Anna Krasnodembskaya

Antibacterial Effect of Human Mesenchymal Stem Cells Is Mediated in Part from Secretion of the Antimicrobial Peptide LL-37

Recent in vivo studies indicate that mesenchymal stem cells (MSCs) may have beneficial effects in the treatment of sepsis induced by bacterial infection. Administration of MSCs in these studies improved survival and enhanced bacterial clearance. The primary objective of this study was to test the hypothesis that human MSCs possessed intrinsic antimicrobial properties. We studied the effect of human MSCs derived from bone marrow on the bacterial growth of Gram‐negative (Escherichia coli and Pseudomonas aeruginosa) and Gram‐positive (Staphylococcus aureus) bacteria. MSCs as well as their conditioned medium (CM) demonstrated marked inhibition of bacterial growth in comparison with control medium or normal human lung fibroblasts (NHLF). Analysis of expression of major antimicrobial peptides indicated that one of the factors responsible for the antimicrobial activity of MSC CM against Gram‐negative bacteria was the human cathelicidin antimicrobial peptide, hCAP‐18/LL‐37. Both m‐RNA and protein expression data showed that the expression of LL‐37 in MSCs increased after bacterial challenge. Using an in vivo mouse model of E. coli pneumonia, intratracheal administration of MSCs reduced bacterial growth (in colony‐forming unit) in the lung homogenates and in the bronchoalveolar lavage (BAL) fluid, and administration of MSCs simultaneously with a neutralizing antibody to LL‐37 resulted in a decrease in bacterial clearance. In addition, the BAL itself from MSC‐treated mice had a greater antimicrobial activity in comparison with the BAL of phosphate buffered saline (PBS)‐treated mice. Human bone marrow‐derived MSCs possess direct antimicrobial activity, which is mediated in part by the secretion of human cathelicidin hCAP‐18/ LL‐37. STEM CELLS 2010;28:2229–2238

Concise Review: Mesenchymal Stem Cells for Acute Lung Injury: Role of Paracrine Soluble Factors

Morbidity and mortality have declined only modestly in patients with clinical acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), despite extensive research into the pathophysiology. Current treatment remains primarily supportive with lung‐protective ventilation and a fluid conservative strategy. Pharmacologic therapies that reduce the severity of lung injury in preclinical models have not yet been translated to effective clinical treatment options. Consequently, further research in translational therapies is needed. Cell‐based therapy with mesenchymal stem cells (MSCs) is one attractive new therapeutic approach. MSCs have the capacity to secrete multiple paracrine factors that can regulate endothelial and epithelial permeability, decrease inflammation, enhance tissue repair, and inhibit bacterial growth. This review will focus on recent studies, which support the potential therapeutic use of MSCs in ALI/ARDS, with an emphasis on the role of paracrine soluble factors. STEM CELLS 2011;29:913–919

Human mesenchymal stem cells reduce mortality and bacteremia in gram-negative sepsis in mice in part by enhancing the phagocytic activity of blood monocytes

The potential therapeutic value of cell-based therapy with mesenchymal stem cells (MSC) has been reported in mouse models of polymicrobial peritoneal sepsis. However, the mechanisms responsible for the beneficial effects of MSC have not been well defined. Therefore, we tested the therapeutic effect of intravenous bone marrow-derived human MSC in peritoneal sepsis induced by gram-negative bacteria. At 48 h, survival was significantly increased in mice treated with intravenous MSC compared with control mice treated with intravenous fibroblasts (3T3) or intravenous PBS. There were no significant differences in the levels of TNF-α, macrophage inflammatory protein 2, or IL-10 in the plasma. However, there was a marked reduction in the number of bacterial colony-forming units of Pseudomonas aeruginosa in the blood of MSC-treated mice compared with the 3T3 and PBS control groups. In addition, phagocytic activity was increased in blood monocytes isolated from mice treated with MSC compared with the 3T3 and PBS groups. Furthermore, levels of C5a anaphylotoxin were elevated in the blood of mice treated with MSC, a finding that was associated with upregulation of the phagocytosis receptor CD11b on monocytes. The phagocytic activity of neutrophils was not different among the groups. There was also an increase in alternately activated monocytes/macrophages (CD163- and CD206-positive) in the spleen of the MSC-treated mice compared with the two controls. Thus intravenous MSC increased survival from gram-negative peritoneal sepsis, in part by a monocyte-dependent increase in bacterial phagocytosis.

Mesenchymal stem cells enhance survival and bacterial clearance in murine Escherichia coli pneumonia

Rationale Bacterial pneumonia is the most common infectious cause of death worldwide and treatment is increasingly hampered by antibiotic resistance. Mesenchymal stem cells (MSCs) have been demonstrated to provide protection against acute inflammatory lung injury; however, their potential therapeutic role in the setting of bacterial pneumonia has not been well studied. Objective This study focused on testing the therapeutic and mechanistic effects of MSCs in a mouse model of Gram-negative pneumonia. Methods and results Syngeneic MSCs from wild-type mice were isolated and administered via the intratracheal route to mice 4 h after the mice were infected with Escherichia coli. 3T3 fibroblasts and phosphate-buffered saline (PBS) were used as controls for all in vivo experiments. Survival, lung injury, bacterial counts and indices of inflammation were measured in each treatment group. Treatment with wild-type MSCs improved 48 h survival (MSC, 55%; 3T3, 8%; PBS, 0%; p<0.05 for MSC vs 3T3 and PBS groups) and lung injury compared with control mice. In addition, wild-type MSCs enhanced bacterial clearance from the alveolar space as early as 4 h after administration, an effect that was not observed with the other treatment groups. The antibacterial effect with MSCs was due, in part, to their upregulation of the antibacterial protein lipocalin 2. Conclusions Treatment with MSCs enhanced survival and bacterial clearance in a mouse model of Gram-negative pneumonia. The bacterial clearance effect was due, in part, to the upregulation of lipocalin 2 production by MSCs.

Mitochondrial Transfer via Tunneling Nanotubes is an Important Mechanism by Which Mesenchymal Stem Cells Enhance Macrophage Phagocytosis in the In Vitro and In Vivo Models of ARDS

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

Expression pattern of arenicins—the antimicrobial peptides of polychaete Arenicola marina

Immune responses of invertebrate animals are mediated through innate mechanisms, among which production of antimicrobial peptides play an important role. Although evolutionary Polychaetes represent an interesting group closely related to a putative common ancestor of other coelomates, their immune mechanisms still remain scarcely investigated. Previously our group has identified arenicins—new antimicrobial peptides of the lugworm Arenicola marina, since then these peptides were thoroughly characterized in terms of their structure and inhibitory potential. In the present study we addressed the question of the physiological functions of arenicins in the lugworm body. Using molecular and immunocytochemical methods we demonstrated that arencins are expressed in the wide range of the lugworm tissues—coelomocytes, body wall, extravasal tissue and the gut. The expression of arenicins is constitutive and does not depend on stimulation of various infectious stimuli. Most intensively arenicins are produced by mature coelomocytes where they function as killing agents inside the phagolysosome. In the gut and the body wall epithelia arenicins are released from producing cells via secretion as they are found both inside the epithelial cells and in the contents of the cuticle. Collectively our study showed that arenicins are found in different body compartments responsible for providing a first line of defense against infections, which implies their important role as key components of both epithelial and systemic branches of host defense.

Aspirin reduces lipopolysaccharide-induced pulmonary inflammation in human models of ARDS

Rationale Platelets play an active role in the pathogenesis of acute respiratory distress syndrome (ARDS). Animal and observational studies have shown aspirins antiplatelet and immunomodulatory effects may be beneficial in ARDS. Objective To test the hypothesis that aspirin reduces inflammation in clinically relevant human models that recapitulate pathophysiological mechanisms implicated in the development of ARDS. Methods Healthy volunteers were randomised to receive placebo or aspirin 75  or 1200 mg (1:1:1) for seven days prior to lipopolysaccharide (LPS) inhalation, in a double-blind, placebo-controlled, allocation-concealed study. Bronchoalveolar lavage (BAL) was performed 6 hours after inhaling 50 µg of LPS. The primary outcome measure was BAL IL-8. Secondary outcome measures included markers of alveolar inflammation (BAL neutrophils, cytokines, neutrophil proteases), alveolar epithelial cell injury, systemic inflammation (neutrophils and plasma C-reactive protein (CRP)) and platelet activation (thromboxane B2, TXB2). Human lungs, perfused and ventilated ex vivo (EVLP) were randomised to placebo or 24 mg aspirin and injured with LPS. BAL was carried out 4 hours later. Inflammation was assessed by BAL differential cell counts and histological changes. Results In the healthy volunteer (n=33) model, data for the aspirin groups were combined. Aspirin did not reduce BAL IL-8. However, aspirin reduced pulmonary neutrophilia and tissue damaging neutrophil proteases (Matrix Metalloproteinase (MMP)-8/-9), reduced BAL concentrations of tumour necrosis factor α and reduced systemic and pulmonary TXB2. There was no difference between high-dose and low-dose aspirin. In the EVLP model, aspirin reduced BAL neutrophilia and alveolar injury as measured by histological damage. Conclusions These are the first prospective human data indicating that aspirin inhibits pulmonary neutrophilic inflammation, at both low and high doses. Further clinical studies are indicated to assess the role of aspirin in the prevention and treatment of ARDS. Trial registration number NCT01659307 Results.

T3 Mitochondrial transfer is an important mechanism by which Mesenchymal Stromal Cells (MSC) facilitate macrophage phagocytosis in the in vitro and in vivo models of Acute Respiratory Distress Syndrome (ARDS)

Background ARDS remains a major cause of respiratory failure in critically ill patients with no specific therapy. MSC based cell therapy is a promising candidate and is being used in clinical trials for ARDS. However the mechanisms of MSC effect in lung injury are not very well understood. Islam et al., 2012 showed mitochondrial transfer from MSC to alveolar epithelial cells was protective in the mouse model of LPS induced pneumonia. Pathophysiology of ARDS is underpinned by dysregulated inflammation and pulmonary macrophages are key cellular mediators of the lung immune response. This study was undertaken to test if MSC could transfer their mitochondria to macrophages and to investigate the effects of MSC mitochondria transfer on macrophage function in the in vivo and in vitro models of ARDS.Abstract T3 Figure 1 Mitochondrial transfer from MSC to macrophages can enhance macrophage phagocytic activity in vivo. (A) MSC use tunnelling nano tubules (TNT) structures to transfer mitochondria (arrows). MSC were pre-stained with MitoTracker Red before co-culture with macrophages, 6 hr later sides were fixed and stained for(blue) to visualise macrophages. Almost allpositive cells demonstrate acquisition of red mitochondria from MSC. (B) In the in vivo model, MSC (MitoTracker)-treated mice BALF was taken and alveolar macrophages assessed for phagocytic activity using fluorescent E.coli bioparticles by flow cytometry. Macrophages that had acquired MSC mitochondria showed a higher phagocytic index in comparison to those without. This was assessed by an increase in Mean fluorescence Intensity (MFI). Some of the materials employed in this work were provided by the Texaz A&M Health Science Centre College of Medicine Institute for Regenerative Medicine at Scott and White through a grant from NCRR of the NIH, Grant #P40RR017447 Methods In vivo studies were performed using a mouse model of E.coli pneumonia induced ARDS. C56BL/6 mice were infected with E.coli, human bone marrow-derived MSC or PBS instilled intra-nasally 4 h after infection. For in vitro studies primary human monocyte-derived macrophages (MDM) were infected with E.coli and co-cultured with MSC in contact. MSC mitochondria were pre-stained with MitoTracker Red and MDM stained for CD45 expression. Double positive cells were visualised with confocal microscopy and quantified using flow cytometry. Phagocytosis was assessed using fluorescent E.coli bioparticles by flow cytometry. Results When co-cultured with MSC >90% of MDMs acquired MitoRed fluorescence, indicating mitochondrial transfer from BM-MSC. Confocal imaging revealed presence of Mito-Red positive tunnelling nanotubules (TNTs) formed by MSC. In vivo >78% of CD11chi/F4–80+ alveolar macrophages retained MSC mitochondria at 24 hr post infection. Alveolar macrophages that had acquired MSC mitochondria had a significantly higher phagocytic index compared to those without suggesting enhancement of phagocytic capacity. Inhibition of TNT formation in MSC resulted in decreased transfer to macrophages by 60%, coupled with significant abrogation of MSC effect on macrophage phagocytosis in vitro and anti-microbial effect seen with MSC in vivo. Conclusions Our findings suggest that anti-microbial activity of macrophages is enhanced at least partially by transfer of BM-MSC mitochondria through TNTs, representing an important mechanism of MSC effect in ARDS. Supported by: MRC MR/L017229/1. Reference 1 Islam MN, Das SR, Emin MT, et al. Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury. Nat Med. 2012;18:759–65

The TLR4-PAR1 Axis Regulates Bone Marrow Mesenchymal Stromal Cell Survival and Therapeutic Capacity in Murine E. coli Pneumonia

Bone marrow derived mesenchymal stromal cells have been shown to have significant therapeutic effects in experimental models of pneumonia and lung injury. The current study examined the roles of the toll like receptor 4 (TLR4) and protease activated receptor 1 (PAR1) pathways on mesenchymal stromal cell (MSC) survival and therapeutic activity in a murine model of pneumonia. MSCs from TLR4 ‐/‐ and R41Q‐PAR1 mutated mice were isolated to test the effect of mutating these specific pathways on MSC survival when exposed to cytotoxic stimuli in vitro. An Escherichia coli pneumonia model was used to assess the effect of these specific pathways on MSC therapeutic activity in vivo. Our results showed that mutation of either the TLR4 or PAR1 pathways in MSCs impaired cell survival under conditions of inflammatory stress in vitro, and eliminated their therapeutic efficacy in vivo. Also, stimulation of the TLR4 pathway on MSCs led to secretion of low levels of prothrombin by MSCs, while disrupting the TLR4 pathway impaired canonical signaling through PAR1 in response to thrombin. Therefore, this study demonstrates that both TLR4 and PAR1 are required for MSC survival under inflammatory conditions in vitro and therapeutic capacity in vivo, and that the TLR4 pathway regulates signaling through PAR1 on MSCs. Stem Cells 2018;36:796–806

The TLR4‐PAR1 Axis Regulates Bone Marrow Mesenchymal Stromal Cell Survival and Therapeutic Capacity in Experimental Bacterial Pneumonia

Bone marrow derived mesenchymal stromal cells have been shown to have significant therapeutic effects in experimental models of pneumonia and lung injury. The current study examined the roles of the toll like receptor 4 (TLR4) and protease activated receptor 1 (PAR1) pathways on mesenchymal stromal cell (MSC) survival and therapeutic activity in a murine model of pneumonia. MSCs from TLR4 ‐/‐ and R41Q‐PAR1 mutated mice were isolated to test the effect of mutating these specific pathways on MSC survival when exposed to cytotoxic stimuli in vitro. An Escherichia coli pneumonia model was used to assess the effect of these specific pathways on MSC therapeutic activity in vivo. Our results showed that mutation of either the TLR4 or PAR1 pathways in MSCs impaired cell survival under conditions of inflammatory stress in vitro, and eliminated their therapeutic efficacy in vivo. Also, stimulation of the TLR4 pathway on MSCs led to secretion of low levels of prothrombin by MSCs, while disrupting the TLR4 pathway impaired canonical signaling through PAR1 in response to thrombin. Therefore, this study demonstrates that both TLR4 and PAR1 are required for MSC survival under inflammatory conditions in vitro and therapeutic capacity in vivo, and that the TLR4 pathway regulates signaling through PAR1 on MSCs. Stem Cells 2018;36:796–806