Sensory neurons promote immune homeostasis in the lung.

Cytokines employ downstream Janus kinases (JAKs) to promote chronic inflammatory diseases. JAK1-dependent type 2 cytokines drive allergic inflammation, and patients with JAK1 gain-of-function (GoF) variants develop atopic dermatitis (AD) and asthma. To explore tissue-specific functions, we inserted a human JAK1 GoF variant (JAK1GoF) into mice and observed the development of spontaneous AD-like skin disease but unexpected resistance to lung inflammation when JAK1GoF expression was restricted to the stroma. We identified a previously unrecognized role for JAK1 in vagal sensory neurons in suppressing airway inflammation. Additionally, expression of Calcb/CGRPß was dependent on JAK1 in the vagus nerve, and CGRPß suppressed group 2 innate lymphoid cell function and allergic airway inflammation. Our findings reveal evolutionarily conserved but distinct functions of JAK1 in sensory neurons across tissues. This biology raises the possibility that therapeutic JAK inhibitors may be further optimized for tissue-specific efficacy to enhance precision medicine in the future.

Patrolling Alveolar Macrophages Conceal Bacteria from the Immune System to Maintain Homeostasis.

During respiration, humans breathe in more than 10,000 liters of non-sterile air daily, allowing some pathogens access to alveoli. Interestingly, alveoli outnumber alveolar macrophages (AMs), which favors alveoli devoid of AMs. If AMs, like most tissue macrophages, are sessile, then this numerical advantage would be exploited by pathogens unless neutrophils from the blood stream intervened. However, this would translate to omnipresent persistent inflammation. Developing in vivo real-time intravital imaging of alveoli revealed AMs crawling in and between alveoli using the pores of Kohn. Importantly, these macrophages sensed, chemotaxed, and, with high efficiency, phagocytosed inhaled bacterial pathogens such as P. aeruginosa and S. aureus, cloaking the bacteria from neutrophils. Impairing AM chemotaxis toward bacteria induced superfluous neutrophil recruitment, leading to inappropriate inflammation and injury. In a disease context, influenza A virus infection impaired AM crawling via the type II interferon signaling pathway, and this greatly increased secondary bacterial co-infection.

E3 Ligase VHL Promotes Group 2 Innate Lymphoid Cell Maturation and Function via Glycolysis Inhibition and Induction of Interleukin-33 Receptor.

Group 2 innate lymphoid cells (ILC2s) are a specialized subset of lymphoid effector cells that are critically involved in allergic responses; however, the mechanisms of their regulation remain unclear. We report that conditional deletion of the E3 ubiquitin ligase VHL in innate lymphoid progenitors minimally affected early-stage bone marrow ILC2s but caused a selective and intrinsic decrease in mature ILC2 numbers in peripheral non-lymphoid tissues, resulting in reduced type 2 immune responses. VHL deficiency caused the accumulation of hypoxia-inducible factor 1α (HIF1α) and attenuated interleukin-33 (IL-33) receptor ST2 expression, which was rectified by HIF1α ablation or inhibition. HIF1α-driven expression of the glycolytic enzyme pyruvate kinase M2 downmodulated ST2 expression via epigenetic modification and inhibited IL-33-induced ILC2 development. Our study indicates that the VHL-HIF-glycolysis axis is essential for the late-stage maturation and function of ILC2s via targeting IL-33-ST2 pathway.

c-Maf enforces cytokine production and promotes memory-like responses in mouse and human type 2 innate lymphoid cells.

Group-2 innate lymphoid cells (ILC2s), which are involved in type 2 inflammatory diseases such as allergy, can exhibit immunological memory, but the basis of this ILC2 "trained immunity" has remained unclear. Here, we found that stimulation with IL-33/IL-25 or exposure to the allergen papain induces the expression of the transcription factor c-Maf in mouse ILC2s. Chronic papain exposure results in high production of IL-5 and IL-13 cytokines and lung eosinophil recruitment, effects that are blocked by c-Maf deletion in ILCs. Transcriptomic analysis revealed that knockdown of c-Maf in ILC2s suppresses expression of type 2 cytokine genes, as well as of genes linked to a memory-like phenotype. Consistently, c-Maf was found highly expressed in human adult ILC2s but absent in cord blood and required for cytokine production in isolated human ILC2s. Furthermore, c-Maf-deficient mouse or human ILC2s failed to exhibit strengthened ("trained") responses upon repeated challenge. Thus, the expression of c-Maf is indispensable for optimal type 2 cytokine production and proper memory-like responses in group-2 innate lymphoid cells.

The breathtaking world of human respiratory in vitro models: Investigating lung diseases and infections in 3D models, organoids, and lung-on-chip.

The COVID-19 pandemic illustrated an urgent need for sophisticated, human tissue models to rapidly test and develop effective treatment options against this newly emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Thus, in particular, the last 3 years faced an extensive boost in respiratory and pulmonary model development. Nowadays, 3D models, organoids and lung-on-chip, respiratory models in perfusion, or precision-cut lung slices are used to study complex research questions in human primary cells. These models provide physiologically relevant systems for studying SARS-CoV-2 and, of course, other respiratory pathogens, but they are, too, suited for studying lung pathologies, such as CF, chronic obstructive pulmonary disease, or asthma, in more detail in terms of viral infection. With these models, the cornerstone has been laid for further advancing the organs by, for example, inclusion of several immune cell types or humoral immune components, combination with other organs in microfluidic organ-on-chip devices, standardization and harmonization of the devices for reliable and reproducible drug and vaccine testing in high throughput.

Sustained innate interferon is an essential inducer of tertiary lymphoid structures.

Tertiary lymphoid structures (TLS) resemble follicles of secondary lymphoid organs and develop in nonlymphoid tissues during inflammation and cancer. Which cell types and signals drive the development of TLS is largely unknown. To investigate early events of TLS development in the lungs, we repeatedly instilled p(I:C) plus ovalbumin (Ova) intranasally. This induced TLS ranging from lymphocytic aggregates to organized and functional structures containing germinal centers. We found that TLS development is independent of FAP+ fibroblasts, alveolar macrophages, or CCL19 but crucially depends on type I interferon (IFN-I). Mechanistically, IFN-I initiates two synergistic pathways that culminate in the development of TLS. On the one hand, IFN-I induces lymphotoxin (LT)α in lymphoid cells, which stimulate stromal cells to produce the B-cell-attracting chemokine CXCL13 through LTßR-signaling. On the other hand, IFN-I is sensed by stromal cells that produce the T-cell-attracting chemokines CXCL9, CXCL10 as well as CCL19 and CCL21 independently of LTßR. Consequently, B-cell aggregates develop within a week, whereas follicular dendritic cells and germinal centers appear after 3 weeks. Thus, sustained production of IFN-I together with an antigen is essential for the induction of functional TLS in the lungs.

Regnase-1 D141N mutation induces CD4+ T cell-mediated lung granuloma formation via upregulation of Pim2.

Regnase-1 is an RNase that plays a critical role in negatively regulating immune responses by destabilizing inflammatory messenger RNAs (mRNAs). Dysfunction of Regnase-1 can be a major cause of various inflammatory diseases with tissue injury and immune cell infiltration into organs. This study focuses on the role of the RNase activity of Regnase-1 in developing inflammatory diseases. We have constructed mice with a single point mutation at the catalytic center of the Regnase-1 RNase domain, which lacks endonuclease activity. D141N mutant mice demonstrated systemic inflammation, immune cell infiltration into various organs, and progressive development of lung granuloma. CD4+ T cells, mainly affected by this mutation, upregulated the mTORC1 pathway and facilitated the autoimmune trait in the D141N mutation. Moreover, serine/threonine kinase Pim2 contributed to lung inflammation in this mutation. Inhibition of Pim2 kinase activity ameliorated granulomatous inflammation, immune cell infiltration, and proliferation in the lungs. Additionally, Pim2 inhibition reduced the expression of adhesion molecules on CD4+ T cells, suggesting a role for Pim2 in facilitating leukocyte adhesion and migration to inflamed tissues. Our findings provide new insights into the role of Regnase-1 RNase activity in controlling immune functions and underscore the therapeutic relevance of targeting Pim2 to modulate abnormal immune responses.

Microbial intestinal dysbiosis drives long-term allergic susceptibility by sculpting an ILC2-B1 cell-innate IgE axis.

BACKGROUND: The abundance and diversity of intestinal commensal bacteria influence systemic immunity with impact on disease susceptibility and severity. For example, loss of short chain fatty acid (SCFA)-fermenting bacteria in early life (humans and mice) is associated with enhanced type 2 immune responses in peripheral tissues including the lung. OBJECTIVE: Our goal was to reveal the microbiome-dependent cellular and molecular mechanisms driving enhanced susceptibility to type 2 allergic lung disease. METHODS: We used low-dose vancomycin to selectively deplete SCFA-fermenting bacteria in wild-type mice. We then examined the frequency and activation status of innate and adaptive immune cell lineages with and without SCFA supplementation. Finally, we used ILC2-deficient and signal transducer and activator of transcription 6 (STAT6)-deficient transgenic mouse strains to delineate the cellular and cytokine pathways leading to enhanced allergic disease susceptibility. RESULTS: Mice with vancomycin-induced dysbiosis exhibited a 2-fold increase in lung ILC2 primed to produce elevated levels of IL-2, -5, and -13. In addition, upon IL-33 inhalation, mouse lung ILC2 displayed a novel ability to produce high levels of IL-4. These expanded and primed ILC2s drove B1 cell expansion and IL-4-dependent production of IgE that in turn led to exacerbated allergic inflammation. Importantly, these enhanced lung inflammatory phenotypes in mice with vancomycin-induced dysbiosis were reversed by administration of dietary SCFA (specifically butyrate). CONCLUSION: SCFAs regulate an ILC2-B1 cell-IgE axis. Early-life administration of vancomycin, an antibiotic known to deplete SCFA-fermenting gut bacteria, primes and amplifies this axis and leads to lifelong enhanced susceptibility to type 2 allergic lung disease.

Interstitial Macrophages Mediate Efferocytosis of Alveolar Epithelium during Influenza Infection.

Efferocytosis is a process whereby apoptotic cells are cleared to maintain tissue homeostasis. In the lungs, efferocytosis has been implicated in several acute and chronic inflammatory diseases. A long-standing method to study efferocytosis in vivo is to instill apoptotic cells into the lungs to evaluate macrophage uptake. However, this approach provides nonphysiologic levels of cells to the airspaces, where there is preferential access to the alveolar macrophages. To circumvent this limitation, we developed a new method to study efferocytosis of damaged alveolar type 2 (AT2) epithelial cells in vivo. A reporter mouse that expresses TdTomato in AT2 epithelial cells was injured with influenza (strain PR8) to induce apoptosis of AT2 cells. We were able to identify macrophages that acquire red fluorescence after influenza injury, indicating efferocytosis of AT2 cells. Furthermore, evaluation of macrophage populations led to the surprising finding that lung interstitial macrophages were the primary efferocyte in vivo. In summary, we present a novel finding that the interstitial macrophage, not the alveolar macrophage, primarily mediates clearance of AT2 cells in the lungs after influenza infection. Our method of studying efferocytosis provides a more physiologic approach in evaluating the spatiotemporal dynamics of apoptotic cell clearance in vivo and opens new avenues to study the mechanisms by which efferocytosis regulates inflammation.

Endothelialized Bronchioalveolar Lung Organoids Model Endothelial Cell Responses to Injury.

Organoid 3D systems are powerful platforms to study development and disease. Recently, the complexity of lung organoid models derived from adult mouse and human stem cells has increased substantially in terms of cellular composition and structural complexity. However, a murine lung organoid system with a clear integrated endothelial compartment is still missing. Here, we describe a novel method that adds another level of intricacy to our published bronchioalveolar lung organoid (BALO) model by microinjection of FACS-sorted lung endothelial cells (ECs) into differentiated organoid cultures. Before microinjection, ECs obtained from the lung homogenate (LH) of young mice expressed typical ECs markers such as CD31 and vascular endothelial (VE)-Cadherin and showed tube formation capacity. Following microinjection, ECs surrounded BALO´s alveolar-like compartment aligning with both alveolar epithelial cells type I (AECI) and type II (AECII), as demonstrated by confocal and electron microscopy. Notably, expression of Car4 and Aplnr was as well detected, suggesting presence of EC microvascular phenotypes in the cultured ECs. Moreover, upon epithelial cell injury by lipopolysaccharides (LPS) and influenza A virus (IV), endothelialized BALO (eBALO) released proinflammatory cytokines leading to the upregulation of the intercellular adhesion molecule 1 (ICAM-1) in ECs. In summary, we characterized for the first time a organoid model that incorporates ECs into the alveolar structures of lung organoids, not only increasing our previous model ́s cellular and structural complexity but also providing a suitable niche to model lung endothelium responses to injury ex vivo.

Cell-Specific Contribution of IL4 Receptor α Signaling Shapes the Overall Manifestation of Allergic Airway Disease.

IL-4 and IL-13 play a critical role in allergic asthma pathogenesis via their common receptor, i.e., IL4Rα. However, the cell-specific role of IL4Rα in mixed allergens (MA)-induced allergic asthma has remained unclear. Therefore, we aimed to identify the cell-specific contribution of IL4Rα signaling in the manifestation of various pathological outcomes in mice with allergic airway disease. We compared MA-induced pathological outcomes between hematopoietic progenitor cells (HPCs)- or non-HPCs-specific IL4Rα-deficient chimera, myeloid cell-specific IL4Rα-deficient (LysMcre+/+/IL4Rαfl/fl), and airway epithelial cell-specific IL4Rα-deficient (CCSP-Cre+ /IL4Rαfl/fl) mice. Chimeric mice with systemic IL4Rα sufficiency displayed hallmark features of allergic asthma, including eosinophilic and lymphocytic infiltration, type 2 (Th2) cytokine/chemokine production, IgE production, and lung pathology. These features were markedly reduced in chimeric mice with systemic IL4Rα deficiency. Non-HPCs-specific IL4Rα-deficient mice displayed typical inflammatory features of allergic asthma but with markedly reduced mucous cell metaplasia (MCM). Deletion of IL4Rα signaling on airway epithelial cells, a subpopulation within the non-HPC lineage, resulted in almost complete absence of MCM. In contrast, all features of allergic asthma except for MCM and mucin production were mitigated in HPCs-specific IL4Rα-deficient chimeric mice. Deleting IL4Rα signaling in myeloid cells, a subpopulation within the HPC lineage, significantly alleviated MA-induced allergic airway inflammatory responses, but similar to the HPCs-specific IL4Rα-deficient chimeric mice, these mice showed significant MCM and mucin production. Our findings demonstrate that the differential allergen responsiveness seen in mice with HPCs-specific and non-HPCs-specific IL4Rα deficiency is predominantly driven by the absence of IL4Rα in myeloid cells and airway epithelial cells, respectively. Our findings also highlight distinct and mutually exclusive roles of IL4Rα signaling in mediating pathological outcomes within the myeloid and airway epithelial cell compartments.

WISP1 induces the expression of macrophage migration inhibitory factor in human lung fibroblasts through Src kinases and EGFR-activated signaling pathways.

Wnt1-inducible signaling protein 1 (WISP1/CCN4) is a secreted matricellular protein that is implicated in lung and airway remodeling. The macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been associated with chronic lung diseases. In this study, we aimed to investigate the WISP1 signaling pathway and its ability to induce the expression of MIF in primary cultures of fibroblasts from normal human lungs (HLFs). Our results showed that WISP1 significantly stimulated the expression of MIF in a concentration- and time-dependent fashion. In WISP1-induced expression of MIF, αvß5-integrin and chondroitin sulfate proteoglycans as well as Src tyrosine kinases, MAP kinases, phosphatidylinositol 3-kinase/Akt, PKC, and NF-κB were involved. WISP1-induced expression of MIF was attenuated in the presence of the Src kinase inhibitor PP2 or the MIF tautomerase activity inhibitor ISO-1. Moreover, WISP1 significantly increased the phosphorylation and activation of EGF receptor (EGFR) through transactivation by Src kinases. WISP1 also induced the expression of MIF receptor CD74 and coreceptor CD44, through which MIF exerts its effects on HLFs. In addition, it was found that MIF induced its own expression, as well as its receptors CD74/CD44, acting in an autocrine manner. Finally, WISP1-induced MIF promoted the expression of cyclooxygenase 2, prostaglandin E2, IL-6, and matrix metalloproteinase-2 demonstrating the regulatory role of WISP1-MIF axis in lung inflammation and remodeling involving mainly integrin αvß5, Src kinases, PKC, NF-κB, and EGFR. The specific signaling pathways involved in WISP1-induced expression of MIF may prove to be excellent candidates for novel targets to control inflammation in chronic lung diseases.NEW & NOTEWORTHY The present study demonstrates for the first time that Wnt1-inducible signaling protein 1 (WISP1) regulates migration inhibitory factor (MIF) expression and activity and identifies the main signaling pathways involved. The newly discovered WISP1-MIF axis may drive lung inflammation and could result in the design of novel targeted therapies in inflammatory lung diseases.

Transcriptomics analysis of allergen-induced inflammatory gene expression in the Four-Core Genotype mouse model.

Sex differences in allergic inflammation have been reported, but the mechanisms underlying these differences remain unknown. Contributions of both sex hormones and sex-related genes to these mechanisms have been previously suggested in clinical and animal studies. Here, Four-Core Genotypes (FCG) mouse model was used to study the inflammatory response to house dust mite (HDM) challenge and identify differentially expressed genes (DEGs) and regulatory pathways in lung tissue. Briefly, adult mice (8-10 wk old) of the FCG (XXM, XXF, XYM, XYF) were challenged intranasally with 25 µg of HDM or vehicle (PBS-control group) 5 days/wk for 5 wk (n = 3/10 group). At 72 h after the last exposure, we analyzed the eosinophils and neutrophils in the bronchoalveolar lavage (BAL) of FCG mice. We extracted lung tissue and determined DEGs using Templated Oligo-Sequencing (TempO-Seq). DEG analysis was performed using the DESeq2 package and gene enrichment analysis was done using Ingenuity Pathway Analysis. A total of 2,863 DEGs were identified in the FCG. Results revealed increased eosinophilia and neutrophilia in the HDM-treated group with the most significantly expressed genes in XYF phenotype and a predominant effect of female hormones vs. chromosomes. Regardless of the sex hormones, mice with female chromosomes had more downregulated genes in the HDM group but this was reversed in the control group. Interestingly, genes associated with inflammatory responses were overrepresented in the XXM and XYF genotypes treated with HDM. Sex hormones and chromosomes contribute to inflammatory responses to HDM challenge, with female hormones exerting a predominant effect mediated by inflammatory DEGs.NEW & NOTEWORTHY Gene expression profiling helps to provide deep insight into the global view of disease-related mechanisms and responses to therapy. Using the Four-Core Genotype mouse model, our findings revealed the influence of sex hormones and sex chromosomes in the gene expression of lungs exposed to an aeroallergen (House Dust Mite) and identified sex-specific pathways to better understand sex disparities associated with allergic airway inflammation.

Anti-inflammatory roles of type I interferon signaling in the lung.

Excessive or persistent inflammation may have detrimental effects on lung structure and function. Currently, our understanding of conserved host mechanisms that control the inflammatory response remains incompletely understood. In this study, we investigated the role of type I interferon signaling in the inflammatory response against diverse clinically relevant stimuli. Using mice deficient in type I interferon signaling (IFNAR1-/-), we demonstrate that the absence of interferon signaling resulted in a robust and persistent inflammatory response against Pseudomonas aeruginosa, lipopolysaccharide, and chemotherapeutic agent bleomycin. The elevated inflammatory response in IFNAR1-/- mice was manifested as elevated myeloid cells, such as macrophages and neutrophils, in the bronchoalveolar lavage. The inflammatory cell response in the IFNAR1-/- mice persisted to 14 days and there is impaired recovery and fibrotic remodeling of the lung in IFNAR1-/- mice after bleomycin injury. In the Pseudomonas infection model, the elevated inflammatory cell response led to improved bacterial clearance in IFNAR1-/- mice, although there was similar lung injury and survival. We performed RNA sequencing of lung tissue in wild-type and IFNAR1-/- mice after LPS and bleomycin injury. Our unbiased analysis identified differentially expressed genes between IFNAR1-/- and wild-type mice, including previously unknown regulation of nucleotide-binding oligomerization domain (NOD)-like receptor signaling, retinoic acid-inducible gene-I (RIG-I) signaling, and necroptosis pathway by type I interferon signaling in both models. These data provide novel insights into the conserved anti-inflammatory mechanisms of the type I interferon signaling.NEW & NOTEWORTHY Type I interferons are known for their antiviral activities. In this study, we demonstrate a conserved anti-inflammatory role of type I interferon signaling against diverse stimuli in the lung. We show that exacerbated inflammatory response in the absence of type I interferon signaling has both acute and chronic consequences in the lung including structural changes.

Aging, brain-derived neurotrophic factor, and allergen-induced pulmonary responses in mice.

Asthma in the elderly is being recognized as more severe, resistant to standard therapies, and having greater morbidity. Therefore, it comes important to understand the impact of aging-associated airway structure and function changes towards pathogenesis of asthma in the elderly. Here, airway smooth muscle plays important roles in airway hyperreactivity and structural remodeling. The role of smooth muscle in asthma can be modulated by growth factors (including neurotrophins such as brain-derived neurotrophic factor (BDNF)) and pro-inflammatory senescence factors. In this study, we investigated aging effects on airway hyperreactivity, structural remodeling, inflammation, and senescence in a mouse model of allergic asthma. C57BL/6J wildtype mice or smooth muscle-specific BDNF knockout mice at 4, 18 and 24 months of age were intranasally exposed to mixed allergens (ovalbumin, aspergillus, Alternaria, and house dust mite) over 4 weeks. Assessing lung function by FlexiVent, we found that compared with 4 month old mice, 18 and 24 month old C57BL/6J mice showed decreased airway resistance and increased airway compliance after PBS or MA treatment. Deletion of smooth muscle BDNF blunted airway hyperreactivity in aged mice. Lung histology analysis revealed that aging increased bronchial airway thickness and decreased lung inflammation. Multiplex assays showed that aging largely reduced allergen-induced lung expression of proinflammatory chemokines and cytokines. By immunohistochemistry staining, we found that aging increased bronchial airway expression of senescence markers, including p21, phospho-p53 and phospho-gH2A.X. Our data suggest that aging associated increase of airway senescence in the context of allergen exposure may contribute to asthma pathology in the elderly.

Tumor necrosis factor-α-dependent inflammation upregulates high mobility group box 1 to induce tumor promotion and anti-programmed cell death protein-1 immunotherapy resistance in lung adenocarcinoma.

Tumor-associated chronic lung inflammation depends on tumor necrosis factor (TNF)-α to activate several cytokines as part of an inflammatory loop, which plays a critical role in tumor progression in lung adenocarcinoma. High mobility group box 1 (HMGB1) is a cytokine that mediates inflammation. Whether TNF-α-induced inflammation regulates HMGB1 to contribute to tumor progression and promotion in lung adenocarcinoma remains unclear. Thus, human samples and a urethane-induced inflammation-driven lung adenocarcinoma (IDLA) mouse model were used to explore the involvement of HMGB1 in tumorigenesis, tumor progression, and efficacy of anti-programmed cell death protein (PD)-1 immunotherapy. High levels of HMGB1 were observed in human lung adenocarcinoma associated with poor overall survival in patients. HMGB1 upregulation was positively correlated with TNF-α-related inflammation and TIM3+ infiltration. TNF-α upregulated intracellular and extracellular HMGB1 expression to contribute to tumor promotion in A549 cells in vitro. Using a urethane-induced IDLA mouse model, we found HMGB1 upregulation was associated with increased TIM3+ T cell infiltration. Blocking TNF-α-dependent inflammation downregulated HMGB1 expression and inhibited tumorigenesis in the IDLA. Anti-PD-1 treatment alone did not inhibit tumor growth in the TNF-α-dependent IDLA, whereas anti-PD-1 combined with TNF-α blockade overcame anti-PD-1 immunotherapy resistance. Furthermore, anti-PD-1 combined with anti-HMGB1 also inhibited tumor growth in IDLA, suggesting increased HMGB1 release by TNF-α contributes to the resistance of anti-PD-1 immunotherapy in IDLA. Thus, tumor-associated TNF-α-dependent inflammation upregulated intracellular and extracellular HMGB1 expression in an inflammatory loop, contributing to tumor promotion and anti-PD-1 immunotherapy resistance in lung adenocarcinoma.

Different bacterial cargo in apoptotic cells drive distinct macrophage phenotypes.

The removal of dead cells (efferocytosis) contributes to the resolution of the infection and preservation of the tissue. Depending on the environment milieu, macrophages may show inflammatory (M1) or anti-inflammatory (M2) phenotypes. Inflammatory leukocytes are recruited during infection, followed by the accumulation of infected and non-infected apoptotic cells (AC). Efferocytosis of non-infected AC promotes TGF-ß, IL-10, and PGE2 production and the polarization of anti-inflammatory macrophages. These M2 macrophages acquire an efficient ability to remove apoptotic cells that are involved in tissue repair and resolution of inflammation. On the other hand, the impact of efferocytosis of infected apoptotic cells on macrophage activation profile remains unknown. Here, we are showing that the efferocytosis of gram-positive Streptococcus pneumoniae-AC (Sp-AC) or gram-negative Klebsiella pneumoniae-AC (Kp-AC) promotes distinct gene expression and cytokine signature in macrophages. Whereas the efferocytosis of Kp-AC triggered a predominant M1 phenotype in vitro and in vivo, the efferocytosis of Sp-AC promoted a mixed M1/M2 activation in vitro and in vivo in a model of allergic asthma. Together, these findings suggest that the nature of the pathogen and antigen load into AC may have different impacts on inducing macrophage polarization.

Therapeutic targeting of full-length interleukin-33 protein levels with cell-permeable decoy peptides attenuates fibrosis in the bleomycin model in vivo.

Interleukin (IL)-33 has been shown to centrally regulate, among other processes, inflammation and fibrosis. Both intracellular full-length (FLIL33) precursor and extracellular mature cytokine (MIL33) forms exert such regulation, albeit differentially. Drug development efforts to target the IL-33 pathway have focused mostly on MIL33 and its specific cell-surface receptor, ST2, with limited attempts to negotiate the pathophysiological contributions from FLIL33. Furthermore, even a successful strategy for targeting MIL33 effects would arguably benefit from a simultaneous attenuation of the levels of FLIL33, which remains the continuous source of MIL33 supply. We therefore sought to develop an approach to depleting FLIL33 protein levels. We previously reported that the steady-state levels of FLIL33 are controlled in part through its proteasomal degradation and that such regulation can be mapped to a segment in the N-terminal portion of FLIL33. We hypothesized that disruption of this regulation would lead to a decrease in FLIL33 levels, thus inducing a beneficial therapeutic effect in an IL-33-dependent pathology. To test this hypothesis, we designed and tested cell-permeable decoy peptides (CPDPs) which mimic the target N-terminal FLIL33 region. We argued that such mimic peptides would compete with FLIL33 for the components of the native FLIL33 production and maintenance molecular machinery. Administered in the therapeutic regimen to bleomycin-challenged mice, the tested CPDPs alleviated the overall severity of the disease by restoring body weight loss and attenuating accumulation of collagen in the lungs. This proof-of-principle study lays the foundation for future work towards the development of this prospective therapeutic approach. Significance Statement An antifibrotic therapeutic approach is proposed and preclinically tested in mice in vivo based on targeting the full-length IL-33 precursor protein. Peptide fusion constructs consisted of a cell-permeable sequence fused with a sequence mimicking an N-terminal segment of IL-33 precursor that is responsible for this protein's stability. Systemic administration of such peptides to mice in either the acute intratracheal or chronic systemic bleomycin challenge models leads to a decrease in the bleomycin-induced elevations of pulmonary IL-33 and collagen.

First-in-Class Mitogen-Activated Protein Kinase p38α: MAPK-Activated Protein Kinase-2 (MK2) Dual Signal Modulator with Anti-inflammatory and Endothelial-stabilizing Properties.

We previously identified a small molecule, UM101, predicted to bind to the substrate-binding groove of p38aMitogen-activated Protein Kinase (MAPK) near the binding site of its proinflammatory substrate, MAPK-activated protein kinase (MK2). UM101 exhibited anti-inflammatory, endothelial-stabilizing, and lung-protective effects. To overcome its limited aqueous solubility and p38a binding affinity, we designed an analog of UM101, GEn-1124, with improved aqueous solubility, stability, and p38a binding affinity. Compared with UM101, GEn-1124 has 18-fold greater p38a-binding affinity as measured by Surface Plasmon Resonance (SPR), 11-fold greater aqueous solubility, enhanced barrier-stabilizing activity in thrombin-stimulated human pulmonary artery endothelial cells (hPAEC) in vitro, and greater lung protection in vivo GEn-1124 improved survival from 10% to 40% in murine acute lung injury (ALI) induced by combined exposure to intratracheal bacterial endotoxin lipopolysaccharide (LPS) instillation and febrile-range hyperthermia (FRH) and from 0% to 50% in a mouse influenza pneumonia model. Gene expression analysis by RNASeq in TNFa-treated hPAEC showed that the gene-modifying effects of GEn-1124 were much more restricted to TNFa-inducible genes than the catalytic site p38 inhibitor, SB203580. Gene expression pathway analysis, confocal immunofluorescence analysis of p38aand MK2 subcellular trafficking, and SPR analysis of phosphorylated p38a:MK2 binding affinity supports a novel mechanism of action. GEn-1124 destabilizes the activated p38a:MK2 complex, dissociates nuclear export of MK2 and p38a, thereby promoting intranuclear retention and enhanced intranuclear signaling by phosphorylated p38a retention, and accelerated inactivation of p38-free cytosolic MK2 by unopposed phosphatases. Significance Statement We describe an analog of our first-in-class small molecule modulator of p38a/MK2 signaling targeted to a pocket near the ED substrate binding domain of p38a, which destabilizes the p38a:MK2 complex without blocking p38 catalytic activity or ablating downstream signaling. The result is a rebalancing of downstream pro- and anti-inflammatory signaling, yielding anti-inflammatory, endothelial-stabilizing, and lung-protective effects with therapeutic potential in ARDS.

Chronic exposure to E-cigarette aerosols potentiates atherosclerosis in a sex-dependent manner.

Despite being designed for smoking cessation, e-cigarettes and their variety of flavors have become increasingly attractive to teens and young adults. This trend has fueled concerns regarding the potential role of e-cigarettes in advancing chronic diseases, notably those affecting the cardiovascular system. E-cigarettes contain a mixture of metals and chemical compounds, some of which have been implicated in cardiovascular diseases like atherosclerosis. Our laboratory has optimized in vivo exposure regimens to mimic human vaping patterns. Using these established protocols in an inducible (AAV-PCSK9) hyperlipidemic mouse model, this study tests the hypothesis that a chronic exposure to e-cigarette aerosols will increase atherosclerotic plaques. The exposures were conducted using the SCIREQ InExpose™ nose-only inhalation system and STLTH or Vuse products for 16 weeks. We observed that only male mice exposed to STLTH or Vuse aerosols had significantly increased plasma total cholesterol, triglycerides, and LDL cholesterol levels compared to mice exposed to system air. Moreover, these male mice also had a significant increase in aortic and sinus plaque area. Male mice exposed to e-cigarette aerosol had a significant reduction in weight gain over the exposure period. Our data indicate that e-cigarette use in young hyperlipidemic male mice increases atherosclerosis in the absence of significant pulmonary and systemic inflammation. These results underscore the need for extensive research to unravel the long-term health effects of e-cigarettes.