Circadian rhythms are known to regulate immune responses in healthy animals, but it is unclear whether they persist during acute illnesses where clock gene expression is disrupted by systemic inflammation. Here, we use a genome-wide approach to investigate circadian gene and metabolite expression in the lungs of endotoxemic mice and find that novel cellular and molecular circadian rhythms are elicited in this setting. The endotoxin-specific circadian program exhibits unique features, including a divergent group of rhythmic genes and metabolites compared to the basal state and a distinct periodicity and phase distribution. At the cellular level endotoxin treatment also alters circadian rhythms of leukocyte counts within the lung in a bmal1-dependent manner, such that granulocytes rather than lymphocytes become the dominant oscillating cell type. Our results show that inflammation produces a complex reorganization of cellular and molecular circadian rhythms that are relevant to early events in lung injury.

Pulmonary arterial (PA) stiffness is associated with increased mortality in patients with pulmonary hypertension (PH); however, the role of PA stiffening in the pathogenesis of PH remains elusive. Here, we show that distal vascular matrix stiffening is an early mechanobiological regulator of experimental PH. We identify cyclooxygenase-2 (COX-2) suppression and corresponding reduction in prostaglandin production as pivotal regulators of stiffness-dependent vascular cell activation. Atomic force microscopy microindentation demonstrated early PA stiffening in experimental PH and human lung tissue. Pulmonary artery smooth muscle cells (PASMC) grown on substrates with the stiffness of remodeled PAs showed increased proliferation, decreased apoptosis, exaggerated contraction, enhanced matrix deposition, and reduced COX-2–derived prostanoid production compared with cells grown on substrates approximating normal PA stiffness. Treatment with a prostaglandin I2 analog abrogated monocrotaline-induced PA stiffening and attenuated stiffness-dependent increases in proliferation, matrix deposition, and contraction in PASMC. Our results suggest a pivotal role for early PA stiffening in PH and demonstrate the therapeutic potential of interrupting mechanobiological feedback amplification of vascular remodeling in experimental PH.

CO increases the survival of mice injured by CLP through systemic enhancement of autophagy and phagocytosis. Taken together, we suggest that CO gas may represent a novel therapy for patients with sepsis.

Supplemental Digital Content is available in the text.

Pulmonary arterial stiffness is an independent risk factor for mortality in pulmonary hypertension (PH) and plays a critical role in PH pathophysiology. Our laboratory has recently demonstrated arterial stiffening early in experimental PH, along with evidence for a mechanobiological feedback loop by which arterial stiffening promotes further cellular remodeling behaviors (Liu F, Haeger CM, Dieffenbach PB, Sicard D, Chrobak I, Coronata AM, Suárez Velandia MM, Vitali S, Colas RA, Norris PC, Marinković A, Liu X, Ma J, Rose CD, Lee SJ, Comhair SA, Erzurum SC, McDonald JD, Serhan CN, Walsh SR, Tschumperlin DJ, Fredenburgh LE. 1: e86987, 2016). Cyclooxygenase-2 (COX-2) and prostaglandin signaling have been implicated in stiffness-mediated regulation, with prostaglandin activity inversely correlated to matrix stiffness and remodeling behaviors in vitro, as well as to disease progression in rodent PH models. The mechanism by which mechanical signaling translates to reduced COX-2 activity in pulmonary vascular cells is unknown. The present work investigated the transcriptional regulators Yes-associated protein (YAP) and WW domain-containing transcription regulator 1 (WWTR1, a.k.a., TAZ), which are known drivers of downstream mechanical signaling, in mediating stiffness-induced changes in COX-2 and prostaglandin activity in pulmonary artery smooth muscle cells (PASMCs). We found that YAP/TAZ activity is increased in PAH PASMCs and experimental PH and is necessary for the development of stiffness-dependent remodeling phenotypes. Knockdown of YAP and TAZ markedly induces COX-2 expression and downstream prostaglandin production by approximately threefold, whereas overexpression of YAP or TAZ reduces COX-2 expression and prostaglandin production to near undetectable levels. Together, our findings demonstrate a stiffness-dependent YAP/TAZ-mediated positive feedback loop that drives remodeling phenotypes in PASMCs via reduced COX-2 and prostaglandin activity. The ability to interrupt this critical mechanobiological feedback loop and enhance local prostaglandin activity via manipulation of YAP/TAZ signaling presents a highly attractive novel strategy for the treatment of PH.

Background Isoflurane may be protective in pre-clinical models of lung injury but its use in patients with lung injury remains controversial and the mechanism of its protective effects remains unclear. We hypothesized that this protection is mediated at the level of alveolar tight junctions and investigated the possibility in a two-hit model of lung injury that mirrors human acute respiratory distress syndrome. Methods Wild-type mice were treated with isoflurane one hour after exposure to nebulized endotoxin (n=8) or saline control (n=9) then allowed to recover for 24 hrs prior to mechanical ventilation (MV, tidal volume 15 mL/kg, 2 hrs) producing ventilator-induced lung injury. Mouse lung epithelial cells were similarly treated with isoflurane one hour after exposure to lipopolysaccharide. Cells were cyclically stretched the following day to mirror the MV protocol used in vivo. Results Mice treated with isoflurane following exposure to inhaled endotoxin and prior to MV exhibited significantly less physiologic lung dysfunction. These effects appeared to be mediated by decreased vascular leak, but not altered inflammatory indices. Mouse lung epithelial cells treated with lipopolysaccharide and cyclic stretch and lungs harvested from mice following treatment with lipopolysaccharide and MV had decreased levels of a key tight junction protein (i.e. zona occludens 1) that was rescued by isoflurane treatment. Conclusions Isoflurane rescued lung injury induced by a two-hit model of endotoxin exposure followed by MV by maintaining the integrity of the alveolar-capillary barrier possibly by modulating the expression of a key tight junction protein.

Oxidative stress resulting from inflammatory responses that occur during acute lung injury and sepsis can initiate changes in mitochondrial function. Autophagy regulates cellular processes in the setting of acute lung injury, sepsis, and oxidative stress by modulating the immune response and facilitating turnover of damaged cellular components. We have shown that mesenchymal stromal cells (MSCs) improve survival in murine models of sepsis by also regulating the immune response. However, the effect of autophagy on MSCs and MSC mitochondrial function during oxidative stress is unknown. This study investigated the effect of depletion of autophagic protein microtubule-associated protein 1 light chain 3B (LC3B) and beclin 1 (BECN1) on the response of MSCs to oxidative stress. MSCs were isolated from wild-type (WT) and LC3B 2/2 or Becn1 1/2 mice. MSCs from the LC3B 2/2 and Becn1 1/2 animals had increased susceptibility to oxidative stress-induced cell death as compared with WT MSCs. The MSCs depleted of autophagic proteins also had impaired mitochondrial function (decreased intracellular ATP, reduced mitochondrial membrane potential, and increased mitochondrial reactive oxygen species production) under oxidative stress as compared with WT MSCs. In WT MSCs, carbon monoxide (CO) preconditioning enhanced autophagy and mitophagy, and rescued the cells from oxidative stress-induced death. CO preconditioning was not able to rescue the decreased survival of MSCs from the LC3B 2/2 and Becn1 1/2 animals, further supporting the tenet that CO exerts its cytoprotective effects via the autophagy pathway.

Sepsis represents a systemic inflammatory response caused by microbial infection in blood. Herein, we present a novel comprehensive approach to mitigate inflammatory responses through broad spectrum removal of pathogens, leukocytes and cytokines based on biomimetic cell margination. Using a murine model of polymicrobial sepsis induced by cecal ligation and puncture (CLP), we performed extracorporeal blood filtration with the developed microfluidic blood margination (µBM) device. Circulating bacteremia, leukocytes and cytokines in blood decreased post-filtration and significant attenuation of immune cell and cytokine responses were observed 3–5 days after intervention, indicating successful long-term immunomodulation. A dose-dependent effect on long-term immune cell count was also achieved by varying filtration time. As proof of concept for human therapy, the µBM device was scaled up to achieve ~100-fold higher throughput (~150mLhr−1). With further multiplexing, the µBM technique could be applied in clinical settings as an adjunctive treatment for sepsis and other inflammatory diseases.