Healthy versus inflamed lung environments differentially affect mesenchymal stromal cells.

BACKGROUND: Despite increased interest in mesenchymal stromal cell (MSC)-based cell therapies for acute respiratory distress syndrome (ARDS), clinical investigations have not yet been successful and our understanding of the potential in vivo mechanisms of MSC actions in ARDS remains limited. ARDS is driven by an acute severe innate immune dysregulation, often characterised by inflammation, coagulation and cell injury. How this inflammatory microenvironment influences MSC functions remains to be determined. AIM: The aim of this study was to comparatively assess how the inflammatory environment present in ARDS lungs versus the lung environment present in healthy volunteers alters MSC behaviour. METHODS: Clinical-grade human bone marrow-derived MSCs (hMSCs) were exposed to bronchoalveolar lavage fluid (BALF) samples obtained from ARDS patients or from healthy volunteers. Following exposure, hMSCs and their conditioned media were evaluated for a broad panel of relevant properties, including viability, levels of expression of inflammatory cytokines, gene expression, cell surface human leukocyte antigen expression, and activation of coagulation and complement pathways. RESULTS: Pro-inflammatory, pro-coagulant and major histocompatibility complex (self-recognition) related gene expression was markedly upregulated in hMSCs exposed ex vivo to BALF obtained from healthy volunteers. These changes were less apparent and often opposite in hMSCs exposed to ARDS BALF samples. CONCLUSION: These data provide new insights into how hMSCs behave in healthy versus inflamed lung environments, and strongly suggest that the inflamed environment in ARDS induces hMSC responses that are potentially beneficial for cell survival and actions. This further highlights the need to understand how different disease environments affect hMSC functions.

Differential effects of the cystic fibrosis lung inflammatory environment on mesenchymal stromal cells.

Growing evidence demonstrates that human mesenchymal stromal cells (MSCs) modify their in vivo anti-inflammatory actions depending on the specific inflammatory environment encountered. Understanding this better is crucial to refine MSC-based cell therapies for lung and other diseases. Using acute exacerbations of cystic fibrosis (CF) lung disease as a model, the effects of ex vivo MSC exposure to clinical bronchoalveolar lavage fluid (BALF) samples, as a surrogate for the in vivo clinical lung environment, on MSC viability, gene expression, secreted cytokines, and mitochondrial function were compared with effects of BALF collected from healthy volunteers. CF BALF samples that cultured positive for Aspergillus sp. (Asp) induced rapid MSC death, usually within several hours of exposure. Further analyses suggested the fungal toxin gliotoxin as a potential mediator contributing to CF BALF-induced MSC death. RNA sequencing analyses of MSCs exposed to either Asp+ or Asp- CF BALF samples identified a number of differentially expressed transcripts, including those involved in interferon signaling, antimicrobial gene expression, and cell death. Toxicity did not correlate with bacterial lung infections. These results suggest that the potential use of MSC-based cell therapies for CF or other lung diseases may not be warranted in the presence of Aspergillus.

Leishmania donovani inhibits inflammasome-dependent macrophage activation by exploiting the negative regulatory proteins A20 and UCP2.

In visceral leishmaniasis, we found that the antileishmanial drug Amp B produces a higher level of IL-1ß over the infected control. Moreover, administering anti-IL-1ß antibody to infected Amp B-treated mice showed significantly less parasite clearance. Investigation revealed that Leishmania inhibits stimuli-induced expression of a multiprotein signaling platform, NLRP3 inflammasome, which in turn inhibits caspase-1 activation mediated maturation of IL-1ß from its pro form. Attenuation of NLRP3 and pro-IL-1ß in infection was found to result from decreased NF-κB activity. Transfecting infected cells with constitutively active NF-κB plasmid increased NLRP3 and pro-IL-1ß expression but did not increase mature IL-1ß, suggesting that IL-1ß maturation requires a second signal, which was found to be reactive oxygen species (ROS). Decreased NF-κB was attributed to increased expression of A20, a negative regulator of NF-κB signaling. Silencing A20 in infected cells restored NLRP3 and pro-IL-1ß expression, but also increased matured IL-1ß, implying an NF-κB-independent A20-modulated IL-1ß maturation. Macrophage ROS is primarily regulated by mitochondrial uncoupling protein 2 (UCP2), and UCP2-silenced infected cells showed an increased IL-1ß level. Short hairpin RNA-mediated knockdown of A20 and UCP2 in infected mice independently documented decreased liver and spleen parasite burden and increased IL-1ß production. These results suggest that Leishmania exploits A20 and UCP2 to impair inflammasome activation for disease propagation.-Gupta, A. K., Ghosh, K., Palit, S., Barua, J., Das, P. K., Ukil, A. Leishmania donovani inhibits inflammasome-dependent macrophage activation by exploiting the negative regulatory proteins A20 and UCP2.

IRAK-M regulates the inhibition of TLR-mediated macrophage immune response during late in vitro Leishmania donovani infection.

Intramacrophage protozoan parasite Leishmania donovani, causative agent of visceral leishmaniasis, escapes Toll-like receptor (TLR) dependent early host immune response by inducing the deubiquitinating enzyme A20, which is sustained up to 6 h postinfection only. Therefore, Leishmania must apply other means to deactivate late host responses. Here, we elucidated the role of IL-1 receptor-associated kinase M (IRAK-M), a negative regulator of TLR signaling, in downregulating macrophage proinflammatory response during late hours of in vitro infection. Our data reveal a sharp decline in IRAK1 and IRAK4 phosphorylation at 24 h postinfection along with markedly reduced association of IRAK1-TNF receptor associated factor 6, which is mandatory for TLR activation. In contrast, IRAK-M was induced after A20 levels decreased and reached a maximum at 24 h postinfection. IRAK-M induction coincided with increased stimulation of TGF-ß, a hallmark cytokine of visceral infection. TGF-ß-dependent signaling-mediated induction of SMAD family of proteins, 2, 3, and 4 plays important roles in transcriptional upregulation of IRAK-M. In infected macrophages, siRNA-mediated silencing of IRAK-M displayed enhanced IRAK1 and IRAK4 phosphorylation with a concomitant increase in downstream NF-κB activity and reduced parasite survival. Taken together, the results suggest that IRAK-M may be targeted by L. donovani to inhibit TLR-mediated proinflammatory response late during in vitro infection.