Directional persistence is essential for efficient immune cell migration in tissues, yet how cytoskeletal systems stabilize migration in complex three-dimensional environments remains unclear. Using intravital subcellular microscopy and quantitative analysis of membrane dynamics, we identify two spatially distinct architectures of non-muscle myosin II (NMII) that coordinate protrusion dynamics during neutrophil migration. In vivo and in collagen matrices, NMII assembles at the leading edge into lattice-like structures that are structurally and functionally distinct from rear contractile actomyosin bundles. Protrusion-resolved analyses reveal that directional persistence correlates strongly with protrusion lifetime and sustained NMII engagement, with rear NMII load showing the strongest association with protrusion persistence. Strikingly, directional migration is not determined by the abundance of favorable protrusions but by their temporal organization during migration. Pharmacological perturbations that redistribute NMII activity disrupt this temporal organization and alter migration trajectories. Together, these findings reveal that spatially distinct NMII architectures coordinate protrusion dynamics across time to stabilize directional migration in complex environments.

Efficient neutrophil chemotaxis requires the integration of mechanical forces and lipid-mediated signaling. While the signaling lipid leukotriene B4 (LTB 4 ) reinforces cellular polarity, how mechanical cues regulate its production remains unclear. We now show that cytosolic phospholipase A2α (cPLA₂α), which is essential for the synthesis of LTB 4 , functions as a nuclear curvosensor. cPLA₂α responds to nuclear constrictions by localizing to ceramide-rich inner nuclear membrane microdomains and incorporating onto the exofacial surface of nuclear envelope-derived exosomes. This unique topology enables localized LTB 4 synthesis, which promotes myosin light chain II phosphorylation, and sustains polarity and directional persistence after constriction. In neutrophils squeezing through small constrictions, loss of cPLA₂α impairs nuclear curvature sensing, exosomal LTB 4 production, and post-constriction motility. These findings uncover a cPLA 2 -dependent mechano-chemical axis linking nuclear architecture to chemotactic efficiency and offering new strategies to modulate inflammatory responses.

ABSTRACT The potential for fibrosis across most organ systems may stem from connections to wound healing and the widespread presence of vascular endothelium. Endothelial cells (ECs) and angiogenesis have been heavily implicated in many organ‐specific fibrotic conditions, but little has been established in terms of how EC phenotype governs tissue healing vs. fibrosis. Here, we examined a murine lung injury model enabling EC lineage tracing and observed the invasion of aberrant ECs from the bronchial microvasculature following injury, along with concurrent densification of surrounding extracellular matrix fibers. To investigate mechanisms governing their appearance, we established a microphysiological system of human microvessels embedded within a tunable stromal matrix and found that heightened fiber density drives endothelial to mesenchymal transition to promote aberrant tip EC (ATEC) invasion into the matrix. ATECs remained adherent to fibrotic matrix and possessed a pro‐inflammatory phenotype that secretes TGF‐β2. Mechanistically, we identify ATEC formation was gated by destabilization of EC adherens junctions upon adhesion to fibrous matrix and associated regulation of TGF‐β signaling through a novel VE‐cadherin – TGF‐βR2 interaction. Altogether, this work identifies how enhanced fiber density associated with fibrogenesis regulates EC phenotype to generate pro‐inflammatory ATECs and suggests new contributions of ECs to fibrotic progression.

Fibrosis is central to numerous fatal conditions including solid cancers, pulmonary fibrosis, cirrhosis and post-infarct cardiac fibrosis amongst many others, thereby collectively contributing to 45% of all deaths in developed nations. The potential for fibrosis across most organ systems may stem from its connections to wound healing and the ubiquitous presence of vascular endothelium. Endothelial cells (ECs) and angiogenesis, cells and associated biological program central to wound healing, have been heavily implicated in many organ-specific fibroses, but the relationship between angiogenesis and fibrogenesis remains debated and little has been established in terms of how the EC phenotype governs tissue healing vs. fibrosis. Here, we examine a murine lung injury model enabling EC lineage tracing and observe the invasion of aberrant ECs from the bronchial microvasculature following lung injury along with concurrent densification of matrix fibers surrounding these vessels. To investigate the underlying mechanisms governing their appearance, we established a microphysiological system (MPS) of arteriole/venule-scale microvessels embedded within a tunable stromal mimetic matrix and find that heightened extracellular matrix fiber density activates ECs, drives endothelial to mesenchymal transition, and promotes aberrant tip EC (ATEC) invasion into the matrix. ATECs remain adherent to fibrotic matrix and possess a pro-inflammatory phenotype that secretes TGF-β2. Notably, our studies establish that the formation of ATECs is gated by destabilization of endothelial adherens junction upon EC adhesion to fibrous matrix, and associated regulation of TGF-β signaling that is mediated by a novel VE-cadherin - TGF-βR2 axis. The current lack of effective anti-fibrotic therapies suggests potential critical involvement of other cell types such as ECs, and our findings suggest new contributions of ECs to fibrotic progression that may better inform future targets for novel anti-fibrotic therapeutics.

Neutrophils exert tumor-promoting roles in breast cancer and are particularly prominent in aggressive breast tumors. The proinflammatory signals TGF-β1 and TNF-α are upregulated in breast tumors and induce epithelial-to-mesenchymal transitions (EMT), a process linked to cancer cell aggressiveness. Here, we investigated the roles of TGF-β1 and TNF-α in the recruitment of neutrophils by breast cancer cells. Dual-treatment with TGF-β1 and TNF-α induces EMT signatures in premalignant M2 cells, which are part of the MCF10A breast cancer progression model. Conditioned media (CM) harvested from M2 cells treated with TGF-β1/TNF-α gives rise to amplified neutrophil chemotaxis compared with CM from vehicle-treated M2 cells. This response correlates with higher levels of the neutrophil chemokines CXCL1 and CXCL8, in a p38MAPK-dependent manner, and is attenuated by CXCL8-neutralizing antibodies. We combined gene editing, immunological, and biochemical assays to show that neutrophil recruitment and EMT are uncoupled in treated M2 cells. Finally, analysis of transcriptomic databases of cancer cell lines revealed a significant correlation between CXCL8 and TGF-β1/TNF-α-regulated or effector genes in breast cancer. These findings establish a novel role for the TGF-β1/TNF-α/p38 MAPK signaling axis in regulating neutrophil recruitment in breast cancer, independent of their profound impact on EMT.

Most adhesion G protein coupled receptors (AGPCRs) are activated by intramolecular binding of a tethered-peptide agonist (TA). Shear force-induced dissociation of the AGPCR N-terminal fragment (NTF) and C-terminal fragment (CTF) exposes the TA. The decrypted TA binds rapidly to its orthosteric site within the CTF to stabilize the active state of the AGPCR. Corticosteroids were previously proposed to be agonists for GPR97/ADGRG3. Later, other steroids and androgens were purported to be selective agonists of additional AGPCRs. Here, we demonstrate that GPR97/ADGRG3 is activated by dissociation of its NTF/CTF and follows the TA mechanism. TA peptidomimetics and the ADGRG subfamily partial agonist 3-acetoxydihydrodeoxygeduin (3-α-DOG), but not corticoids, stimulated GPR97/ADGRG3 in cell-based luciferase reporter assays and receptor/G protein reconstitution assays. GPR97 was defined as a promiscuous AGPCR that couples to G13, Gs and Gi, but not Gq. GPR97 is highly expressed in human polymorphonuclear neutrophils (hPMNs). Neutrophils undergo actin polymerization-induced cell shape changes and polarization and migrate upon activation. We found that GPR97 activation via TA peptidomimetics or 3-α-DOG robustly stimulated hPMN and mouse bone-marrow neutrophil (mBMN) polarization. Furthermore, GPR97 TA peptidomimetics and 3-α-DOG, but not beclomethasone, induced hPMN and mBMN chemotaxis. Together, our results demonstrate that GPR97/ADGRG3 utilizes a tethered agonist mechanism to activate G protein signaling and induce neutrophil polarization and migration. One Sentence Summary:GPR97/ADGRG3 tethered agonism regulates G protein signaling in neutrophils.

Alveolar macrophages (AMs) are specialized lung-resident immune cells that clear inhaled bacteria and debris to maintain tissue homeostasis. AMs secrete eicosanoid lipid mediators such as prostaglandin E2 (PGE2) maintaining an anti-inflammatory environment, whereas during infection AMs release leukotriene B4 (LTB4) driving neutrophil infiltration. PGE2 and LTB4 are synthesized by cyclooxygenase 2 and 5-lipoxygenase (5LO), respectively. While lipid droplets (LDs) facilitate eicosanoid secretion by storing eicosanoid precursors such as triacylglycerol (TAG) and arachidonic acid (AA), the cellular mechanisms segregating PGE2 and LTB4 biogenesis remain unclear. We investigated these pathways in rat AMs whose eicosanoid profile closely resembles that of the human AM. In ionophore-activated AMs, we observed an early LTB4 spike, in contrast to a delayed bump in basal PGE2 levels. High-throughput microscopy revealed that in rat AMs, 5LO-activating protein (FLAP) mediates de novo LD biogenesis for LTB4 secretion, without affecting PGE2; which we validated in human PBMC-derived AM-like cells. FLAP also interacts with the LD biogenesis protein Perilipin 1 in an AA-dependent manner. We propose that activated AMs use a FLAP-dependent, AA-rich LD biogenesis pathway for LTB4 synthesis, distinct from steady-state TAG-rich LDs driving PGE2 production. This study highlights LDs’ role in AM production of functionally opposing eicosanoids, offering insights into respiratory immune disorders. NIAID 1R01AI152517; NHLBI 5R35HL144979 Mucosal and Regional Immunology (MUC)

Acute inflammation, characterized by a rapid influx of neutrophils, is a protective response that can lead to chronic inflammatory diseases when left unresolved. We previously showed that secretion of LTB4-containing exosomes via nuclear envelope-derived multivesicular bodies is required for effective neutrophil infiltration during inflammation. Here we report that the co-secretion of these exosomes with nuclear DNA facilitates the resolution of the neutrophil infiltrate in a mouse skin model of sterile inflammation. Activated neutrophils exhibit rapid and repetitive DNA secretion as they migrate directionally using a mechanism distinct from suicidal neutrophil extracellular trap release and cell death. Packaging of DNA in the lumen of nuclear envelope-multivesicular bodies is mediated by lamin B receptor and chromatin decondensation. These findings advance our understanding of neutrophil functions during inflammation and the physiological relevance of DNA secretion.

The stress-induced keratin intermediate filament gene/protein KRT16 (K16) is spatially restricted to the suprabasal compartment of the epidermis and extensively used as a biomarker for psoriasis, hidradenitis suppurativa, atopic dermatitis and other inflammatory disorders. However, its role in these conditions remains poorly defined. Here we show that K16 negatively regulates type-I interferon (IFN) signaling and innate immune responses. In mouse skin in vivo, loss of Krt16 leads to exacerbation of imiquimod-induced psoriasiform disease and heightened recruitment of neutrophils in a phorbol ester-induced model of acute sterile inflammation. In KRT16 null human keratinocytes, loss of K16 amplifies IFN signaling including phospho-IRF7 and ISG15 after treatment with synthetic dsRNA poly(I:C). Mechanistically, K16 interacts with effectors of the RIG-I-like receptor (RLR) pathway, including 14-3-3ɛ, and inhibits the 14-3-3ɛ:RIG-I interaction upstream of IFN activation in vivo and ex vivo . These findings uncover a new paradigm for keratin-dependent regulation of innate immunity, with significant implications for our understanding of inflammatory skin diseases.

Neutrophils are the first immune cells to reach inflamed sites and contribute to the pathogenesis of chronic inflammatory skin diseases. Yet, little is known about the pattern of neutrophil infiltration in inflamed skin in vivo and the mechanisms mediating their recruitment. Here, we provide insight into the dynamics of neutrophil infiltration in skin in response to acute or repeated inflammatory stress, highlighting a novel keratinocyte- and keratin 17 (K17)-dependent mechanism that regulates neutrophil recruitment to inflamed skin. We used the phorbol ester TPA and UVB, alone or in combination, to induce sterile inflammation in mouse skin. A single TPA treatment results in a neutrophil influx in the dermis that peaks at 12 h and resolves within 24 h. A subsequent TPA treatment or a UVB challenge, when applied 24 h but not 48 h later, accelerates, amplifies, and prolongs neutrophil infiltration. This transient amplification response (TAR) is mediated by local signals in inflamed skin, can be recapitulated in ex vivo culture, and involves the K17-dependent sustainment of protein kinase Cα (PKCα) activity and release of chemoattractants by stressed keratinocytes. K17 binds RACK1, a scaffold protein essential for PKCα activity. The N-terminal head domain of K17 is crucial for its association with RACK1 and regulation of PKCα activity. Analysis of RNAseq data reveals a signature consistent with TAR and PKCα activation in inflammatory skin diseases. These findings uncover a novel, keratin-dependent mechanism that amplifies neutrophil recruitment in skin under stress, with direct implications for inflammatory skin disorders.

Neutrophils contribute to the pathogenesis of chronic inflammatory skin diseases. Little is known about the source and identity of the signals mediating their recruitment in inflamed skin. We used the phorbol ester TPA and UVB, alone or in combination, to induce sterile inflammation in mouse skin and assess whether keratinocyte-derived signals impact neutrophil recruitment. A single TPA treatment results in a neutrophil influx in the dermis that peaks at 12h and resolves within 24h. A second TPA treatment or a UVB challenge, when applied at 24h but not 48h later, accelerates, amplifies, and prolongs neutrophil infiltration. This transient amplification response (TAR) is mediated by local signals in inflamed skin, can be recapitulated in ex vivo culture, and involves the K17-dependent sustainment of protein kinase Cα (PKCα) activity and release of neutrophil chemoattractants by stressed keratinocytes. We show that K17 binds RACK1, a scaffold essential for PKCα activity. Finally, analyses of RNAseq data reveal the presence of a transcriptomic signature consistent with TAR and PKCα activation in chronic inflammatory skin diseases. These findings uncover a novel, transient, and keratin-dependent mechanism that amplifies neutrophil recruitment to the skin under stress, with direct implications for inflammatory skin disorders.

Neutrophils have tumor-promoting roles in breast cancer and are detected in higher numbers in aggressive breast tumors. How aggressive breast tumors recruit neutrophils remains undefined. Here, we investigated the roles of TGFβ1 and TNFα in the regulation of neutrophil recruitment by breast cancer cells. TGFβ1 and TNFα are pro-inflammatory factors upregulated in breast tumors and induce epithelial to mesenchymal transitions (EMT), a process linked to cancer cell aggressiveness. We report that, as expected, dual treatment with TGFβ1 and TNFα induces EMT signatures in premalignant M2 cells, which are part of the MCF10A breast cancer progression model. Conditioned media (CM) harvested from M2 cells treated with TGFβ1/TNFα gives rise to amplified neutrophil chemotaxis compared to CM from control M2 cells. This response correlates with higher levels of the neutrophil chemokines CXCL1, CXCL2, and CXCL8 and is significantly attenuated in the presence of a CXCL8-neutralizing antibody. Furthermore, we found that secretion of CXCL1 and CXCL8 from treated M2 cells depends on p38MAPK activity. By combining gene editing, immunological and biochemical approaches, we show that the regulation of neutrophil recruitment and EMT signatures are not mechanistically linked in treated M2 cells. Finally, analysis of publicly available cancer cell line transcriptomic databases revealed a significant correlation between CXCL8 and TGFβ1/TNFα-regulated or effector genes in breast cancer. Together, our findings establish a novel role for the TGFβ1/TNFα/p38 MAPK signaling axis in regulating neutrophil recruitment in breast cancer, independent of TGFβ1/TNFα regulated EMT.

Collective cell migration is critical for proper embryonic development, wound healing, and cancer cell invasion. However, much of our knowledge of cell migration has been performed using flat surfaces that lack topographical features and do not recapitulate the complex fibrous architecture of the extracellular matrix (ECM). The recent availability of synthetic fibrous networks designed to mimic in vivo ECM has been key to identify the topological features that dictate cell migration patterns as well as to determine the underlying mechanisms that regulate topography-sensing. Recent studies have underscored the prevalence of collective cell migration during cancer invasion, and these observations present a compelling need to understand the mechanisms controlling contact guidance within migratory, multicellular groups. Therefore, we designed an integrated migration analysis platform combining tunable electrospun fibers that recapitulate aspects of the biophysical properties of the ECM, and computational approaches to investigate collective cell migration. To quantitatively assess migration as a function of matrix topography, we developed an automated MATLAB code that quantifies cell migration dynamics, including speed, directionality, and the number of detached cells. This platform enables live cell imaging while providing enough cells for biochemical, proteomic, and genomic analyses, making our system highly adaptable to multiple experimental investigations.

Tumor-associated neutrophils are found in many types of cancer and are often reported to contribute to negative outcomes. The presence of transforming growth factor-beta (TGF-β) in the tumor microenvironment reportedly contributes to the skewing of neutrophils to a more pro-tumor phenotype. The effects of TGF-β on neutrophil signaling and migration are, however, unclear. We sought to characterize TGF-β signaling in both primary human neutrophils and the neutrophil-like cell line HL-60 and determine whether it directly induces neutrophil migration. We found that TGF-β1 does not induce neutrophil chemotaxis in transwell or underagarose migration assays. TGF-β1 does activate canonical signaling through SMAD3 and noncanonical signaling through ERK1/2 in neutrophils in a time-and dose-dependent manner. Additionally, TGF-β1 present in the tumor-conditioned media (TCM) of invasive breast cancer cells results in SMAD3 activation. We discovered that TCM induces neutrophils to secrete leukotriene B 4 (LTB 4 ), which is a lipid mediator important for amplifying the range of neutrophil recruitment. However, TGF-β1 alone does not induce secretion of LTB 4 . RNA-sequencing revealed that TGF-β1 and TCM alter gene expression in HL-60 cells, including the mRNA levels of the pro-tumor oncostatin M ( OSM ) and vascular endothelial growth factor A ( VEGFA ). These new insights into the role and impact of TGF-β1 on neutrophil signaling, migration, and gene expression have significant implications in the understanding of the changes in neutrophils that occur in the tumor microenvironment.

Exosomes are specialized cargo delivery vesicles secreted from cells by fusion of multivesicular bodies (MVBs) with the plasma membrane (PM). While the function of exosomes during physiological and pathological events has been extensively reported, there remains a lack of understanding of the mechanisms that regulate exosome biogenesis, secretion, and internalization. Recent technological and methodological advances now provide details about MVB/exosome structure as well as the pathways of exosome biogenesis, secretion, and uptake. In this review, we outline our current understanding of these processes and highlight outstanding questions following on recent discoveries in the field.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease characterized by collagen deposition within the lung interstitium. Bacterial infection is associated with increased morbidity and more rapid mortality in IPF patient populations, and pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) are commonly isolated from the lungs of hospitalized patients with IPF. Despite this, the effects of fibrotic lung injury on critical immune responses to infection remain unknown. In the present study, we show that, like humans with IPF, fibrotic mice infected with MRSA exhibit increased morbidity and mortality compared with uninfected fibrotic mice. We determine that fibrosis conferred a defect in MRSA clearance compared with nonfibrotic mice, resulting from blunted innate immune responses. We show that fibrosis inhibited neutrophil intracellular killing of MRSA through impaired neutrophil elastase release and oxidative radical production. Additionally, we demonstrate that lung macrophages from fibrotic mice have impaired phagocytosis of MRSA. Our study describes potentially novel impairments of antimicrobial responses upon pulmonary fibrosis development, and our findings suggest a possible mechanism for why patients with IPF are at greater risk of morbidity and mortality related to infection.

Neutrophils migrating towards chemoattractant gradients amplify their recruitment range by releasing the secondary chemoattractant leukotriene B4(LTB4)1,2. We previously demonstrated that LTB4and its synthesizing enzymes, the 5-lipoxygenase (5-LO), 5-LO activating protein (FLAP), and leukotriene A4hydrolase (LTA4H), are packaged and released in exosomes3. We now report that the biogenesis of the LTB4-containing exosomes is initiated at the nuclear envelope (NE) of activated neutrophils. We show that the neutral sphingomyelinase 1 (nSMase1)-mediated generation of ceramide enriched lipid-ordered microdomains initiates the clustering of the LTB4-synthesizing enzymes on the NE. We isolated and analyzed exosomes from activated neutrophils and established that the FLAP/5-LO-positive exosome population is distinct from that of the CD63-positive exosome population. Furthermore, we observed a strong co-localization between ALIX and FLAP at the periphery of nuclei and within cytosolic vesicles. We propose that the initiation of NE curvature and bud formation is mediated by nSMase1-dependent ceramide generation, which leads to FLAP and ALIX recruitment. Together, these observations elucidate the mechanism for LTB4secretion and identify a novel pathway for exosome generation.