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Macrophages can exhibit pro-inflammatory or pro-reparatory functions, contingent upon their specific activation state. This dynamic behavior empowers macrophages to engage in immune reactions and contribute to tissue homeostasis. Understanding the intricate interplay between macrophage motility and activation status provides valuable insights into the complex mechanisms that govern their diverse functions. In a recent study, we developed a classification method based on morphology, which demonstrated that movement characteristics, including speed and displacement, can serve as distinguishing factors for macrophage subtypes. In this study, we develop a deep learning model to explore the potential of classifying macrophage subtypes based solely on raw trajectory patterns. The classification model relies on the time series of x-y coordinates, as well as the distance traveled and net displacement. We begin by investigating the migratory patterns of macrophages to gain a deeper understanding of their behavior. Although this analysis does not directly inform the deep learning model, it serves to highlight the intricate and distinct dynamics exhibited by different macrophage subtypes, which cannot be easily captured by a finite set of motility metrics. Our study uses cell trajectories to classify three macrophage subtypes: M0, M1, and M2. This advancement holds promising implications for the future, as it suggests the possibility of identifying macrophage subtypes without relying on shape analysis. Consequently, it could potentially eliminate the necessity for high-quality imaging techniques and provide more robust methods for analyzing inherently blurry images.

Electrical signaling plays a crucial role in the cellular response to tissue injury in wound healing and an external electric field (EF) may expedite the healing process. Here, we have developed a standalone, wearable, and programmable electronic device to administer a well-controlled exogenous EF, aiming to accelerate wound healing in an in vivo mouse model to provide pre-clinical evidence. We monitored the healing process by assessing the re-epithelization rate and the ratio of M1/M2 macrophage phenotypes through histology staining. Following three days of treatment, the M1/M2 macrophage ratio decreased by 30.6% and the re-epithelization in the EF-treated wounds trended towards a non-statically significant 24.2% increase compared to the control. These findings provide point towards the effectiveness of the device in shortening the inflammatory phase by promoting reparative macrophages over inflammatory macrophages, and in speeding up re-epithelialization. Our wearable device supports the rationale for the application of programmed EFs for wound management in vivo and provides an exciting basis for further development of our technology based on the modulation of macrophages and inflammation to better wound healing.

Summary: The composite material-like extracellular matrix (ECM) in the sinoatrial node (SAN) supports the native pacemaking cardiomyocytes (PCMs). To test the roles of SAN ECM in the PCM phenotype and function, we engineered reconstructed-SAN heart tissues (rSANHTs) by recellularizing porcine SAN ECMs with hiPSC-derived PCMs. The hiPSC-PCMs in rSANHTs self-organized into clusters resembling the native SAN and displayed higher expression of pacemaker-specific genes and a faster automaticity compared with PCMs in reconstructed-left ventricular heart tissues (rLVHTs). To test the protective nature of SAN ECMs under strain, rSANHTs and rLVHTs were transplanted onto the murine thoracic diaphragm to undergo constant cyclic strain. All strained-rSANHTs preserved automaticity, whereas 66% of strained-rLVHTs lost their automaticity. In contrast to the strained-rLVHTs, PCMs in strained-rSANHTs maintained high expression of key pacemaker genes (HCN4, TBX3, and TBX18). These findings highlight the promotive and protective roles of the composite SAN ECM and provide valuable insights for pacemaking tissue engineering.

Abstract Many cell types migrate in response to naturally generated electric fields. Furthermore, it has been suggested that the external application of an electric field may be used to intervene in and optimize natural processes such as wound healing. Precise cell guidance suitable for such optimization may rely on predictive models of cell migration, which do not generalize. Here, we present a machine learning model that can forecast directedness of cell migration given a timeseries of previous directedness and electric field values. This model is trained using time series galvanotaxis data of mammalian cranial neural crest cells obtained through time-lapse microscopy of cells cultured at 37 °C in a galvanotaxis chamber at ambient pressure. Next, we show that our modeling approach can be used for a variety of cell types and experimental conditions with very limited training data using transfer learning methods. We adapt the model to predict cell behavior for keratocytes (room temperature, ~ 18–20 °C) and keratinocytes (37 °C) under similar experimental conditions with a small dataset (~ 2–5 cells). Finally, this model can be used to perform in silico studies by simulating cell migration lines under time-varying and unseen electric fields. We demonstrate this by simulating feedback control on cell migration using a proportional–integral–derivative (PID) controller. This data-driven approach provides predictive models of cell migration that may be suitable for designing electric field based cellular control mechanisms for applications in precision medicine such as wound healing.

Directed cardiomyogenesis from human induced pluripotent stem cells (hiPSCs) has been greatly improved in the last decade but directed differentiation to pacemaking cardiomyocytes (CMs) remains incompletely understood. In this study, we demonstrated that inhibition of NODAL signaling by a specific NODAL inhibitor (SB431542) in the cardiac mesoderm differentiation stage downregulated PITX2c, a transcription factor that is known to inhibit the formation of the sinoatrial node in the left atrium during cardiac development. The resulting hiPSC-CMs were smaller in cell size, expressed higher pro-pacemaking transcription factors, TBX3 and TBX18, and exhibited pacemaking-like electrophysiological characteristics compared to control hiPSC-CMs differentiated from established Wnt-based protocol. The pacemaker-like subtype increased up to 2.4-fold in hiPSC-CMs differentiated with the addition of SB431542 relative to the control. Hence, Nodal inhibition in the cardiac mesoderm stage promoted pacemaker-like CM differentiation from hiPSCs. Improving the yield of human pacemaker-like CMs is a critical first step in the development of functional human cell-based biopacemakers.

The pathogenesis of the Brucella-induced inflammatory response in the bovine placenta is not completely understood. In this study we evaluated the role of the B. abortus Type IV secretion system and the anti-inflammatory factor BtpB in early interactions with bovine placental tissues. Transcription profiles of chorioallantoic membrane (CAM) explants inoculated with wild type (strain 2308), ΔvirB2 or ΔbtpB Brucella abortus were compared by microarray analysis at 4 hours post infection. Transcripts with significant variation (>2 fold change; P

Macrophages can exhibit pro-inflammatory or pro-reparatory functions, contingent upon their specific activation state. This dynamic behavior empowers macrophages to engage in immune reactions and contribute to tissue homeostasis. Understanding the intricate interplay between macrophage motility and activation status provides valuable insights into the complex mechanisms that govern their diverse functions. In a recent study, we developed a classification method based on morphology, which demonstrated that movement characteristics, including speed and displacement, can serve as distinguishing factors for macrophage subtypes. In this study, we develop a deep learning model to explore the potential of classifying macrophage subtypes based solely on raw trajectory patterns. The classification model relies on the time series of x-y coordinates, as well as the distance traveled and net displacement. We begin by investigating the migratory patterns of macrophages to gain a deeper understanding of their behavior. Although this analysis does not directly inform the deep learning model, it serves to highlight the intricate and distinct dynamics exhibited by different macrophage subtypes, which cannot be easily captured by a finite set of motility metrics. Our study uses cell trajectories to classify three macrophage subtypes: M0, M1, and M2. This advancement holds promising implications for the future, as it suggests the possibility of identifying macrophage subtypes without relying on shape analysis. Consequently, it could potentially eliminate the necessity for highquality imaging techniques and provide more robust methods for analyzing inherently blurry images. [ABSTRACT FROM AUTHOR]

Damage to the respiratory epithelium is one of the most critical steps to many life-threatening diseases, such as acute respiratory distress syndrome and chronic obstructive pulmonary disease. The mechanisms underlying repair of the damaged epithelium have not yet been fully elucidated. Here we provide experimental evidence suggesting a novel mechanism for wound repair: endogenous electric currents. It is known that the airway epithelium maintains a voltage difference referred to as the transepithelial potential. Using a noninvasive vibrating probe, we demonstrate that wounds in the epithelium of trachea from rhesus monkeys generate significant outward electric currents. A small slit wound produced an outward current (1.59 µA/cm²), which could be enhanced (nearly doubled) by the ion transport stimulator aminophylline. In addition, inhibiting cystic fibrosis transmembrane conductance regulator (CFTR) with CFTR(Inh)-172 significantly reduced wound currents (0.17 µA/cm²), implicating an important role of ion transporters in wound induced electric potentials. Time-lapse video microscopy showed that applied electric fields (EFs) induced robust directional migration of primary tracheobronchial epithelial cells from rhesus monkeys, towards the cathode, with a threshold of <23 mV/mm. Reversal of the field polarity induced cell migration towards the new cathode. We further demonstrate that application of an EF promoted wound healing in a monolayer wound healing assay. Our results suggest that endogenous electric currents at sites of tracheal epithelial injury may direct cell migration, which could benefit restitution of damaged airway mucosa. Manipulation of ion transport may lead to novel therapeutic approaches to repair damaged respiratory epithelium. [ABSTRACT FROM AUTHOR]

Bacterial flagellins are potent inducers of innate immunity. Three signaling pathways have been implicated in the sensing of flagellins; these involve toll-like receptor 5 (TLR5) and the cytosolic proteins Birc1e/Naip5 and Ipaf. Although the structural basis of TLR5-flagellin interaction is known, little is known about how flagellin enters the host cell cytosol to induce signaling via Birc1e/Naip5 and Ipaf. Here we demonstrate for the first time the translocation of bacterial flagellin into the cytosol of host macrophages by the vacuolar pathogen, Salmonella enterica serotype Typhimurium. Translocation of flagellin into the host cell cytosol was directly demonstrated using β-lactamase reporter constructs. Flagellin translocation required the Salmonella Pathogenicity Island 1 Type III secretion system (SPL-1 T3SS) but not the flagellar T3SS. [ABSTRACT FROM AUTHOR]

Neisseria meningitidis is an important cause of septicaemia and meningitis. To cause disease, the bacterium must acquire essential nutrients for replication in the systemic circulation, while avoiding exclusion by host innate immunity. Here we show that the utilization of carbon sources by N. meningitidis determines its ability to withstand complement-mediated lysis, through the intimate relationship between metabolism and virulence in the bacterium. The gene encoding the lactate permease, IctP, was identified and disrupted. The IctP mutant had a reduced growth rate in cerebrospinal fluid compared with the wild type, and was attenuated during bloodstream infection through loss of resistance against complement-mediated killing. The link between lactate and complement was demonstrated by the restoration of virulence of the IcfP mutant in complement (C3-/-)-deficient animals. The underlying mechanism for attenuation is mediated through the sialic acid biosynthesis pathway, which is directly connected to central carbon metabolism. The findings highlight the intimate relationship between bacterial physiology and resistance to innate immune killing in the meningococcus. [ABSTRACT FROM AUTHOR]

We have identified genes required for competence of Neisseria meningitidis, a naturally transformable human pathogen. Although not comprehensive, our analysis identified competence-defective mutants with transposon insertions in genes not previously implicated in this process in Neisseria. [ABSTRACT FROM AUTHOR]