutoff (0·274) to classify phenotype, five (63%) of eight patients with the hyperinflammatory phenotype and 12 (39%) of 31 with the hypoinflammatory phenotype died. Compared with matched patients recruited to HARP-2, levels of IL-6 were similar in our cohort, whereas soluble TNFR1 was significantly lower in patients with COVID-19-associated ARDS. Interpretation In this exploratory analysis of 39 patients, ARDS due to COVID-19 was not associated with higher systemic inflammation and was associated with a lower prevalence of the hyperinflammatory phenotype than that observed in historical ARDS data. This finding suggests that the excess mortality observed in COVID-19-related ARDS is unlikely to be due to the upregulation of inflammatory pathways described by the parsimonious model. Funding US National Institutes of Health, Innovate UK, and Randox.