One of the early events in the progression of LPS-mediated acute lung injury in mice is the disruption of the pulmonary endothelial barrier resulting in lung edema. However, the molecular mechanisms by which the endothelial barrier becomes compromised remain unresolved. The SRY (sex-determining region on the Y chromosome)–related high-mobility group box (Sox) group F family member, SOX18, is a barrier-protective protein through its ability to increase the expression of the tight junction protein CLDN5. Thus, the purpose of this study was to determine if downregulation of the SOX18-CLDN5 axis plays a role in the pulmonary endothelial barrier disruption associated with LPS exposure. Our data indicate that both SOX18 and CLDN5 expression is decreased in two models of in vivo LPS exposure (intraperitoneal, intratracheal). A similar downregulation was observed in cultured human lung microvascular endothelial cells (HLMVECs) exposed to LPS. SOX18 overexpression in HLMVECs or in the mouse lung attenuated the LPS-mediated vascular barrier disruption. Conversely, reduced CLDN5 expression (siRNA) reduced the HLMVEC barrier-protective effects of SOX18 overexpression. The mechanism by which LPS decreases SOX18 expression was identified as transcriptional repression through binding of NF-κB (p65) to a SOX18 promoter sequence located between −1,082 and −1,073 bp with peroxynitrite contributing to LPS-mediated NF-κB activation. We conclude that NF-κB–dependent decreases in the SOX18-CLDN5 axis are essentially involved in the disruption of human endothelial cell barrier integrity associated with LPS-mediated acute lung injury.

Macrophages stand at the forefront of both innate and adapted immunity through their capacities to recognize, engulf, and eliminate foreign particles, and to stimulate adapted immune cells. They are also involved in controlling pro- and anti-inflammatory pathways. Macrophage activity against Mycobacterium tuberculosis (M. tuberculosis) has been shown to involve Toll-like receptor (TLR) activation and ROS production. Previous studies have shown that lipopolysaccharide (LPS), through TLR4, could activate macrophages, improve their bactericidal ROS production, and facilitate anti-infective immune responses. We sought to better understand the role of the TLR4-NOX2 axis in macrophage activation during M. tuberculosis infection. THP-1 macrophages and PMA primed THP-1 macrophages [THP-1(A)] were treated with LPS and infected by M. tuberculosis. Cells were analyzed by flow cytometry for TLR4 expression, ROS production, phagocytosis, and killing of M. tuberculosis. Western blotting was used to analyze NOX2 expression. Inhibitors of the TLR4-NOX2 pathway were used to assess this pathway’s role in these processes, and their role in LPS activation of macrophages. We found that THP1-derived macrophages or PMA primed THP-1 macrophages exhibit higher surface TLR4 levels and increased NOX2 expression levels following LPS treatment. M. tuberculosis infection reduced these levels, but LPS was able to limit the negative effects of M.tb. Additionally, LPS increases THP-1(A) cells’ bactericidal activities including phagocytosis, ROS production, and destruction of M. tuberculosis. Significantly, all of these activities are impaired when TLR4 or NOX2 are inhibited. These studies demonstrate the importance of the TLR4-NOX2 axis in M. tuberculosis elimination by macrophages and may lead to novel therapies for tuberculosis and other bacterial infections.

Mechanical ventilation (MV) is a therapeutic intervention widely used in the clinic to assist patients that have difficulty breathing due to lung edema, trauma, or general anesthesia. However, MV causes ventilator-induced lung injury (VILI), a condition characterized by increased permeability of the alveolar-capillary barrier that results in edema, hemorrhage and neutrophil infiltration, leading to exacerbated lung inflammation and oxidative stress. This study explored the feasibility of using bixin, a canonical NRF2 inducer identified during the current study, to ameliorate lung damage in a murine VILI model. In vitro, bixin was found to activate the NRF2 signaling pathway through blockage of ubiquitylation and degradation of NRF2 in a KEAP1-C151 dependent manner; intraperitoneal (IP) injection of bixin led to pulmonary upregulation of the NRF2 response in vivo. Remarkably, IP administration of bixin restored normal lung morphology and attenuated inflammatory response and oxidative DNA damage following MV. This observed beneficial effect of bixin derived from induction of the NRF2 cytoprotective response since it was only observed in Nrf2^+/+ but not in Nrf2^−/− mice. This is the first study providing proof-of-concept that NRF2 activators can be developed into pharmacological agents for clinical use to prevent patients from lung injury during MV treatment.

Nuclear factor, erythroid 2-like 2 (NFE2L2), a transcription factor also known as NF-E2-related factor 2 (Nrf2), is a key cytoprotective gene that regulates critical antioxidant and stress-responsive genes. Nrf2 has been demonstrated to be a promising therapeutic target and useful biomarker in malignant disease. We hypothesized that NFE2L2-mediated gene expression would reflect cancer severity and progression. We conducted a meta-analysis of microarray data for 240 NFE2L2-mediated genes that were enriched in tumor tissues. We then developed a risk scoring system based on NFE2L2 gene expression profiling and designated 50 tumor-associated genes as the NFE2L2-associated molecular signature (NAMS). We tested the relationship between this gene expression signature and both recurrence-free survival and overall survival in lung cancer patients. We find that NAMS predicts clinical outcome in the training cohort and in 12 out of 20 validation cohorts. Cox proportional hazard regressions indicate that NAMS is a robust prognostic gene signature, independent of other clinical and pathological factors including patient age, gender, smoking, gene alteration, MYC level and cancer stage. NAMS is an excellent predictor of recurrence-free survival and overall survival in human lung cancer. This gene signature represents a promising prognostic biomarker in human lung cancer.

Nicotinamide phosphoribosyltransferase (NAMPT) exists as both intracellular NAMPT and extracellular NAMPT (eNAMPT) proteins. eNAMPT is secreted into the blood and functions as a cytokine/enzyme (cytozyme) that activates NF-κB signaling via ligation of Toll-like receptor 4 (TLR4), further serving as a biomarker for inflammatory lung disorders such as acute respiratory distress syndrome. In contrast, intracellular NAMPT is involved in nicotinamide mononucleotide synthesis and has been implicated in the regulation of cellular apoptosis, although the exact mechanisms for this regulation are poorly understood. We examined the role of NAMPT in TNF-α–induced human lung endothelial cell (EC) apoptosis and demonstrated that reduced NAMPT expression (siRNA) increases EC susceptibility to TNF-α–induced apoptosis as reflected by PARP-1 cleavage and caspase-3 activation. In contrast, overexpression of NAMPT served to reduce degrees of TNF-α–induced EC apoptosis. Inhibition of nicotinamide mononucleotide synthesis by FK866 (a selective NAMPT enzymatic inhibitor) failed to alter TNF-α–induced human lung EC apoptosis, suggesting that NAMPT-dependent NAD+ generation is unlikely to be involved in regulation of TNF-α–induced EC apoptosis. We next confirmed that TNF-α–induced EC apoptosis is attributable to NAMPT secretion into the EC culture media and subsequent eNAMPT ligation of TLR4 on the EC membrane surface. Silencing of NAMPT expression, direct neutralization of secreted eNAMPT by an NAMPT-specific polyclonal antibody (preventing TLR4 ligation), or direct TLR4 antagonism all served to significantly increase EC susceptibility to TNF-α–induced EC apoptosis. Together, these studies provide novel insights into NAMPT contributions to lung inflammatory events and to novel mechanisms of EC apoptosis regulation.

Mycobacterium tuberculosis (M. tuberculosis) is the pathogen which causes tuberculosis (TB), a significant human public health threat. Co-infection of M. tuberculosis and the human immunodeficiency virus (HIV), emergence of drug resistant M. tuberculosis, and failure to develop highly effective TB vaccines have limited control of the TB epidemic. Trained immunity is an enhanced innate immune response which functions independently of the adaptive/acquired immune system and responds non-specifically to reinfection with invading agents. Recently, several studies have found trained immunity has the capability to control and eliminate M. tuberculosis infection. Over the past decades, however, the consensus was adaptive immunity is the only protective mechanism by which hosts inhibit M. tuberculosis growth. Furthermore, autophagy plays an essential role in the development of trained immunity. Further investigation of trained immunity, M. tuberculosis infection, and the role of autophagy in this process provide new possibilities for vaccine development. In this review, we present the general characteristics of trained immunity and autophagy. We additionally summarize several examples where initiation of trained immunity contributes to the prevention of M. tuberculosis infection and propose future directions for research in this area.