Proinflammatory CD14highCD16low monocytes/macrophages prevail in Treponema phagedenis-associated bovine digital dermatitis.

Digital dermatitis (DD) is a skin disease in cattle characterized by painful inflammatory ulcerative lesions in the feet, mostly associated with local colonization by Treponema spp., including Treponema phagedenis. The reason why most DD lesions remain actively inflamed and progress to chronic conditions despite antibiotic treatment remains unknown. Herein, we show an abundant infiltration of proinflammatory (CD14highCD16low) monocytes/macrophages in active DD lesions, a skin response that was not mitigated by topical treatment with oxytetracycline. The associated bacterium, T. phagedenis, isolated from DD lesions in cattle, when injected subcutaneously into mice, induced abscesses with a local recruitment of Ly6G+ neutrophils and proinflammatory (Ly6ChighCCR2+) monocytes/macrophages, which appeared at infection onset (4 days post challenge) and persisted for at least 7 days post challenge. When exploring the ability of macrophages to regulate inflammation, we showed that bovine blood-derived macrophages challenged with live T. phagedenis or its structural components secreted IL-1ß via a mechanism dependent on the NLRP3 inflammasome. This study shows that proinflammatory characteristics of monocytes/macrophages and neutrophils dominate active non-healing ulcerative lesions in active DD, thus likely impeding wound healing after antibiotic treatment.

Blockade of Proteinase-Activated Receptor 2 (PAR2) Attenuates Neuroinflammation in Experimental Autoimmune Encephalomyelitis.

Proteinase-activated receptor-2 (PAR2), which modulates inflammatory responses, is elevated in the central nervous system in multiple sclerosis (MS) and in its murine model, experimental autoimmune encephalomyelitis (EAE). In PAR2-null mice, disease severity of EAE is markedly diminished. We therefore tested whether inhibiting PAR2 activation in vivo might be a viable strategy for the treatment of MS. Using the EAE model, we show that a PAR2 antagonist, the pepducin palmitoyl-RSSAMDENSEKKRKSAIK-amide (P2pal-18S), attenuates EAE progression by affecting immune cell function. P2pal-18S treatment markedly diminishes disease severity and reduces demyelination, as well as the infiltration of T-cells and macrophages into the central nervous system. Moreover, P2pal-18S decreases granulocyte-macrophage colony-stimulating factor (GM-CSF) production and T-cell activation in cultured splenocytes and prevents macrophage polarization in vitro. We conclude that PAR2 plays a key role in regulating neuroinflammation in EAE and that PAR2 antagonists represent promising therapeutic agents for treating MS and other neuroinflammatory diseases. SIGNIFICANCE STATEMENT: Proteinase-activated receptor-2 modulates inflammatory responses and is increased in multiple sclerosis lesions. We show that the proteinase-activated receptor-2 antagonist palmitoyl-RSSAMDENSEKKRKSAIK-amide reduces disease in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis by inhibiting T-cell and macrophage activation and infiltration into the central nervous system, making it a potential treatment for multiple sclerosis.

Endothelial PAR2 activation evokes resistance artery relaxation.

Protease-activated receptor-1 & -2 (PAR1 and PAR2) are expressed widely in cardiovascular tissues including endothelial and smooth muscle cells. PAR1 and PAR2 may regulate blood pressure via changes in vascular contraction or relaxation mediated by endothelial Ca2+ signaling, but the mechanisms are incompletely understood. By using single-cell Ca2+ imaging across hundreds of endothelial cells in intact blood vessels, we explored PAR-mediated regulation of blood vessel function using PAR1 and PAR2 activators. We show that PAR2 activation evoked multicellular Ca2+ waves that propagated across the endothelium. The PAR2-evoked Ca2+ waves were temporally distinct from those generated by muscarinic receptor activation. PAR2 activated distinct clusters of endothelial cells, and these cells were different from those activated by muscarinic receptor stimulation. These results indicate that distinct cell clusters facilitate spatial segregation of endothelial signal processing. We also demonstrate that PAR2 is a phospholipase C-coupled receptor that evokes Ca2+ release from the IP3 -sensitive store in endothelial cells. A physiological consequence of this PAR2 signaling system is endothelium-dependent relaxation. Conversely, PAR1 activation did not trigger endothelial cell Ca2+ signaling nor relax or contract mesenteric arteries. Neither did PAR1 activators alter the response to PAR2 or muscarinic receptor activation. Collectively, these results suggest that endothelial PAR2 but not PAR1 evokes mesenteric artery relaxation by evoking IP3 -mediated Ca2+ release from the internal store. Sensing mediated by PAR2 receptors is distributed to spatially separated clusters of endothelial cells.

The roles of proteases in prostate cancer.

Since the proposition of the pro-invasive activity of proteolytic enzymes over 70 years ago, several roles for proteases in cancer progression have been established. About half of the 473 active human proteases are expressed in the prostate and many of the most well-characterized members of this enzyme family are regulated by androgens, hormones essential for development of prostate cancer. Most notably, several kallikrein-related peptidases, including KLK3 (prostate-specific antigen, PSA), the most well-known prostate cancer marker, and type II transmembrane serine proteases, such as TMPRSS2 and matriptase, have been extensively studied and found to promote prostate cancer progression. Recent findings also suggest a critical role for proteases in the development of advanced and aggressive castration-resistant prostate cancer (CRPC). Perhaps the most intriguing evidence for this role comes from studies showing that the protease-activated transmembrane proteins, Notch and CDCP1, are associated with the development of CRPC. Here, we review the roles of proteases in prostate cancer, with a special focus on their regulation by androgens.

Protease allergens as initiators-regulators of allergic inflammation.

Tremendous progress in the last few years has been made to explain how seemingly harmless environmental proteins from different origins can induce potent Th2-biased inflammatory responses. Convergent findings have shown the key roles of allergens displaying proteolytic activity in the initiation and progression of the allergic response. Through their propensity to activate IgE-independent inflammatory pathways, certain allergenic proteases are now considered as initiators for sensitization to themselves and to non-protease allergens. The protease allergens degrade junctional proteins of keratinocytes or airway epithelium to facilitate allergen delivery across the epithelial barrier and their subsequent uptake by antigen-presenting cells. Epithelial injuries mediated by these proteases together with their sensing by protease-activated receptors (PARs) elicit potent inflammatory responses resulting in the release of pro-Th2 cytokines (IL-6, IL-25, IL-1ß, TSLP) and danger-associated molecular patterns (DAMPs; IL-33, ATP, uric acid). Recently, protease allergens were shown to cleave the protease sensor domain of IL-33 to produce a super-active form of the alarmin. At the same time, proteolytic cleavage of fibrinogen can trigger TLR4 signaling, and cleavage of various cell surface receptors further shape the Th2 polarization. Remarkably, the sensing of protease allergens by nociceptive neurons can represent a primary step in the development of the allergic response. The goal of this review is to highlight the multiple innate immune mechanisms triggered by protease allergens that converge to initiate the allergic response.

Legumain Induces Oral Cancer Pain by Biased Agonism of Protease-Activated Receptor-2.

Oral squamous cell carcinoma (OSCC) is one of the most painful cancers, which interferes with orofacial function including talking and eating. We report that legumain (Lgmn) cleaves protease-activated receptor-2 (PAR2) in the acidic OSCC microenvironment to cause pain. Lgmn is a cysteine protease of late endosomes and lysosomes that can be secreted; it exhibits maximal activity in acidic environments. The role of Lgmn in PAR2-dependent cancer pain is unknown. We studied Lgmn activation in human oral cancers and oral cancer mouse models. Lgmn was activated in OSCC patient tumors, compared with matched normal oral tissue. After intraplantar, facial or lingual injection, Lgmn evoked nociception in wild-type (WT) female mice but not in female mice lacking PAR2 in NaV1.8-positive neurons (Par2Nav1.8), nor in female mice treated with a Lgmn inhibitor, LI-1. Inoculation of an OSCC cell line caused mechanical and thermal hyperalgesia that was reversed by LI-1. Par2Nav1.8 and Lgmn deletion attenuated mechanical allodynia in female mice with carcinogen-induced OSCC. Lgmn caused PAR2-dependent hyperexcitability of trigeminal neurons from WT female mice. Par2 deletion, LI-1, and inhibitors of adenylyl cyclase or protein kinase A (PKA) prevented the effects of Lgmn. Under acidified conditions, Lgmn cleaved within the extracellular N terminus of PAR2 at Asn30↓Arg31, proximal to the canonical trypsin activation site. Lgmn activated PAR2 by biased mechanisms in HEK293 cells to induce Ca2+ mobilization, cAMP formation, and PKA/protein kinase D (PKD) activation, but not ß-arrestin recruitment or PAR2 endocytosis. Thus, in the acidified OSCC microenvironment, Lgmn activates PAR2 by biased mechanisms that evoke cancer pain.SIGNIFICANCE STATEMENT Oral squamous cell carcinoma (OSCC) is one of the most painful cancers. We report that legumain (Lgmn), which exhibits maximal activity in acidic environments, cleaves protease-activated receptor-2 (PAR2) on neurons to produce OSCC pain. Active Lgmn was elevated in OSCC patient tumors, compared with matched normal oral tissue. Lgmn evokes pain-like behavior through PAR2 Exposure of pain-sensing neurons to Lgmn decreased the current required to generate an action potential through PAR2 Inhibitors of adenylyl cyclase and protein kinase A (PKA) prevented the effects of Lgmn. Lgmn activated PAR2 to induce calcium mobilization, cAMP formation, and activation of protein kinase D (PKD) and PKA, but not ß-arrestin recruitment or PAR2 endocytosis. Thus, Lgmn is a biased agonist of PAR2 that evokes cancer pain.

Metformin Prevents Hyperglycemia-Associated, Oxidative Stress-Induced Vascular Endothelial Dysfunction: Essential Role for the Orphan Nuclear Receptor Human Nuclear Receptor 4A1 (Nur77).

Vascular pathology is increased in diabetes because of reactive-oxygen-species (ROS)-induced endothelial cell damage. We found that in vitro and in a streptozotocin diabetes model in vivo, metformin at diabetes-therapeutic concentrations (1-50 µM) protects tissue-intact and cultured vascular endothelial cells from hyperglycemia/ROS-induced dysfunction typified by reduced agonist-stimulated endothelium-dependent, nitric oxide-mediated vasorelaxation in response to muscarinic or proteinase-activated-receptor 2 agonists. Metformin not only attenuated hyperglycemia-induced ROS production in aorta-derived endothelial cell cultures but also prevented hyperglycemia-induced endothelial mitochondrial dysfunction (reduced oxygen consumption rate). These endothelium-protective effects of metformin were absent in orphan-nuclear-receptor Nr4a1-null murine aorta tissues in accord with our observing a direct metformin-Nr4a1 interaction. Using in silico modeling of metformin-NR4A1 interactions, Nr4a1-mutagenesis, and a transfected human embryonic kidney 293T cell functional assay for metformin-activated Nr4a1, we identified two Nr4a1 prolines, P505/P549 (mouse sequences corresponding to human P501/P546), as key residues for enabling metformin to affect mitochondrial function. Our data indicate a critical role for Nr4a1 in metformin's endothelial-protective effects observed at micromolar concentrations, which activate AMPKinase but do not affect mitochondrial complex-I or complex-III oxygen consumption rates, as does 0.5 mM metformin. Thus, therapeutic metformin concentrations requiring the expression of Nr4a1 protect the vasculature from hyperglycemia-induced dysfunction in addition to metformin's action to enhance insulin action in patients with diabetes. SIGNIFICANCE STATEMENT: Metformin improves diabetic vasodilator function, having cardioprotective effects beyond glycemic control, but its mechanism to do so is unknown. We found that metformin at therapeutic concentrations (1-50µM) prevents hyperglycemia-induced endothelial dysfunction by attenuating reactive oxygen species-induced damage, whereas high metformin (>250 µM) impairs vascular function. However, metformin's action requires the expression of the orphan nuclear receptor NR4A1/Nur77. Our data reveal a novel mechanism whereby metformin preserves diabetic vascular endothelial function, with implications for developing new metformin-related therapeutic agents.

Oxytocin: much more than childbirth and milk letdown.

This commentary deals with the new observations that dendritic cell (DC) oxytocin receptors play a role in the inflammatory response generated in murine animal models of colitis. The overview provides a context of the discovery of oxytocin (OT), its chemical synthesis and the cell biology of its neurohypophysial synthesis and secretion. This perspective provides insight and raises questions to be answered related to the impact of OT in the gastrointestinal tract and to further the exploration of OT as a potentially locally synthesised regulator of intestinal inflammatory pathophysiology.

Protease-Activated Receptor 2 Agonist as Adjuvant: Augmenting Development of Protective Memory CD8 T Cell Responses Induced by Influenza Virosomes.

Protease-activated receptor 2 (PAR-2) is expressed in various tissues, including lung, and when activated, promotes inflammation, differentiation, and migration of dendritic cells. We found that combining influenza virosomes containing hemagglutinin and neuraminidase with a PAR-2 agonist peptide (PAR-2AP) in an intranasal prime boost approach increased survival of mice challenged weeks later with lethal influenza virus over that by virosome or PAR-2AP prime boost alone. No weight loss occurred from influenza challenge after virosome-plus-PAR-2AP prime boost compared with either virosomes or PAR-2AP alone. Thus, virosomes plus PAR-2AP prevented morbidity as well as mortality. Through adoptive transfer, CD8+ lung T cells but not CD4+ T cells from virosomes plus PAR-2AP-primed mice protected from lethal influenza virus challenge and enhanced survival with less weight loss and faster recovery. Virosome-plus-PAR-2AP prime boost resulted in greater percentages of T effector memory phenotype cells (Tem) in lung, and higher frequencies of CD8 Tem and T central memory cells displayed effector functions in response to virus challenge in vivo. Virosome-plus-PAR-2AP prime boost also resulted in greater percentages of Ag-specific CD8+ T cells, both Tem and T central memory cells, in lungs of animals subsequently challenged with live influenza virus. Our findings indicate that PAR-2AP, a short peptide, may be a new and useful mucosal adjuvant.

Physician Scientists Of Yesterday, Today And Tomorrow - Published 44(3) September 2021.

In this issue, Ryan Kirkpatrick and Gordon Boyd speculated on the reasons for the dwindling number of physician scientists in Canada. To help stimulate discussion on this important issue, Clinical and Investigative Medicine invited two distinguished scientists to present their views on this issue.

Proceedings From The 11th Annual University Of Calgary Leaders In Medicine Research Symposium.

On November 8th, 2019, the Cumming School of Medicine at the University of Calgary hosted the 11th annual Leaders in Medicine (LIM) Research Symposium. Dr. Donald A. Redelmeier, Professor at the University of Toronto and Canada Research Chair in Medical Decision Sciences, served as the keynote speaker with a talk entitled "Pitfalls of Reasoning and Clinical Medicine". In addition, there were five oral and 64 poster presentations. These presentations covered topics ranging from health promotion to neuroimaging. The event celebrated the continuing success and diversity of the LIM program and the training of clinician-scientists at the University of Calgary.

Why the endothelium? The endothelium as a target to reduce diabetes-associated vascular disease.

Over the past 66 years, our knowledge of the role of the endothelium in the regulation of cardiovascular function and dysfunction has advanced from the assumption that it is a single layer of cells that serves as a barrier between the blood stream and vascular smooth muscle to an understanding of its role as an essential endocrine-like organ. In terms of historical contributions, we pay particular credit to (1) the Canadian scientist Dr. Rudolf Altschul who, based on pathological changes in the appearance of the endothelium, advanced the argument in 1954 that "one is only as old as one's endothelium" and (2) the American scientist Dr. Robert Furchgott, a 1998 Nobel Prize winner in Physiology or Medicine, who identified the importance of the endothelium in the regulation of blood flow. This review provides a brief history of how our knowledge of endothelial function has advanced and now recognize that the endothelium produces a plethora of signaling molecules possessing paracrine, autocrine, and, arguably, systemic hormone functions. In addition, the endothelium is a therapeutic target for the anti-diabetic drugs metformin, glucagon-like peptide I (GLP-1) receptor agonists, and inhibitors of the sodium-glucose cotransporter 2 (SGLT2) that offset the vascular disease associated with diabetes.

Can a virtual on-line nation-wide mentorship-matching process meet the need?

In Homer's 8th century BC Greek epic poem "Odyssey", Odysseus, upon leaving town to do battle for an extended period, could not have done better than to leave his son, Telemachus, in the care of a trusted friend, Mentor. Thus, "mentorship" can be seen as a key process, whereby a more experienced individual takes on an advisor role for a less-experienced colleague.

Granulocyte-colony-stimulating factor (G-CSF) signaling in spinal microglia drives visceral sensitization following colitis.

Pain is a main symptom of inflammatory diseases and often persists beyond clinical remission. Although we have a good understanding of the mechanisms of sensitization at the periphery during inflammation, little is known about the mediators that drive central sensitization. Recent reports have identified hematopoietic colony-stimulating factors as important regulators of tumor- and nerve injury-associated pain. Using a mouse model of colitis, we identify the proinflammatory cytokine granulocyte-colony-stimulating factor (G-CSF or Csf-3) as a key mediator of visceral sensitization. We report that G-CSF is specifically up-regulated in the thoracolumbar spinal cord of colitis-affected mice. Our results show that resident spinal microglia express the G-CSF receptor and that G-CSF signaling mediates microglial activation following colitis. Furthermore, healthy mice subjected to intrathecal injection of G-CSF exhibit pronounced visceral hypersensitivity, an effect that is abolished by microglial depletion. Mechanistically, we demonstrate that G-CSF injection increases Cathepsin S activity in spinal cord tissues. When cocultured with microglia BV-2 cells exposed to G-CSF, dorsal root ganglion (DRG) nociceptors become hyperexcitable. Blocking CX3CR1 or nitric oxide production during G-CSF treatment reduces excitability and G-CSF-induced visceral pain in vivo. Finally, administration of G-CSF-neutralizing antibody can prevent the establishment of persistent visceral pain postcolitis. Overall, our work uncovers a DRG neuron-microglia interaction that responds to G-CSF by engaging Cathepsin S-CX3CR1-inducible NOS signaling. This interaction represents a central step in visceral sensitization following colonic inflammation, thereby identifying spinal G-CSF as a target for treating chronic abdominal pain.

The pregnane X receptor and its microbiota-derived ligand indole 3-propionic acid regulate endothelium-dependent vasodilation.

We proposed that circulating metabolites generated by the intestinal microbiota can affect vascular function. One such metabolite, indole 3-propionic acid (IPA), can activate the pregnane X receptor(PXR), a xenobiotic-activated nuclear receptor present in many tissues, including the vascular endothelium. We hypothesized that IPA could regulate vascular function by modulating PXR activity. To test this, Pxr+/+ mice were administered broad-spectrum antibiotics for 2 wk with IPA supplementation. Vascular function was evaluated by bioassay using aorta and pulmonary artery ring tissue from antibiotic-treated Pxr+/+ and Pxr-/-mice, supplemented with IPA, and using aorta tissue maintained in organ culture for 24 h in the presence of IPA. Endothelium-dependent, nitric oxide(NO)-mediated muscarinic and proteinase-activated receptor 2(PAR2)-stimulated vasodilation was assessed. Endothelial nitric oxide synthase (eNOS) abundance was evaluated in intact tissue or in aorta-derived endothelial cell cultures from Pxr+/+ and Pxr-/- mice, and vascular Pxr levels were assessed in tissues obtained from Pxr+/+ mice treated with antibiotics and supplemented with IPA. Antibiotic-treated Pxr+/+ mice exhibited enhanced agonist-induced endothelium-dependent vasodilation, which was phenocopied by tissues from either Pxr-/- or germ-free mice. IPA exposure reduced the vasodilatory responses in isolated and cultured vessels. No effects of IPA were observed for tissues obtained from Pxr-/- mice. Serum nitrate levels were increased in antibiotic-treated Pxr+/+and Pxr-/- mice. eNOS abundance was increased in aorta tissues and cultured endothelium from Pxr-/- mice. PXR stimulation reduced eNOS expression in cultured endothelial cells from Pxr+/+ but not Pxr-/- mice. The microbial metabolite IPA, via the PXR, plays a key role in regulating endothelial function. Furthermore, antibiotic treatment changes PXR-mediated vascular endothelial responsiveness by upregulating eNOS.

Proceedings from the 9th Annual University of Calgary Leaders in Medicine Research Symposium.

On November 3, 2017 the Leaders in Medicine (LIM) program at the University of Calgary's Cumming School of Medicine hosted the 9th Annual Leaders in Medicine (LIM) Symposium. This year's event commemorated 20 years of the LIM program and its dedication to the training of clinician- scientists.

Proceedings from the 8th Annual University of Calgary Leaders in Medicine Research Symposium.

On November 14, 2016, the Leaders in Medicine (LIM) program at the Cumming School of Medicine, University of Calgary hosted its 8th Annual Research Symposium. Professor Stephen Sawcer, Professor of Neurological Genetics at the University of Cambridge and an Honorary Consultant Neurologist at Addenbrooke's Hospital, was the keynote speaker and presented a lecture entitled, "Multiple sclerosis genetics - prospects and pitfalls". This was not only a cutting edge address on genetics but also a thoughtful overview on Dr. Sawcer's career and career choices. We were extremely grateful for the opportunity to have Dr. Sawcer participate in our annual symposium.

Proteinases, Their Extracellular Targets, and Inflammatory Signaling.

Given that over 2% of the human genome codes for proteolytic enzymes and their inhibitors, it is not surprising that proteinases serve many physiologic-pathophysiological roles. In this context, we provide an overview of proteolytic mechanisms regulating inflammation, with a focus on cell signaling stimulated by the generation of inflammatory peptides; activation of the proteinase-activated receptor (PAR) family of G protein-coupled receptors (GPCR), with a mechanism in common with adhesion-triggered GPCRs (ADGRs); and by proteolytic ion channel regulation. These mechanisms are considered in the much wider context that proteolytic mechanisms serve, including the processing of growth factors and their receptors, the regulation of matrix-integrin signaling, and the generation and release of membrane-tethered receptor ligands. These signaling mechanisms are relevant for inflammatory, neurodegenerative, and cardiovascular diseases as well as for cancer. We propose that the inflammation-triggering proteinases and their proteolytically generated substrates represent attractive therapeutic targets and we discuss appropriate targeting strategies.

Protease-Activated Receptor 2 Facilitates Bacterial Dissemination in Pneumococcal Pneumonia.

Streptococcus pneumoniae is the most common causative pathogen in community-acquired pneumonia. Protease-activated receptor 2 (PAR2) is expressed by different cell types in the lungs and can mediate inflammatory responses. We sought to determine the role of PAR2 during pneumococcal pneumonia. Pneumococcal pneumonia or sepsis was induced in wild-type and PAR2 knock-out (Par2-/-) mice by infection with viable S. pneumoniae. Par2-/- mice demonstrated improved host defense, a largely preserved lung barrier integrity, and reduced mortality during pneumococcal pneumonia. PAR2 deficiency did not influence bacterial growth after intravenous infection. Inhibition of the endogenous PAR2 activating proteases tissue factor/factor VIIa or tryptase did not impact on bacterial burdens during pneumonia. In a PAR2 reporter cell line it was demonstrated that S. pneumoniae-derived proteases are able to cleave PAR2. These results show that S. pneumoniae is able to cleave and exploit PAR2 to disseminate systemically from the airways.

Protease-activated receptor-2 signaling through ß-arrestin-2 mediates Alternaria alkaline serine protease-induced airway inflammation.

Alternaria alternata is a fungal allergen associated with severe asthma and asthma exacerbations. Similarly to other asthma-associated allergens, Alternaria secretes a serine-like trypsin protease(s) that is thought to act through the G protein-coupled receptor protease-activated receptor-2 (PAR2) to induce asthma symptoms. However, specific mechanisms underlying Alternaria-induced PAR2 activation and signaling remain ill-defined. We sought to determine whether Alternaria-induced PAR2 signaling contributed to asthma symptoms via a PAR2/ß-arrestin signaling axis, identify the protease activity responsible for PAR2 signaling, and determine whether protease activity was sufficient for Alternaria-induced asthma symptoms in animal models. We initially used in vitro models to demonstrate Alternaria-induced PAR2/ß-arrestin-2 signaling. Alternaria filtrates were then used to sensitize and challenge wild-type, PAR2-/- and ß-arrestin-2-/- mice in vivo. Intranasal administration of Alternaria filtrate resulted in a protease-dependent increase of airway inflammation and mucin production in wild-type but not PAR2-/- or ß-arrestin-2-/- mice. Protease was isolated from Alternaria preparations, and select in vitro and in vivo experiments were repeated to evaluate sufficiency of the isolated Alternaria protease to induce asthma phenotype. Administration of a single isolated serine protease from Alternaria, Alternaria alkaline serine protease (AASP), was sufficient to fully activate PAR2 signaling and induce ß-arrestin-2-/--dependent eosinophil and lymphocyte recruitment in vivo. In conclusion, Alternaria filtrates induce airway inflammation and mucus hyperplasia largely via AASP using the PAR2/ß-arrestin signaling axis. Thus, ß-arrestin-biased PAR2 antagonists represent novel therapeutic targets for treating aeroallergen-induced asthma.