Brant M. Wagener

Fibroblast survival and dysregulated activation drive fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). During physiological wound repair, fibroblasts are transiently activated to restore tissue integrity and are subsequently cleared by programmed cell death. In fibrotic disease, however, fibroblasts evade apoptosis and persist in a pathologically activated state. Although S100A4 has been implicated in fibrotic lung disease, the mechanisms by which S100A4 governs fibroblast fate and sustains profibrotic behavior remain unclear. Here, we identify S100A4 as a key regulator of apoptosis‐resistant, profibrotically activated fibroblasts through engagement of extracellular signal‐regulated kinase (ERK) signaling. In primary murine lung fibroblasts, S100A4 activates ERK, resulting in a coordinated program of fibroblast activation, including increased migration, extracellular matrix (ECM) contractility, stress fiber formation, and alpha‐smooth muscle actin (α‐SMA) induction. Functionally, S100A4 confers resistance to apoptosis induced by pro‐apoptotic and oxidative stress stimuli, as evidenced by reduced cleaved caspase‐3 and preserved cell viability. Pharmacological blockade of ERK signaling attenuates these responses, supporting ERK as an important downstream mediator of S100A4‐driven fibroblast activation and survival programs. Extending these findings to disease‐relevant contexts, bleomycin (BLM)‐induced lung injury in mice induces robust fibrotic remodeling, excessive collagen deposition, and transcriptional upregulation of S100A4. Consistently, primary lung fibroblasts from IPF patients exhibit elevated S100A4 expression, enhanced ERK activation, and increased α‐SMA expression, demonstrating conservation of this signaling axis across experimental models and human disease. Importantly, siRNA‐mediated knockdown of S100A4 in IPF fibroblasts suppresses ERK activation and attenuates expression of key profibrotic genes, indicating that S100A4 contributes to maintaining the fibrotic program in IPF fibroblasts. Collectively, these findings define a mechanistic link between S100A4‐mediated fibroblast survival and activation that drives pathological matrix remodeling and identify S100A4 and ERK as potential therapeutic targets in pulmonary fibrosis.

Background/Objectives: G protein-coupled receptors (GPCRs) can promote ligand-biased signaling, yet the mechanisms that promote bias are not well understood. We have shown that C-C Chemokine Ligand 19 (CCL19) and CCL21 promote ligand-biased internalization and signaling of C-C Chemokine Receptor 7 (CCR7) in T cells. The roles of GPCR kinases (GRKs) in regulating biased CCR7 internalization and biased signaling in T cells are unclear. GRK2 is a serine/threonine kinase that phosphorylates GPCRs in response to ligand binding and is recruited to the plasma membrane via its C-terminal pleckstrin homology domain to phosphatidylinositol 4,5-bisphosphate (PIP₂). Methods: Human embryonic kidney cells (HEK293) transfected to express wild-type and mutant GRK2 and human CCR7, human T cell lines harboring heterozygous deletions of GRK2, and naïve primary T cells from GRK2 heterozygous (GRK2^+/−) or GRK2^f/f CD4-Cre mice were used to examine the effects of GRK2 on ligand-induced CCR7 signaling in T cells. We used flow cytometry to assay the effect of GRK2 on CCR7 internalization, Fluorescence Resonance Energy Transfer (FRET) to define the effect of GRK2 on CCR7 activation of Gα_i isoforms and transwell migration assays to examine the effect of GRK2 on chemotaxis. Since chemotaxis via CCR7 is mediated by phospholipase Cγ1 (PLCγ1), Western blot assays were used to measure the effect of GRK2 during downstream signaling via phosphorylation of PLCγ1. Results: We found that following CCL19 binding, GRK2 promoted kinase-dependent CCR7 recruitment of arrestin-3, rapid CCR7 internalization and Gα_i3 recruitment to CCR7. In contrast, following binding of CCL21 to CCR7, GRK2 slowed CCR7 internalization, induced recruitment of Gα_i2 to the activated receptor_, and promoted chemotaxis. Since we have shown that CCL21 promotes chemotaxis via PLCγ1, we examined the effect of GRK2 on PLCγ1 activation and found that GRK2 had no effect on CCL21-mediated PLCγ1 phosphorylation. Conclusions: GRK2 promotes differential signaling downstream of CCR7 activation by CCL19 and CCL21 and provides a model for biased signaling downstream of a GPCR driven by GRK2.

Background: Despite low mortality for elective procedures in the United States and developed countries, some patients have unexpected care escalations (UCE) following post-anesthesia care unit (PACU) discharge. Studies indicate patient risk factors for UCE, but determining which factors are most important is unclear. Machine learning (ML) can predict clinical events. We hypothesized that ML could predict patient UCE after PACU discharge in surgical patients and identify specific risk factors. Methods: We conducted a single center, retrospective analysis of all patients undergoing non-cardiac surgery (elective and emergent). We collected data from pre-operative visits, intra-operative records, PACU admissions, and the rate of UCE. We trained a ML model with this data and tested the model on an independent data set to determine its efficacy. Finally, we evaluated the individual patient and clinical factors most likely to predict UCE risk. Results: Our study revealed that ML could predict UCE risk which was approximately 5% in both the training and testing groups. We were able to identify patient risk factors such as patient vital signs, emergent procedure, ASA Status, and non-surgical anesthesia time as significant variable. We plotted Shapley values for significant variables for each patient to help determine which of these variables had the greatest effect on UCE risk. Of note, the UCE risk factors identified frequently by ML were in alignment with anesthesiologist clinical practice and the current literature. Conclusions: We used ML to analyze data from a single-center, retrospective cohort of non-cardiac surgical patients, some of whom had an UCE. ML assigned risk prediction for patients to have UCE and determined perioperative factors associated with increased risk. We advocate to use ML to augment anesthesiologist clinical decision-making, help decide proper disposition from the PACU, and ensure the safest possible care of our patients.

Lung endothelia in the arteries, capillaries, and veins are heterogeneous in structure and function. Lung capillaries in particular represent a unique vascular niche, with a thin yet highly restrictive alveolar-capillary barrier that optimizes gas exchange. Capillary endothelium surveys the blood while simultaneously interpreting cues initiated within the alveolus and communicated via immediately adjacent type I and type II epithelial cells, fibroblasts, and pericytes. This cell-cell communication is necessary to coordinate the immune response to lower respiratory tract infection. Recent discoveries identify an important role for the microtubule-associated protein tau that is expressed in lung capillary endothelia in the host-pathogen interaction. This endothelial tau stabilizes microtubules necessary for barrier integrity, yet infection drives production of cytotoxic tau variants that are released into the airways and circulation, where they contribute to end-organ dysfunction. Similarly, beta-amyloid is produced during infection. Beta-amyloid has antimicrobial activity, but during infection it can acquire cytotoxic activity that is deleterious to the host. The production and function of these cytotoxic tau and amyloid variants are the subject of this review. Lung-derived cytotoxic tau and amyloid variants are a recently discovered mechanism of end-organ dysfunction, including neurocognitive dysfunction, during and in the aftermath of infection.

Background: Bacterial pneumonia and sepsis are both common causes of end-organ dysfunction, especially in immunocompromised and critically ill patients. Pre-clinical data demonstrate that bacterial pneumonia and sepsis elicit the production of cytotoxic tau and amyloids from pulmonary endothelial cells, which cause lung and brain injury in naïve animal subjects, independent of the primary infection. The contribution of infection-elicited cytotoxic tau and amyloids to end-organ dysfunction has not been examined in the clinical setting. We hypothesized that cytotoxic tau and amyloids are present in the bronchoalveolar lavage fluid of critically ill patients with bacterial pneumonia and that these tau/amyloids are associated with end-organ dysfunction. Methods: Bacterial culture-positive and culture-negative mechanically ventilated patients were recruited into a prospective, exploratory observational study. Levels of tau and Aβ₄₂ in, and cytotoxicity of, the bronchoalveolar lavage fluid were measured. Cytotoxic tau and amyloid concentrations were examined in comparison with patient clinical characteristics, including measures of end-organ dysfunction. Results: Tau and Aβ₄₂ were increased in culture-positive patients (n = 49) compared to culture-negative patients (n = 50), independent of the causative bacterial organism. The mean age of patients was 52.1 ± 16.72 years old in the culture-positive group and 52.78 ± 18.18 years old in the culture-negative group. Males comprised 65.3% of the culture-positive group and 56% of the culture-negative group. Caucasian culture-positive patients had increased tau, boiled tau, and Aβ₄₂ compared to both Caucasian and minority culture-negative patients. The increase in cytotoxins was most evident in males of all ages, and their presence was associated with end-organ dysfunction. Conclusions: Bacterial infection promotes the generation of cytotoxic tau and Aβ₄₂ within the lung, and these cytotoxins contribute to end-organ dysfunction among critically ill patients. This work illuminates an unappreciated mechanism of injury in critical illness.

Pulmonary microvascular endothelial cells contribute to the integrity of the lung gas exchange interface, and they are highly glycolytic. Although glucose and fructose represent discrete substrates available for glycolysis, pulmonary microvascular endothelial cells prefer glucose over fructose, and the mechanisms involved in this selection are unknown. 6-Phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) is an important glycolytic enzyme that drives glycolytic flux against negative feedback and links glycolytic and fructolytic pathways. We hypothesized that PFKFB3 inhibits fructose metabolism in pulmonary microvascular endothelial cells. We found that PFKFB3 knockout cells survive better than wild-type cells in fructose-rich medium under hypoxia. Seahorse assays, lactate and glucose measurements, and stable isotope tracing showed that PFKFB3 inhibits fructose–hexokinase–mediated glycolysis and oxidative phosphorylation. Microarray analysis revealed that fructose upregulates PFKFB3, and PFKFB3 knockout cells increase fructose-specific GLUT5 (glucose transporter 5) expression. Using conditional endothelial-specific PFKFB3 knockout mice, we demonstrated that endothelial PFKFB3 knockout increases lung tissue lactate production after fructose gavage. Last, we showed that pneumonia increases fructose in BAL fluid in mechanically ventilated ICU patients. Thus, PFKFB3 knockout increases GLUT5 expression and the hexokinase-mediated fructose use in pulmonary microvascular endothelial cells that promotes their survival. Our findings indicate that PFKFB3 is a molecular switch that controls glucose versus fructose use in glycolysis and help better understand lung endothelial cell metabolism during respiratory failure.

Background: The vagus nerve affects innate immune responses by activating spleen-projecting sympathetic neurons, which modulate leukocyte function. Recent basic and clinical research investigating vagus nerve stimulation to engage the cholinergic anti-inflammatory pathway (CAP) has shown promising therapeutic results for a variety of inflammatory diseases. Abundant sympathetic innervation occurs in rodent spleens, and use of these species has dominated mechanistic research investigating the CAP. However, previous neuroanatomical studies of human spleen found a more restricted pattern of innervation compared to rodents. Therefore, our primary goal was to establish the full extent of sympathetic innervation of human spleens using donor tissue with the shortest procurement to fixation time. Parallel studies of porcine spleen, a large animal model, were performed as a positive control and for comparison.Methods: Human and porcine spleen tissue were fixed immediately after harvest and prepared for immunohistochemistry. Human heart and porcine spleen were stained in conjunction as positive controls. Several immunohistochemical protocols were compared for best results. Tissue was stained for tyrosine hydroxylase (TH), a noradrenergic marker, using VIP purple chromogen. Consecutive tissue slices were stained for neuropeptide Y (NPY), which often co-localizes with TH, or double-labelled for TH and CD3, a T cell marker. High-magnification images and full scans of the tissue were obtained and analyzed for qualitative differences between species.Results: TH had dominant perivascular localization in human spleen, with negligible innervation of parenchyma, but such nerves were abundant throughout ventricular myocardium. In marked contrast, noradrenergic innervation was abundant in all regions of porcine spleen, with red pulp having more nerves than white pulp. NPY stain results were consistent with this pattern. In human spleen, noradrenergic nerves only ran close to T cells at the boundary of the periarterial lymphatic sheath and arteries. In porcine spleen, noradrenergic nerves were closely associated with T cells in both white and red pulp as well as other leukocytes in red pulp.Conclusion: Sympathetic innervation of the spleen varies between species in both distribution and abundance, with humans and pigs being at opposite extremes. This has important implications for sympathetic regulation of neuroimmune interactions in the spleen of different species and focused targeting of the CAP in humans.