A Pilot Study To Assess the Suitability of Riboflavin As a Surrogate Marker of Breast Cancer Resistance Protein in Healthy Participants.

We recently showed that riboflavin is a selected substrate of breast cancer resistance protein (BCRP) over P-glycoprotein (P-gp) and demonstrated its prediction performance in preclinical drug-drug interaction (DDI) studies. The aim of this study was to investigate the suitability of riboflavin to assess BCRP inhibition in humans. First, we assessed the substrate potential of riboflavin toward other major drug transporters using established transfected cell systems. Riboflavin is a substrate for organic anion transporter (OAT)1, OAT3, and multidrug and toxin extrusion protein (MATE)2-K, with uptake ratios ranging from 2.69 to 11.6, but riboflavin is not a substrate of organic anion-transporting polypeptide (OATP)1B1, OATP1B3, organic cation transporter (OCT)2, and MATE1. The effects of BMS-986371, a potent in vitro inhibitor of BCRP (IC 50 0.40 µM), on the pharmacokinetics of riboflavin, isobutyryl carnitine, and arginine were then examined in healthy male adults (N = 14 or 16) after oral administration of methotrexate (MTX) (7.5 mg) and enteric-coated (EC) sulfasalazine (SSZ) (1000 mg) alone or in combination with BMS-986371 (150 mg). Oral administration of BMS-986371 increased the area under the plasma concentration-time curves (AUCs) of rosuvastatin and immediate-release (IR) SSZ to 1.38- and 1.51-fold, respectively, and significantly increased AUC(0-4h), AUC(0-24h), and C max of riboflavin by 1.25-, 1.14-, and 1.11-fold (P-values of 0.003, 0.009, and 0.025, respectively) compared with the MTX/SSZ EC alone group. In contrast, BMS-986371 did not significantly influence the AUC(0-24h) and C max values of isobutyryl carnitine and arginine (0.96- to 1.07-fold, respectively; P > 0.05). Overall, these data indicate that plasma riboflavin is a promising biomarker of BCRP that may offer a possibility to assess drug candidate as a BCRP modulator in early drug development. SIGNIFICANCE STATEMENT: Endogenous compounds that serve as biomarkers for clinical inhibition of breast cancer resistance protein (BCRP) are not currently available. This study provides the initial evidence that riboflavin is a promising BCRP biomarker in humans. For the first time, the value of leveraging the substrate of BCRP with acceptable prediction performance in clinical studies is shown. Additional clinical investigations with known BCRP inhibitors are needed to fully validate and showcase the utility of this biomarker.

Neutrophils promote clearance of nuclear debris following acid-induced lung injury.

Neutrophils are critical mediators of host defense in pathogen-induced and sterile inflammation. Excessive neutrophil activation has been associated with increased host pathology through collateral organ damage. The beneficial aspects of neutrophil activation, particularly in sterile inflammation, are less well defined. We observed accumulation of nuclear debris in the lungs of neutropenic mice exposed to acid-induced injury compared with wild type. Size analysis of DNA debris showed that neutropenic mice were unable to degrade extracellular DNA fragments. In addition, we found that neutrophils are able to differentially express DNA-degrading and repair-associated genes and proteins. Once neutrophils are at sites of lung inflammation, they are able to phagocytose and degrade extracellular DNA. This neutrophil-dependent DNA degradation occurs in a MyD88-dependent pathway. The increased DNA debris in neutropenic mice was associated with dysregulated alveolar repair and the phenotype is rescued by intratracheal administration of DNase I. Thus, we show a novel mechanism as part of the inflammatory response, in which neutrophils engulf and degrade extracellular DNA fragments and allow for optimal organ repair.

A core viral protein binds host nucleosomes to sequester immune danger signals.

Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important part in innate immune responses. Viral-encoded core basic proteins compact viral genomes, but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles, it is unknown whether protein VII affects cellular chromatin. Here we show that protein VII alters cellular chromatin, leading us to hypothesize that this has an impact on antiviral responses during adenovirus infection in human cells. We find that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter the protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in the chromatin of members of the high-mobility-group protein B family (HMGB1, HMGB2 and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together, our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling.

Mechanisms that determine nanocarrier targeting to healthy versus inflamed lung regions.

Inflamed organs display marked spatial heterogeneity of inflammation, with patches of inflamed tissue adjacent to healthy tissue. To investigate how nanocarriers (NCs) distribute between such patches, we created a mouse model that recapitulates the spatial heterogeneity of the inflammatory lung disease ARDS. NCs targeting the epitope PECAM strongly accumulated in the lungs, but were shunted away from inflamed lung regions due to hypoxic vasoconstriction (HVC). In contrast, ICAM-targeted NCs, which had lower whole-lung uptake than PECAM/NCs in inflamed lungs, displayed markedly higher NC levels in inflamed regions than PECAM/NCs, due to increased regional ICAM. Regional HVC, epitope expression, and capillary leak were sufficient to predict intra-organ of distribution of NCs, antibodies, and drugs. Importantly, these effects were not observable with traditional spatially-uniform models of ARDS, nor when examining only whole-organ uptake. This study underscores how examining NCs' intra-organ distribution in spatially heterogeneous animal models can guide rational NC design.

Neutrophils promote alveolar epithelial regeneration by enhancing type II pneumocyte proliferation in a model of acid-induced acute lung injury.

Alveolar epithelial regeneration is essential for resolution of the acute respiratory distress syndrome (ARDS). Although neutrophils have traditionally been considered mediators of epithelial damage, recent studies suggest they promote type II pneumocyte (AT2) proliferation, which is essential for regenerating alveolar epithelium. These studies did not, however, evaluate this relationship in an in vivo model of alveolar epithelial repair following injury. To determine whether neutrophils influence alveolar epithelial repair in vivo, we developed a unilateral acid injury model that creates a severe yet survivable injury with features similar to ARDS. Mice that received injections of the neutrophil-depleting Ly6G antibody had impaired AT2 proliferation 24 and 72 h after acid instillation, which was associated with decreased reepithelialization and increased alveolar protein concentration 72 h after injury. As neutrophil depletion itself may alter the cytokine response, we questioned the contribution of neutrophils to alveolar epithelial repair in neutropenic granulocyte-colony stimulating factor (G-CSF)-/- mice. We found that the loss of G-CSF recapitulated the neutrophil response of Ly6G-treated mice and was associated with defective alveolar epithelial repair, similar to neutrophil-depleted mice, and was reversed by administration of exogenous G-CSF. To approach the mechanisms, we employed an unbiased protein analysis of bronchoalveolar lavage fluid from neutrophil-depleted and neutrophil-replete mice 12 h after inducing lung injury. Pathway analysis identified significant differences in multiple signaling pathways that may explain the differences in epithelial repair. These data emphasize an important link between the innate immune response and tissue repair in which neutrophils promote alveolar epithelial regeneration.

A polymorphism that delays fibrosis in hepatitis C promotes alternative splicing of AZIN1, reducing fibrogenesis.

UNLABELLED: Among several single-nucleotide polymorphisms (SNPs) that correlate with fibrosis progression in chronic HCV, an SNP in the antizyme inhibitor (AzI) gene is most strongly associated with slow fibrosis progression. Our aim was to identify the mechanism(s) underlying this observation by exploring the impact of the AzI SNP on hepatic stellate cell (HSC) activity. Seven novel AZIN1 splice variants ("SV2-8") were cloned by polymerase chain reaction from the LX2 human HSC line. Expression of a minigene in LX2 containing the AZIN1 slow-fibrosis SNP yielded a 1.67-fold increase in AZIN1 splice variant 2 (AZIN1 SV2) messenger RNA (mRNA) (P = 0.05). In healthy human leukocytes, the SNP variant also correlated with significantly increased SV2 mRNA. Cells (293T) transfected with short hairpin RNA (shRNA) complementary to the exonic splicing chaperone SRp40 expressed 30% less SRp40 (P = 0.044) and 43% more AzI SV2 (P = 0.021) than control shRNA-expressing cells, mimicking the effect of the sequence variant. LX2 cells transfected with AZIN1 full-length complementary DNA expressed 35% less collagen I mRNA (P = 0.09) and 18% less α-smooth muscle actin mRNA (P = 0.09). Transient transfection of AZIN1 SV2 complementary DNA into LX2 cells reduced collagen I gene expression by 64% (P = 0.001) and α-smooth muscle actin by 43% (P = 0.005) compared to vector-transfected controls, paralleling changes in protein expression. Both AZIN1 and AZIN-SV2 mRNAs are detectable in normal human liver and reduced in HCV cirrhotic livers. The AZIN1-SV2 acts via a polyamine-independent pathway, as it neither interacts with antizyme nor affects the ability of AZIN1 lacking this variant to neutralize antizyme. CONCLUSION: An SNP variant in the AZIN1 gene leads to enhanced generation of a novel alternative splice form that modifies the fibrogenic potential of HSCs.

Type II alveolar cell MHCII improves respiratory viral disease outcomes while exhibiting limited antigen presentation.

Type II alveolar cells (AT2s) are critical for basic respiratory homeostasis and tissue repair after lung injury. Prior studies indicate that AT2s also express major histocompatibility complex class II (MHCII) molecules, but how MHCII expression by AT2s is regulated and how it contributes to host defense remain unclear. Here we show that AT2s express high levels of MHCII independent of conventional inflammatory stimuli, and that selective loss of MHCII from AT2s in mice results in modest worsening of respiratory virus disease following influenza and Sendai virus infections. We also find that AT2s exhibit MHCII presentation capacity that is substantially limited compared to professional antigen presenting cells. The combination of constitutive MHCII expression and restrained antigen presentation may position AT2s to contribute to lung adaptive immune responses in a measured fashion, without over-amplifying damaging inflammation.

STAT3-BDNF-TrkB signalling promotes alveolar epithelial regeneration after lung injury.

Alveolar epithelial regeneration is essential for recovery from devastating lung diseases. This process occurs when type II alveolar pneumocytes (AT2 cells) proliferate and transdifferentiate into type I alveolar pneumocytes (AT1 cells). We used genome-wide analysis of chromatin accessibility and gene expression following acute lung injury to elucidate repair mechanisms. AT2 chromatin accessibility changed substantially following injury to reveal STAT3 binding motifs adjacent to genes that regulate essential regenerative pathways. Single-cell transcriptome analysis identified brain-derived neurotrophic factor (Bdnf) as a STAT3 target gene with newly accessible chromatin in a unique population of regenerating AT2 cells. Furthermore, the BDNF receptor tropomyosin receptor kinase B (TrkB) was enriched on mesenchymal alveolar niche cells (MANCs). Loss or blockade of AT2-specific Stat3, Bdnf or mesenchyme-specific TrkB compromised repair and reduced Fgf7 expression by niche cells. A TrkB agonist improved outcomes in vivo following lung injury. These data highlight the biological and therapeutic importance of the STAT3-BDNF-TrkB axis in orchestrating alveolar epithelial regeneration.

Using selective lung injury to improve murine models of spatially heterogeneous lung diseases.

Many lung diseases, such as the acute respiratory distress syndrome (ARDS), display significant regional heterogeneity with patches of severely injured tissue adjacent to apparently healthy tissue. Current mouse models that aim to mimic ARDS generally produce diffuse injuries that cannot reproducibly generate ARDS's regional heterogeneity. This deficiency prevents the evaluation of how well therapeutic agents reach the most injured regions and precludes many regenerative medicine studies since it is not possible to know which apparently healing regions suffered severe injury initially. Finally, these diffuse injury models must be relatively mild to allow for survival, as their diffuse nature does not allow for residual healthy lung to keep an animal alive long enough for many drug and regenerative medicine studies. To solve all of these deficiencies in current animal models, we have created a simple and reproducible technique to selectively induce lung injury in specific areas of the lung. Our technique, catheter-in-catheter selective lung injury (CICSLI), involves guiding an inner catheter to a particular area of the lung and delivering an injurious agent mixed with nanoparticles (fluorescently and/or radioactively labeled) that can be used days later to track the location and extent of where the initial injury occurred. Furthermore, we demonstrate that CICSLI can produce a more severe injury than diffuse models, yet has much higher survival since CICSLI intentionally leaves lung regions undamaged. Collectively, these attributes of CICSLI will allow investigators to better study how drugs act within heterogeneous lung pathologies and how regeneration occurs in severely damaged lung tissue, thereby aiding the development of new therapies for ARDS and other heterogenous lung diseases.

Mesenchyme-free expansion and transplantation of adult alveolar progenitor cells: steps toward cell-based regenerative therapies.

Alveolar type-2 (AT2) cells are necessary for the lung's regenerative response to epithelial insults such as influenza. However, current methods to expand these cells rely on mesenchymal co-culture, complicating the possibility of transplantation following acute injury. Here we developed several mesenchyme-free culture conditions that promote growth of murine AT2 organoids. Transplanting dissociated AT2 organoids into influenza-infected mice demonstrated that organoids engraft and either proliferate as AT2 cells or unexpectedly adopt a basal cell-like fate associated with maladaptive regeneration. Alternatively, transplanted primary AT2 cells also robustly engraft, maintaining their AT2 lineage while replenishing the alveolar type-1 (AT1) cell population in the epithelium. Importantly, pulse oximetry revealed significant increase in blood-oxygen saturation in primary AT2 recipients, indicating that transplanted cells also confer increased pulmonary function after influenza. We further demonstrated that both acid installation and bleomycin injury models are also amenable to AT2 transplantation. These studies provide additional methods to study AT2 progenitor potential, while serving as proof-of-principle for adoptive transfer of alveolar progenitors in potential therapeutic applications.

An inflammatory pulmonary insult post-traumatic brain injury worsens subsequent spatial learning and neurological outcomes.

BACKGROUND: Severe traumatic brain injury (TBI) patients are at high risk for early aspiration and pneumonia. How pneumonia impacts neurological recovery after TBI is not well characterized. We hypothesized that, independent of the cerebral injury, pneumonia after TBI delays and worsens neurological recovery and cognitive outcomes. METHODS: Fifteen CD1 male mice were randomized to sham craniotomy or severe TBI (controlled cortical impact [CCI] - velocity 6 m/s, depth 1.0 mm) ± intratracheal lipopolysaccharide (LPS-2 mg/kg in 0.1 mL saline) as a pneumonia bioeffector. Neurological functional recovery by Garcia Neurologic Testing (GNT) and body weight loss were recorded daily for 14 days. On Days 6-14, animals underwent Morris Water Maze learning and memory testing with cued trials (platform visible), spatial learning trials (platform invisible, spatial cues present), and probe (memory) trials (platform removed, spatial clues present). Intergroup differences were assessed by the Kruskal-Wallis test with Bonferroni correction (p < 0.05). RESULTS: Weight loss was greatest in the CCI + LPS group (maximum 24% on Day 3 vs. 8% [Sham], 7% [CCI], both on Day 1). GNT was lowest in CCI + LPS during the first week. Morris Water Maze testing demonstrated greater spatial learning impairment in the CCI + LPS group vs. Sham or CCI counterparts. Cued learning and long-term memory were worse in CCI + LPS and CCI as compared to Sham. CONCLUSION: A pneumonia bioeffector insult after TBI worsens weight loss and mortality in a rodent model. Not only is spatial learning impaired, but animals are more debilitated and have worse neurologic performance. Understanding the adverse effects of pneumonia on TBI recovery is the first step d patients.

Tumor suppressor activity of KLF6 mediated by downregulation of the PTTG1 oncogene.

The tumor suppressor Kruppel-like factor 6 (KLF6) is frequently inactivated in hepatocellular carcinoma (HCC). To unearth downstream transcriptional targets of KLF6, cDNA microarray analysis of whole liver was compared between KLF6+/+ and KLF6+/- mice. Pituitary tumor transforming gene 1 (PTTG1), an oncogene, was the most up-regulated transcript in KLF6+/- liver. In human HCCs, KLF6 mRNA was significantly decreased, associated with increased PTTG1. In HepG2, KLF6 transcriptionally repressed PTTG1 by direct promoter interaction. Whereas KLF6 downregulation by siRNA increased HepG2 proliferation, siRNA to PTTG1 was anti-proliferative. PTTG1 downregulation represents a novel tumor suppressor pathway of KLF6.