We investigated the genetic and epigenetic regulation of the UBASH3A gene and its association with early-onset sepsis. Using matched whole blood DNA methylation, gene expression, genotypes, and immune cell counts from the EPIC-HIPC newborn cohort, we report that promoter methylation was negatively correlated (Pearson r = −0.5, P < 2.2 × 10−16) with ontogenetic changes in UBASH3A gene expression and circulating CD3+ T-cell numbers. Higher promoter methylation at birth was associated with lower UBASH3A expression and reduced early-onset sepsis risk (odds ratio, 0.26; P = .015). Genetic variation significantly influenced variations in baseline UBASH3A methylation (132 cis-meQTL, false discovery rate <0.05).

ABSTRACT The MgtC virulence factor is important during the intramacrophage stage in both classical intracellular pathogens, such as Salmonella Typhimurium, and in extracellular bacteria that transiently encounter intracellular environments during infection, such as Pseudomonas aeruginosa . In these different pathogens, mgtC expression is induced in vitro by magnesium ion depletion, a condition reported to mimic the macrophage environment. Here, we developed an unstable GFP reporter system to monitor in real time the transcriptional activation of the P. aeruginosa mgtC promoter. After in vitro validation in magnesium-defined media, this reporter system allowed visualization of the mgtC promoter induction in a subset of bacteria when P. aeruginosa localized inside cultured macrophages. In addition, although rare under our experimental conditions, in vivo activation of the mgtC promoter was observed for the first time within macrophages of live, infected zebrafish larvae, a cutting-edge vertebrate model for real-time imaging. While MgtC regulation in Salmonella is mediated by the magnesium-responsive PhoPQ two-component system, its regulation in P. aeruginosa remained unknown. The use of mutant strains for two-component regulatory systems revealed that the PhoP regulator, but not by its cognate sensor PhoQ, was required to activate P. aeruginosa MgtC expression in vitro . Unexpectedly, CbrAB, a two-component system specific to Pseudomonas species, was also involved in P. aeruginosa MgtC regulation. Both PhoP and CbrB regulatory proteins were found to directly bind the mgtC promoter, supporting a dual transcriptional control. These findings reveal substantial differences in mgtC gene regulation in different bacterial pathogens, reflecting distinct strategies to drive appropriate expression of a shared virulence factor involved in macrophage adaptation. IMPORTANCE The adaptation of bacterial pathogens to the host intracellular microenvironment requires tight and rapid regulation of specific genes, and investigating the in vivo transcriptional dynamics of such genes is a major challenge. Here, we focused on the expression of mgtC , a gene important for adaptation to the intramacrophage environment in classical intracellular pathogens, such as Salmonella Typhimurium, and bacteria with a transient intracellular lifestyle, such as Pseudomonas aeruginosa . An unstable GFP reporter system was designed to monitor the transcriptional dynamics of P. aeruginosa mgtC . The use of this reporter system in a state-of-the-art vertebrate model for live imaging, the zebrafish embryo, allowed in vivo tracking of P. aeruginosa mgtC promoter activation inside macrophages in a living host. Furthermore, the expression of P. aeruginosa mgtC was found to be regulated through a mechanism distinct from that of Salmonella MgtC, since it involves the PhoP regulatory protein, but not the PhoQ sensor, and the Pseudomonas -specific CbrAB two-component system, reflecting diverse, finely tuned strategies to control a virulence factor shared by several major human pathogens.

Increasing concerns regarding prolonged antibiotic usage have spurred the search for alternative treatments. Antimicrobial peptides (AMPs), first discovered in the 1980s, have exhibited significant potential against a broad range of bacteria. Short-sequenced AMPs are abundant in nature and present across various organisms. Recently, machine learning technologies, such as Quantitative Structure-Activity Relationships (QSAR), have enabled the expedited discovery of potential AMPs with broad-spectrum antibacterial activity as the amount of available AMP training data increases. Among these, Deep QSAR has recently emerged as a distinct type of application that utilizes conventional molecular descriptors in conjunction with more powerful deep learning (DL) models. Here, we demonstrate the power of Deep QSAR in predicting broad-spectrum AMP activity. Using a recurrent neural network-based QSAR model, we achieved nearly 90% 5-fold cross-validated accuracy in classifying AMP activity. Using the developed approach, we designed 100 novel peptides, of which 44 experimentally demonstrated more effective antibiofilm activity, and 31 peptides exhibited stronger antimicrobial activity compared to the well-characterized host defense peptide IDR-1018, which was demonstrated to possess broad-spectrum antibiofilm activity against a wide range of bacterial pathogens. . Additionally, a previous computer-aided peptide design study employing IDR-1018 derivatives successfully identified novel peptides with enhanced antibiofilm activity. Notably, 29 of these peptides demonstrated improvements of both antimicrobial and, particularly, antibiofilm properties, making them suitable prototypes for preclinical development and demonstrating the efficacy of DeepQSAR modeling in identifying novel and potent AMPs.

Staphylococcus aureus relies on the (p)ppGpp-mediated stringent response to adapt to nutrient limitation and other environmental stress, with important consequences for metabolism, antibiotic tolerance, and virulence. In this study, we identified that disruption of (p)ppGpp synthesis altered the small RNA (sRNA) cargo of extracellular vesicles (EVs) produced by S. aureus. We characterized EV-associated sRNAs from wild-type and stringent response mutant (rshsyn) strains, revealing the presence of SprX2 in mutant EVs. The rshsyn mutant also showed modestly enhanced survival under high concentrations of vancomycin and oxacillin in late stationary phase. Transcriptomic analysis under mupirocin-induced amino acid limitation demonstrated broad ppGpp-dependent remodeling of gene expression, including changes in amino acid biosynthesis, translation-associated functions, adhesion factors, and virulence-associated genes. Treatment with the synthetic cationic peptide DJK-5 reduced intracellular ppGpp levels, suppressed the expression of genes involved in adhesion (fnbA, fnbB, clfB, emp), cytolytic toxins (psmβ1, psmβ2, hla, lukED), and exoenzymes (lip, geh, sspA, aur, sspB), and reduced methicillin-resistant S. aureus (MRSA)-mediated toxicity toward eukaryotic cells. These findings suggest that targeting the stringent response impairs both intracellular regulatory networks and EV-mediated signaling, offering a promising approach to attenuate S. aureus virulence and enhance efficacy against resistant infections.IMPORTANCEThe stringent response helps Staphylococcus aureus survive nutrient stress, adapt its metabolism, and regulate virulence. This study shows that disrupting this pathway not only alters intracellular gene expression but is also associated with changes in the small RNA (sRNA) cargo of extracellular vesicles. In addition, the synthetic peptide DJK-5 weakens the pathogen's ability to regulate biofilm formation, its virulence, and reduces its communication through small RNA molecules, which are critical for stress adaptation and infection. These findings introduce an approach to weakening bacterial defenses and suggest that interfering with stress responses could enhance the effectiveness of existing antibiotics against antibiotic-resistant bacteria, offering a promising strategy for combating bacterial infections and reducing antibiotic resistance.

Although myriads of potential antiviral agents have been tested against SARS-CoV-2, only a handful have proven to be effective in clinical trials. During the COVID-19 pandemic, many known or novel peptides were evaluated for their ability to inhibit SARS-CoV-2 replication; however, testing of D-enantiomers that resist body and viral proteases has been limited. Here, we characterized the ability of D-3006, a D-enantiomeric synthetic host defense peptide, to inhibit SARS-CoV-2 replication in vitro. A battery of authentic SARS-CoV-2 variants (ancestral, Mu, Delta, and Omicron BA.1) and a comprehensive panel of β-coronavirus spike pseudotyped lentiviruses were used to demonstrate that D-3006 safely (CC50value = 430 µg/mL) blocked spike-mediated entry (EC50 values ranging from 1.57 to 5.37 µg/mL) and also had synergistic anti-SARS-CoV-2 activity in vitro when combined with the viral polymerase inhibitor remdesivir. We also showed that D-3006 inhibited influenza A virus (H1N1) replication in vitro, suggesting that this synthetic host defense peptide could have potential broad antiviral activity against multiple enveloped viruses. These data, together with negative-stain transmission electron microscopy analysis, suggest that the mechanism of action of D-3006 is associated with non-specific binding to the viral membrane, most likely causing virus aggregation and interfering with virus attachment and entry. The potential broad-spectrum antiviral activity of D-3006, its innate resistance to host proteases, as well as the possibility of being used in combination with other antiviral drugs suggest that this host synthetic peptide could be developed as a candidate for the treatment of SARS-CoV-2 and/or other respiratory viral infections.

Intracellular pathogens such as Mycobacterium tuberculosis (Mtb) evade host defence mechanisms to infect and survive within host cells. Host-directed therapy (HDT) offers a promising alternative to antibiotics and may overcome antimicrobial resistance. Using high-content screening, we identified benztropine (BZT), an approved Parkinson’s disease drug, as a potent inhibitor of intracellular Mtb. BZT is active in both human and murine macrophages but is inactive in broth. In an aerosol Mtb mouse infection model, oral administration of BZT reduced the burden of Mtb in the lungs by up to 70%. BZT was also active against Salmonella enterica serovar Typhimurium (STm) in an abscess model of infection, significantly reducing size and bacterial load. Chemical competition assays, CRISPR knockouts, and siRNA silencing assays revealed that BZT’s activity against Mtb is mediated via macrophage histamine receptor 1 (HRH1). Our findings establish BZT as a promising repurposed candidate and a lead compound for developing HRH1-targeting antibacterial HDTs.

Sepsis is a life-threatening organ dysfunction due to a dysfunctional response to infection. Delays in diagnosis have substantial impact on survival. Herein, blood samples from 586 in-house patients with suspected sepsis are used in conjunction with machine learning and cross-validation to define a six-gene expression signature of immune cell reprogramming, termed Sepset, to predict clinical deterioration within the first 24 h (h) of clinical presentation. Prediction accuracy (~90% in early intensive care unit (ICU) and 70% in emergency room patients) is validated in 3178 patients from existing independent cohorts. A RT-PCR-based Sepset detection test shows a 94% sensitivity in 248 patients to predict worsening of the sequential organ failure assessment scores within the first 24 h. A stand-alone centrifugal microfluidic instrument that automates whole-blood Sepset classifier detection is tested, showing a sensitivity of 92%, and specificity of 89% in identifying the risk of clinical deterioration in patients with suspected sepsis.

Sepsis is an abnormal, life-threatening response to infection that leads to (multi-)organ dysfunction and failure. It causes ~20% of deaths worldwide each year, and most deaths related to severe COVID-19 share various molecular features with sepsis. Current treatment approaches (antimicrobials and supportive care) do not address the complexity of sepsis or its mechanistic heterogeneity between and within patients over time. Systems immunology methods, including multiomics (notably RNA sequencing transcriptomics), machine learning, and network biology analysis, have the potential to transform the management paradigm toward precision approaches. Immune dysfunctions evident very early in sepsis drive the development of novel diagnostic gene expression signatures (e.g., cellular reprogramming) that could inform early therapy. Sepsis patients can now be categorized into “endotypes” based on unique immune dysfunction mechanisms corresponding to varying severity and mortality rates, raising the prospect of endotype-specific diagnostics and patient-specific immune-directed therapy. Longitudinal within-patient analyses can also reveal mechanisms (including epigenetics) that drive differential sepsis trajectories over time, enabling the prospect of disease stage-specific therapy during and after hospitalization, including for post-sepsis and long COVID syndromes. Achieving this transformation will require addressing barriers to systems immunology research, including its cost and resource-intensiveness, the relatively low volume of available data, and lack of suitable animal models; it will also require a change in the mindset of healthcare providers toward precision approaches. This should be prioritized in multistakeholder collaborations involving research communities, healthcare providers/systems, patients, and governments to reduce the current high disease burden from sepsis and to mitigate against future pandemics.

Sepsis is a life-threatening reaction to an infection in which the immune system, which usually helps fight infections, reacts abnormally and can cause organs to stop working. About 20% of deaths worldwide are attributed to sepsis—more than any type of heart disease or cancer. Sepsis can be difficult for doctors to recognize because symptoms start out similar to many other medical conditions, and it is hard to treat because the bodily “malfunctions” that cause sepsis vary between patients. To recognize sepsis early and understand differences between patients, researchers are looking at many parts of the immune system at once, collecting lots of data on patients’ genes and proteins. Computers are used to analyze the data, to identify unique patterns or connections. By doing so, scientists have identified unique groups of sepsis patients that differ in their immune responses. This knowledge can help doctors choose the best treatment for each person and might even help protect people from severe COVID-19 or future pandemics.

Chronic infections represent a significant global health and economic challenge. Biofilms, which are bacterial communities encased in an extracellular polysaccharide matrix, contribute to approximately 80% of these infections. In particular, pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus are frequently co-isolated from the sputum of patients with cystic fibrosis and are commonly found in chronic wound infections. Within biofilms, bacteria demonstrate a remarkable increase in resistance and tolerance to antimicrobial treatment. We investigated the efficacy of combining the last-line antibiotic colistin with a membrane- and stringent stress response-targeting anti-biofilm peptide DJK-5 against co-biofilms comprised of multidrug-resistant P. aeruginosa and methicillin-resistant S. aureus (MRSA). Colistin lacks canonical activity against S. aureus. However, our study revealed that under co-biofilm conditions, the antibiofilm peptide DJK-5 synergized with colistin against S. aureus. Similar enhancement was observed when daptomycin, a cyclic lipopeptide against Gram-positive bacteria, was combined with DJK-5, resulting in increased activity against P. aeruginosa. The combinatorial treatment induced morphological changes in both P. aeruginosa and S. aureus cell shape and size within co-biofilms. Importantly, our findings also demonstrate synergistic activity against both P. aeruginosa and S. aureus in a murine subcutaneous biofilm-like abscess model. In conclusion, combinatorial treatments with colistin or daptomycin and the anti-biofilm peptide DJK-5 show significant potential for targeting co-biofilm infections. These findings offer promising avenues for developing new therapeutic approaches to combat complex chronic infections.

Bioactive peptides such as anticancer peptides (ACPs) offer a promising therapeutic alternative to small molecules due to their efficiency and selectivity against tumors and minimal toxicity towards healthy human cells. However, their rational discovery requires navigating a vast chemical space using computationally demanding in silico tools. Herein, we present a computational method enabling cost-efficient exploration of large peptide libraries using reinforcement learning and posterior sampling. Practical application of the developed approach results in identification of membranolytic peptides with therapeutic potential. The developed computational method reduces the search space by over 90% compared to exhaustive library screening and enables effective balancing between dataset's exploration and exploitation. We demonstrate the scalability of this method by screening a focused library of 36 million structurally resolved helical peptides curated from the Protein Data Bank. When screened in in vitro assays, 15 of the top 100 selected candidates exhibit cytotoxic activity against breast cancer cells including drug resistant triple-negative breast cancer, with the three lead compounds further characterizing as non-toxic towards healthy human cells. This study highlights the potential of using deep reinforcement learning to expedite bioactive peptide discovery, offering a promising path for developing new peptide-based cancer therapies.

Syphilis, caused by Treponema pallidum subsp. pallidum, is an urgent global public health threat. Syphilis vaccine development has been impeded by limited understanding of the molecular mechanisms that enable T. pallidum to establish and maintain infection. The vascular endothelium is critical for T. pallidum attachment, dissemination, and host immune response initiation; however, the molecular details of T. pallidum-endothelial interactions are incompletely understood. To enhance understanding, we performed time-course transcriptomic profiling on T. pallidum-exposed brain microvascular endothelial cells. These analyses showed T. pallidum exposure altered pathways related to extracellular matrix, growth factors, integrins, and Rho GTPases. The induced transcriptional response was consistent with endothelial to mesenchymal transition, a process involved in fetal development and vascular dysfunction. In cells exposed to T. pallidum, the primary transcription factor associated with this process (Snail) was increased at both the transcript and protein levels, and microscopy analyses demonstrate F-actin cellular contraction. This study provides a comprehensive understanding of the molecular responses of endothelial cells to T. pallidum and identified the host pathways that might cause syphilis disease symptoms, information that could aid in syphilis vaccine design.

The emergence of antibiotic resistance, biofilm formation, and internalization by host cells contribute to a high risk of chronic infections, highlighting the necessity to develop novel therapeutic strategies. Identification of natural host defense peptides (HDPs) with promising antimicrobial and antibiofilm activities led to the development of synthetic peptides with broad-spectrum efficacy. However, few studies have examined their effect on anaerobic bacterial species. This study aimed to test the effect of synthetic HDPs on Cutibacterium acnes, an anaerobe species involved in 10% of prosthesis joint infections (PJI). A preliminary screen identified three peptides (DJK5, AB009-D, and AB101-D) with promising activity against four C. acnes strains (two of which were isolated from PJI). A bactericidal effect was observed for the three peptides with 50% of planktonic bacteria killing for AB009-D and AB101-D after only 3 hours of contact. DJK5 and AB009-D inhibited the C. acnes adhesion on plastic and titanium supports with a 2-log decrease in bacterial cells. In the presence of peptides, the morphology of C. acnes cells was altered with an increase in cell length observed, especially for one of the non-PJI-related strains. Against mature biofilms, AB101-D was the most effective with an approximate 2-log decrease in adhered CFUs, indicating the induction of bacterial dispersion or death. DJK5 also inhibited C. acnes internalization by osteoblasts, with a reduction of the internalized bacteria quantity for three strains. Overall, this study demonstrates that synthetic HDPs are effective against anaerobic bacteria and hold promise as novel therapeutic candidates to prevent or treat C. acnes PJIs.IMPORTANCEThe emergence of antibiotic tolerance highlights the necessity to develop novel therapeutic strategies with promising antimicrobial but also antibiofilm activities. In this study, we tested the effect of synthetic host defense peptides (HDPs) on Cutibacterium acnes, an anaerobic species, rarely studied, whereas involved in 10% of prosthesis joint infections (PJI). In our study, we demonstrate that the selected synthetic HDPs are effective against this anaerobic bacteria, both as a preventive treatment (effect on planktonic growth, bacterial adhesion, and biofilm formation) and against internalization of C. acnes by osteoblasts, revealing that these peptides are promising as novel therapeutic candidates to prevent or treat C. acnes PJIs.

Antimicrobial peptides have emerged as a potential alternative to traditional small-molecule antimicrobials. They possess broad-spectrum efficacy and increasingly confront the challenges of bacterial resistance, especially the adaptive resistance of biofilms. However, advanced rational peptide design methods are still required to ensure optimal property profiles of such peptides, while limiting the cost of their synthesis and screening. Here, we present a computational pipeline for the rational de novo design of antimicrobial and antibiofilm peptides based on an explainable artificial intelligence (XAI) framework. The developed framework combines a Wasserstein Autoencoder (WAE) and a nonlinear dimensionality reduction method─generative topographic mapping (GTM). The WAE was used to learn the latent representation of the peptide space, while the GTM guided the generation of novel AMPs through an illustrative depiction of the latent space in the form of 2D maps. The generated peptides were subjected to screening by machine learning models, resulting in the final hit list based on their predicted activity. The efficacy of the peptides generated with the developed pipeline was experimentally verified by synthesis and testing for activity against methicillin-resistant Staphylococcus aureus (MRSA), achieving a 100% hit rate in targeting biofilms. Notably, the most potent antibiofilm peptide developed in this study demonstrated almost one order of magnitude improvement in IC50 value compared with the potent antibiofilm peptide reference "1018", used as a positive control. The developed pipeline is readily extendable for the optimization of additional peptide properties, including cytotoxicity, tendency to aggregate, and proteolytic stability, underscoring its potential utility for rational design of the peptide-based therapeutics.

Persistent bacterial infections evade host immunity and resist antibiotic treatments through various mechanisms that are difficult to evaluate in a living host. Pseudomonas aeruginosa is a main cause of chronic infections in patients with cystic fibrosis (CF) and wounds. Here, by immersing wounded zebrafish embryos in a suspension of P. aeruginosa isolates from CF patients, we established a model of persistent infection that mimics a murine chronic skin infection model. Live and electron microscopy revealed persisting aggregated P. aeruginosa inside zebrafish cells, including macrophages, at unprecedented resolution. Persistent P. aeruginosa exhibited adaptive resistance to several antibiotics, host cell permeable drugs being the most efficient. Moreover, persistent bacteria could be partly re-sensitized to antibiotics upon addition of anti-biofilm molecules that dispersed the bacterial aggregates in vivo. Collectively, this study demonstrates that an intracellular location protects persistent P. aeruginosa in vivo in wounded zebrafish embryos from host innate immunity and antibiotics, and provides new insights into efficient treatments against chronic infections.

Understanding of newborn immune ontogeny in the first week of life will enable age-appropriate strategies for safeguarding vulnerable newborns against infectious diseases. Here we conducted an observational study exploring the immunological profile of infants longitudinally throughout their first week of life. Our Expanded Program on Immunization - Human Immunology Project Consortium (EPIC-HIPC) studies the epigenetic regulation of systemic immunity using small volumes of peripheral blood samples collected from West African neonates on days of life (DOL) 0, 1, 3, and 7. Genome-wide DNA methylation and single nucleotide polymorphism markers are examined alongside matched transcriptomic and flow cytometric data. Integrative analysis reveals that a core network of transcription factors mediates dynamic shifts in neutrophil-to-lymphocyte ratios (NLR), which are underpinned by cell-type specific methylation patterns in the two cell types. Genetic variants are associated with lower NLRs at birth, and healthy newborns with lower NLRs at birth are more likely to subsequently develop sepsis. These findings provide valuable insights into the early-life determinants of immune system development.

Innate defense regulator-1002 (IDR-1002) is a synthetic peptide with promising immunomodulatory and antibiofilm properties. An appreciable body of work exists around its mechanism of action at the cellular and molecular level, along with its efficacy across several infection and inflammation models. However, little is known about its absorption, distribution, and excretion in live organisms. Here, we performed a comprehensive biodistribution assessment with a gallium-67 radiolabeled derivative of IDR-1002 using nuclear tracing techniques. Various dose levels of the radiotracer (2-40 mg/kg) were administered into the blood, peritoneal cavity, and subcutaneous tissue, or instilled into the lungs. The peptide was well tolerated at all subcutaneous and intraperitoneal doses, although higher levels were associated with delayed absorption kinetics and precipitation of the peptide within the tissues. Low intratracheal doses were rapidly absorbed systemically, and small increases in the dose level were lethal. Intravenous doses were rapidly cleared from the blood at lower levels, and upon escalation, were toxic with a high proportion of the dose accumulating within the lung tissue. To improve biocompatibility and prolong its circulation within the blood, IDR-1002 was further formulated onto high molecular weight hyperbranched polyglycerol (HPG) polymers. Constructs prepared at 5:1 and 10:1 peptide-to-polymer ratios were colloidally stable, maintained the biological profile of the peptide payload and helped reduce red blood cell lysis. The 5:1 construct circulated well in the blood, but higher peptide loading was associated with rapid clearance by the reticuloendothelial system. Many peptides face pharmacokinetic and biocompatibility challenges, but formulations such as those with HPG have the potential to overcome these limitations.

Importance Appendicitis is the most common indication for urgent surgery in the pediatric population, presenting across a range of severity and with variable complications. Differentiating simple appendicitis (SA) and perforated appendicitis (PA) on presentation may help direct further diagnostic workup and appropriate therapy selection, including antibiotic choice and timing of surgery. Objective To provide a mechanistic understanding of the differences in disease severity of appendicitis with the objective of developing improved diagnostics and treatments, specifically for the pediatric population. Design, Setting, and Participants The Gene Expression Profiling of Pediatric Appendicitis (GEPPA) study was a single-center prospective exploratory diagnostic study with transcriptomic profiling of peripheral blood collected from a cohort of children aged 5 to 17 years with abdominal pain and suspected appendicitis between November 2016 and April 2017 at the Alberta Children’s Hospital in Calgary, Alberta, Canada, with data analysis reported in August 2023. There was no patient follow-up in this study. Exposure SA, PA, or nonappendicitis abdominal pain. Main Outcomes and Measures Blood transcriptomics was used to develop a hypothesis of underlying mechanistic differences between SA and PA to build mechanistic hypotheses and blood-based diagnostics. Results Seventy-one children (mean [SD] age, 11.8 [3.0] years; 48 [67.6%] male) presenting to the emergency department with abdominal pain and suspected appendicitis were investigated using whole-blood transcriptomics. A central role for immune system pathways was revealed in PA, including a dampening of major innate interferon responses. Gene expression changes in patients with PA were consistent with downregulation of immune response and inflammation pathways and shared similarities with gene expression signatures derived from patients with sepsis, including the most severe sepsis endotypes. Despite the challenges in identifying early biomarkers of severe appendicitis, a 4-gene signature that was predictive of PA compared to SA, with an accuracy of 85.7% (95% CI, 72.8-94.1) was identified. Conclusions This study found that PA was complicated by a dysregulated immune response. This finding should inform improved diagnostics of severity, early management strategies, and prevention of further postsurgical complications.

The development of atopic dermatitis (AD) in infancy, and subsequent allergic rhinitis, food allergies, and asthma in later childhood, is known as the atopic march. The mechanism is largely unknown, yet the course of disease indicates the contribution of inter-epithelial crosstalk, through to the onset of inflammation in the skin and progression to another mucosal epithelium. Here, we investigated if and how skin-lung epithelial crosstalk could contribute to the development of the atopic march. First, we emulated this inter-epithelial crosstalk through indirect co-culture of bio-engineered atopic-like skin disease models and three-dimensional bronchial epithelial models trig-gering an asthma-like phenotype in the latter. A subsequent secretome analysis identified thrombos-pondin-1, CD44, complement factor C3, fibronectin, and syndecan-4 as potentially relevant skin-derived mediators. As these mediators are extracellular matrix (ECM)-related proteins, we then stud-ied the involvement of the ECM, unveiling distinct proteomic, transcriptomic, and ultrastructural dif-ferences in atopic samples. The latter indicated ECM remodeling triggering the release of the above-mentioned mediators. In addition to pro-inflammatory effects in lung tissue, the ECM mediators also exert distinct effects on CD4+ T cells. In vivo mouse data showed that exposure to these mediators over seven days dysregulated activated circadian clock genes which have been previously dis-cussed in the context of atopic diseases and asthma development. We hypothesize the existence of a skin-lung axis that could contribute to the atopic march driven by skin ECM remodeling.