Mediator complex subunit 1 architects a tumorigenic Treg cell program independent of inflammation.

While immunotherapy has revolutionized cancer treatment, its safety has been hampered by immunotherapy-related adverse events. Unexpectedly, we show that Mediator complex subunit 1 (MED1) is required for T regulatory (Treg) cell function specifically in the tumor microenvironment. Treg cell-specific MED1 deletion does not predispose mice to autoimmunity or excessive inflammation. In contrast, MED1 is required for Treg cell promotion of tumor growth because MED1 is required for the terminal differentiation of effector Treg cells in the tumor. Suppression of these terminally differentiated Treg cells is sufficient for eliciting antitumor immunity. Both human and murine Treg cells experience divergent paths of differentiation in tumors and matched tissues with non-malignant inflammation. Collectively, we identify a pathway promoting the differentiation of a Treg cell effector subset specific to tumors and demonstrate that suppression of a subset of Treg cells is sufficient for promoting antitumor immunity in the absence of autoimmune consequences.

Tissue-specific reprogramming leads to angiogenic neutrophil specialization and tumor vascularization in colorectal cancer.

Neutrophil (PMN) tissue accumulation is an established feature of ulcerative colitis (UC) lesions and colorectal cancer (CRC). To assess the PMN phenotypic and functional diversification during the transition from inflammatory ulceration to CRC we analyzed the transcriptomic landscape of blood and tissue PMNs. Transcriptional programs effectively separated PMNs based on their proximity to peripheral blood, inflamed colon, and tumors. In silico pathway overrepresentation analysis, protein-network mapping, gene signature identification, and gene-ontology scoring revealed unique enrichment of angiogenic and vasculature development pathways in tumor-associated neutrophils (TANs). Functional studies utilizing ex vivo cultures, colitis-induced murine CRC, and patient-derived xenograft models demonstrated a critical role for TANs in promoting tumor vascularization. Spp1 (OPN) and Mmp14 (MT1-MMP) were identified by unbiased -omics and mechanistic studies to be highly induced in TANs, acting to critically regulate endothelial cell chemotaxis and branching. TCGA data set and clinical specimens confirmed enrichment of SPP1 and MMP14 in high-grade CRC but not in patients with UC. Pharmacological inhibition of TAN trafficking or MMP14 activity effectively reduced tumor vascular density, leading to CRC regression. Our findings demonstrate a niche-directed PMN functional specialization and identify TAN contributions to tumor vascularization, delineating what we believe to be a new therapeutic framework for CRC treatment focused on TAN angiogenic properties.

Phenotypic and Functional Diversity of Neutrophils in Gut Inflammation and Cancer.

Neutrophils [polymorphonuclear leukocytes (PMNs)] execute important effector functions protecting the host against invading pathogens. However, their activity in tissue can exacerbate inflammation and inflammation-associated tissue injury and tumorigenesis. Until recently, PMNs were considered to be short-lived, terminally differentiated phagocytes. However, this view is rapidly changing with the emerging evidence of increased PMN lifespan in tissues, PMN plasticity, and phenotypic heterogeneity. Specialized PMN subsets have been identified in inflammation and in developing tumors, consistent with both beneficial and detrimental functions of PMNs in these conditions. Because PMN and tumor-associated neutrophil activity and the resulting beneficial/detrimental impacts primarily occur after homing to inflamed tissue/tumors, studying the underlying mechanisms of PMN/tumor-associated neutrophil trafficking is of high interest and clinical relevance. This review summarizes some of the key findings from over a decade of work from my laboratory and others on the regulation of PMN recruitment and identification of phenotypically and functionally diverse PMN subtypes as they pertain to gut inflammation and colon cancer.

Macrophage-endothelial cell crosstalk orchestrates neutrophil recruitment in inflamed mucosa.

Neutrophil (PMN) mobilization to sites of insult is critical for host defense and requires transendothelial migration (TEM). TEM involves several well-studied sequential adhesive interactions with vascular endothelial cells (ECs); however, what initiates or terminates this process is not well-understood. Here, we describe what we believe to be a new mechanism where vessel-associated macrophages through localized interactions primed EC responses to form ICAM-1 "hot spots" to support PMN TEM. Using real-time intravital microscopy of LPS-inflamed intestines in CX3CR1-EGFP macrophage-reporter mice, complemented by whole-mount tissue imaging and flow cytometry, we found that macrophage vessel association is critical for the initiation of PMN-EC adhesive interactions, PMN TEM, and subsequent accumulation in the intestinal mucosa. Anti-colony stimulating factor 1 receptor Ab-mediated macrophage depletion in the lamina propria and at the vessel wall resulted in elimination of ICAM-1 hot spots impeding PMN-EC interactions and TEM. Mechanistically, the use of human clinical specimens, TNF-α-KO macrophage chimeras, TNF-α/TNF receptor (TNF-α/TNFR) neutralization, and multicellular macrophage-EC-PMN cocultures revealed that macrophage-derived TNF-α and EC TNFR2 axis mediated this regulatory mechanism and was required for PMN TEM. As such, our findings identified clinically relevant mechanisms by which macrophages regulate PMN trafficking in inflamed mucosa.

Atovaquone attenuates experimental colitis by reducing neutrophil infiltration of colonic mucosa.

Ulcerative colitis (UC) is a chronic relapsing disease featuring aberrant accumulation of neutrophils in colonic mucosa and the luminal space. Although significant advances in UC therapy have been made with the development of novel biologics and small molecules targeting immune responses, success of most current therapies is still limited, with significant safety concerns. Thus, there is a need to develop additional safe and effective therapies for the treatment of UC. Antimalarial drugs have been safely used for many years to resolve tissue inflammation and the associated pathologies. Atovaquone is a recent FDA-approved antimalarial drug that has shown anti-viral and tumor-suppressive properties in vitro however, its role in mucosal inflammation has not been evaluated. Using pre-clinical murine DSS-induced colitis model combined with complementary in vivo peritonitis and ex vivo human neutrophil activation and chemotaxis assays we investigated functional and mechanistic impacts of atovaquone on disease resolution and neutrophil trafficking. We demonstrate that atovaquone promotes resolution of DSS-induced murine colitis by reducing neutrophil accumulation in the inflamed colonic mucosa. Mechanistically, we show that atovaquone suppressed induction of CD11b expression in neutrophils, reducing their polarization and migratory ability. Thus, our findings identify a new role of atovaquone in promoting resolution of mucosal inflammation, supporting the idea of potential repurposing of this FDA-approved drug as UC therapeutic.

Non-Canonical Functions of Myeloperoxidase in Immune Regulation, Tissue Inflammation and Cancer.

Myeloperoxidase (MPO) is one of the most abundantly expressed proteins in neutrophils. It serves as a critical component of the antimicrobial defense system, facilitating microbial killing via generation of reactive oxygen species (ROS). Interestingly, emerging evidence indicates that in addition to the well-recognized canonical antimicrobial function of MPO, it can directly or indirectly impact immune cells and tissue responses in homeostatic and disease states. Here, we highlight the emerging non-canonical functions of MPO, including its impact on neutrophil longevity, activation and trafficking in inflammation, its interactions with other immune cells, and how these interactions shape disease outcomes. We further discuss MPO interactions with barrier forming endothelial and epithelial cells, specialized cells of the central nervous system (CNS) and its involvement in cancer progression. Such diverse function and the MPO association with numerous inflammatory disorders make it an attractive target for therapies aimed at resolving inflammation and limiting inflammation-associated tissue damage. However, while considering MPO inhibition as a potential therapy, one must account for the diverse impact of MPO activity on various cellular compartments both in health and disease.

Neutrophils and micronuclei: An emerging link between genomic instability and cancer-driven inflammation.

Two recent studies by Bui and Butin-Israeli et al. have established the novel contribution of neutrophils to genomic instability induction and aberrant shaping of the DNA repair landscape, particularly observed in patients with inflammatory bowel diseases (IBD) and/or progressive colorectal cancer (CRC). In addition, these back-to-back studies uncovered a sharp increase in the numbers of micronuclei and lagging chromosomes in pre-cancerous and cancerous epithelium in response to prolonged PMN exposure. Given the emerging link between neutrophils and micronuclei as well as the established role of micronuclei in cGAS/STING activation, this special commentary aims to elaborate on the mechanisms by which CRC cells may adapt to neutrophil-driven genomic instability while concurrently sustain an inflamed tumor niche. We postulate that such tumor microenvironment with constant immune cell presence, inflammatory milieu, and cumulative DNA damage can drive tumor adaptation and resistance to therapeutic interventions. Finally, we discuss potential novel therapeutic approaches that can be leveraged to target this emerging neutrophil-micronuclei pathological axis, thereby preventing perpetual CRC inflammation and unwanted tumor adaptation.

A microRNA panel that regulates proinflammatory cytokines as diagnostic and prognosis biomarkers in colon cancer.

Colon cancer (CC) is the third most common neoplasm and the fourth cause of cancer-related death worldwide in both sexes. It has been established that inflammation plays a critical role in tumorigenesis and progression of CC. Immune, stromal and tumor cells supply the tumor microenvironment with pro-inflammatory cytokines such as interleukin 1ß, TNFα, IL-6 and IL-11, to hyperactivate signaling pathways linked to cancerous processes. Recent findings suggest a putative role of microRNAs (miRNAs) in the progression and management of the inflammatory response in intestinal diseases. Moreover, miRNAs are able to regulate expression of molecular mediators that are linking inflammation and cancer. In this work a miRNA panel differentially expressed between healthy, inflammatory bowel disease (IBD) and CC tissue was established. Identified miRNAs regulate signaling pathways related to inflammation and cancer progression. An inflammation associated-miRNA panel composed of 11-miRNAs was found to be overexpressed in CC but not in inflamed or normal tissues (miR-21-5p, miR-304-5p, miR-577, miR-335-5p, miR-21-3p, miR-27b-5p, miR-335-3p, miR-215-5p, miR-30b-5p, miR-192-5p, miR-3065-5p). The association of top hit miRNAs, miR-3065-5p and miR-30b-5p expression with overall survival of CC patients was demonstrated using Kaplan-Meier tests. Finally, differential miRNA expression was validated using an inflammation-associated CC model induced by Azoxymethane/Dextran Sodium Sulfate (AOM/DSS) to compare miRNA expression in normal and inflamed tissue versus CC tissues. Based on these findings we propose the identified inflammatory miRNA panel as a potent diagnostic tool for CC determination.

Tumor-associated neutrophils: orchestrating cancer pathobiology and therapeutic resistance.

Introduction: Neutrophils or polymorphonuclear cells (PMNs) account for a considerable portion of the tumor immune stroma. Emerging single-cell transcriptomic analyses have elucidated the striking cellular heterogeneity of PMNs during homeostasis and pathologic conditions and have established their diverse roles in cancer. PMNs have emerged as important players in cancer pathobiology and therapeutic resistance. Tumor-associated neutrophils (TANs) effector functions influence tumor development and resistance or response to therapy.Areas covered: This review focuses on PMN heterogeneity and functional diversity in the context of carcinogenesis. TANs, by activating diverse signaling pathways, contribute to cancer progression and resistance to therapies. Mechanisms by which TANs impact therapeutic resistance include alterations of the tumoral DNA damage response, angiogenesis, reactivation of cancer dormancy, enhancement of tumor cell proliferation/survival and immune evasion.Expert opinion: With the emerging phenotypic and function heterogeneity of TANs, targeting specific TAN functions in developing tumors can lead to translatable therapeutic approaches and limit drug resistance. We propose that combining specific targeting of TAN activity with standard cancer therapy can help patients achieving a complete response and prevent cancer relapse.

Neutrophils Alter DNA Repair Landscape to Impact Survival and Shape Distinct Therapeutic Phenotypes of Colorectal Cancer.

BACKGROUND & AIMS: Tumor-infiltrating neutrophils (polymorphonuclear neutrophils [PMNs]) are a prominent feature of colorectal cancer (CRC), where they can promote cytotoxicity or exacerbate disease outcomes. We recently showed that in acute colon injury, PMNs can increase DNA double-strand break (DSB) burden and promote genomic instability via microRNA-dependent inhibition of homologous recombination (HR) repair. In this study, we aimed to establish whether in inflamed colon, neutrophils shape the DSB-repair responses to impact CRC progression and sensitivity/resistance to DNA-repair targeted therapy. METHODS: Human sporadic CRC biopsies, The Cancer Genome Atlas gene expression analyses, tumor xenografts, and murine CRC models, as well as small-molecule inhibition of key DSB-repair factors were leveraged to investigate changes in the DSB-repair landscape and identify unique CRC responses with/without tumor infiltration by PMNs. RESULTS: We reveal that neutrophils exert a functional dualism in cancer cells, driving temporal modulation of the DNA damage landscape and resolution of DSBs. PMNs were found to promote HR deficiency in low-grade CRC by miR-155-dependent downregulation of RAD51, thus attenuating tumor growth. However, neutrophil-mediated genotoxicity due to accumulation of DSBs led to the induction of non-homologous end-joining (NHEJ), allowing for survival and growth of advanced CRC. Our findings identified a PMN-induced HR-deficient CRC phenotype, featuring low RAD51 and low Ku70 levels, rendering it susceptible to synthetic lethality induced by clinically approved PARP1 inhibitor Olaparib. We further identified a distinct PMN-induced HR-deficient CRC phenotype, featuring high Ku70 and heightened NHEJ, which can be therapeutically targeted by specific inhibition of NHEJ. CONCLUSIONS: Our work delineates 2 mechanism-based translatable therapeutic interventions in sporadic CRC.

Diaphanous-related formin mDia2 regulates beta2 integrins to control hematopoietic stem and progenitor cell engraftment.

Bone marrow engraftment of the hematopoietic stem and progenitor cells (HSPCs) involves homing to the vasculatures and lodgment to their niches. How HSPCs transmigrate from the vasculature to the niches is unclear. Here, we show that loss of diaphanous-related formin mDia2 leads to impaired engraftment of long-term hematopoietic stem cells and loss of competitive HSPC repopulation. These defects are likely due to the compromised trans-endothelial migration of HSPCs since their homing to the bone marrow vasculatures remained intact. Mechanistically, loss of mDia2 disrupts HSPC polarization and induced cytoplasmic accumulation of MAL, which deregulates the activity of serum response factor (SRF). We further reveal that beta2 integrins are transcriptional targets of SRF. Knockout of beta2 integrins in HSPCs phenocopies mDia2 deficient mice. Overexpression of SRF or beta2 integrins rescues HSPC engraftment defects associated with mDia2 deficiency. Our findings show that mDia2-SRF-beta2 integrin signaling is critical for HSPC lodgment to the niches.

ICAM-1: A master regulator of cellular responses in inflammation, injury resolution, and tumorigenesis.

ICAM-1 is a cell surface glycoprotein and an adhesion receptor that is best known for regulating leukocyte recruitment from circulation to sites of inflammation. However, in addition to vascular endothelial cells, ICAM-1 expression is also robustly induced on epithelial and immune cells in response to inflammatory stimulation. Importantly, ICAM-1 serves as a biosensor to transduce outside-in-signaling via association of its cytoplasmic domain with the actin cytoskeleton following ligand engagement of the extracellular domain. Thus, ICAM-1 has emerged as a master regulator of many essential cellular functions both at the onset and at the resolution of pathologic conditions. Because the role of ICAM-1 in driving inflammatory responses is well recognized, this review will mainly focus on newly emerging roles of ICAM-1 in epithelial injury-resolution responses, as well as immune cell effector function in inflammation and tumorigenesis. ICAM-1 has been of clinical and therapeutic interest for some time now; however, several attempts at inhibiting its function to improve injury resolution have failed. Perhaps, better understanding of its beneficial roles in resolution of inflammation or its emerging function in tumorigenesis will spark new interest in revisiting the clinical value of ICAM-1 as a potential therapeutic target.

Intercellular Adhesion Molecule 1 Functions as an Efferocytosis Receptor in Inflammatory Macrophages.

Intercellular adhesion molecule-1 (ICAM-1) is up-regulated during inflammation by several cell types. ICAM-1 is best known for its role in mediating leukocyte adhesion to endothelial cells and guiding leukocytes across the vascular wall. Recently, macrophages have been shown to express ICAM-1, however, their role in macrophage function is unclear. We found that ICAM-1 expression was induced during inflammatory macrophage polarization and high numbers of ICAM-1-expressing macrophages were noted in inflamed colon tissue in a murine colitis model and in human inflammatory bowel disease. Because tissue macrophages play a critical role in removing apoptotic/necrotic cells in inflammation and injury, a process termed efferocytosis, it was examined whether ICAM-1 contributes to this process. Genetic deletion (ICAM-1 knockout mice) or siRNA-mediated knockdown of ICAM-1 in isolated murine and human macrophages significantly impaired apoptotic cell (AC) engulfment. Impairment in the engulfment of Jurkat T cells, neutrophils, and epithelial cells was confirmed ex vivo by inflammatory macrophages and in vivo by thioglycolate-recruited peritoneal macrophages. Decreased efferocytosis was also seen in vitro and in vivo with inhibition of ICAM-1 adhesive interactions, using a function blocking anti-ICAM-1 antibody. Mechanistically, it was found that ICAM-1 actively redistributes to cluster around engulfed ACs to facilitate macrophage-AC binding. Our findings define a new role for ICAM-1 in promoting macrophage efferocytosis, a critical process in the resolution of inflammation and restoration of tissue homeostasis.

Neutrophil-Derived Reactive Oxygen Orchestrates Epithelial Cell Signaling Events during Intestinal Repair.

Recent evidence has demonstrated that reactive oxygen (eg, hydrogen peroxide) can activate host cell signaling pathways that function in repair. We show that mice deficient in their capacity to generate reactive oxygen by the NADPH oxidase 2 holoenzyme, an enzyme complex highly expressed in neutrophils and macrophages, have disrupted capacity to orchestrate signaling events that function in mucosal repair. Similar observations were made for mice after neutrophil depletion, pinpointing this cell type as the source of the reactive oxygen driving oxidation-reduction protein signaling in the epithelium. To simulate epithelial exposure to high levels of reactive oxygen produced by neutrophils and gain new insight into this oxidation-reduction signaling, epithelial cells were treated with hydrogen peroxide, biochemical experiments were conducted, and a proteome-wide screen was performed using isotope-coded affinity tags to detect proteins oxidized after exposure. This analysis implicated signaling pathways regulating focal adhesions, cell junctions, and maintenance of the cytoskeleton. These pathways are also known to act via coordinated phosphorylation events within proteins that constitute the focal adhesion complex, including focal adhesion kinase and Crk-associated substrate. We identified the Rho family small GTP-binding protein Ras-related C3 botulinum toxin substrate 1 and p21 activated kinases 2 as operational in these signaling and localization pathways. These data support the hypothesis that reactive oxygen species from neutrophils can orchestrate epithelial cell-signaling events functioning in intestinal repair.

Progressing from Recurring Tissue Injury to Genomic Instability: A New Mechanism of Neutrophil Pathogenesis.

Aberrant neutrophil (PMN) infiltration of the intestinal mucosa is a hallmark of inflammatory bowel diseases, including Crohn's disease and ulcerative colitis. While the genotoxic function of PMNs and its implications in carcinogenesis have been primarily associated with oxidative stress, recent work by Butin-Israeli and colleagues has defined a novel mechanism where PMN-derived microparticles through the delivery and activity of specific miRNAs promoted formation of double-strand breaks (DSBs), and in parallel, suppressed DSB repair through the downregulation of lamin B1 and Rad51. Respective downregulation of these two proteins compromised the nuclear envelope and high-fidelity repair by homologous recombination, increasing DSB accumulation and aneuploidy. This discovery defined a novel mode of action where PMN-mediated suppression of DSB repair leading to genomic instability in the injured mucosa may facilitate progression toward colorectal cancer.

Neutrophil-induced genomic instability impedes resolution of inflammation and wound healing.

Neutrophil (PMN) infiltration of the intestinal mucosa is a hallmark of tissue injury associated with inflammatory bowel diseases (IBDs). The pathological effects of PMNs are largely attributed to the release of soluble mediators and reactive oxygen species (ROS). We identified what we believe is a new, ROS-independent mechanism whereby activated tissue-infiltrating PMNs release microparticles armed with proinflammatory microRNAs (miR-23a and miR-155). Using IBD clinical samples, and in vitro and in vivo injury models, we show that PMN-derived miR-23a and miR-155 promote accumulation of double-strand breaks (DSBs) by inducing lamin B1-dependent replication fork collapse and inhibition of homologous recombination (HR) by targeting HR-regulator RAD51. DSB accumulation in injured epithelium led to impaired colonic healing and genomic instability. Targeted inhibition of miR-23a and miR-155 in cultured intestinal epithelial cells and in acutely injured mucosa decreased the detrimental effects of PMNs and enhanced tissue healing responses, suggesting that this approach can be used in therapies aimed at resolution of inflammation, in wound healing, and potentially to prevent neoplasia.

In vivo imaging reveals unique neutrophil transendothelial migration patterns in inflamed intestines.

Neutrophil (PMN) infiltration of the intestinal mucosa is a hallmark of gastrointestinal inflammation, with significant implications for host defense, injury and repair. However, phenotypic and mechanistic aspects of PMN recruitment in inflamed intestines have not been explored in vivo. Using novel epithelial/PMN fluorescence reporter mice, advanced intravital imaging and 3D reconstruction analysis, we mapped the microvasculature architecture across the intestinal layers and determined that in response to Salmonella/endotoxin-induced inflammation, PMN transendothelial migration (TEM) was restricted to submucosal vessels. PMN TEM was not observed in villus or crypt vessels, proximal to the epithelium that underlies the intestinal lumen, and was partially dependent on (C-X-C motif) ligands 1 (CXCL1) and 2 (CXCL2) expression, which was found to be elevated in the submucosa layer. Restricted PMN extravasation at the submucosa and subsequent PMN interstitial migration may serve as a novel regulatory step of PMN effector function and recruitment to the luminal space in inflamed intestines.

Extracellular vesicles regulate immune responses and cellular function in intestinal inflammation and repair.

Tightly controlled communication among the various resident and recruited cells in the intestinal tissue is critical for maintaining tissue homeostasis, re-establishment of the barrier function and healing responses following injury. Emerging evidence convincingly implicates extracellular vesicles (EVs) in facilitating this important cell-to-cell crosstalk by transporting bioactive effectors and genetic information in healthy tissue and disease. While many aspects of EV biology, including release mechanisms, cargo packaging, and uptake by target cells are still not completely understood, EVs contribution to cellular signaling and function is apparent. Moreover, EV research has already sparked a clinical interest, as a potential diagnostic, prognostic and therapeutic tool. The current review will discuss the function of EVs originating from innate immune cells, namely, neutrophils, monocytes and macrophages, as well as intestinal epithelial cells in healthy tissue and inflammatory disorders of the intestinal tract. Our discussion will specifically emphasize the contribution of EVs to the regulation of vascular and epithelial barrier function in inflamed intestines, wound healing, as well as trafficking and activity of resident and recruited immune cells.

Loss of pleckstrin-2 reverts lethality and vascular occlusions in JAK2V617F-positive myeloproliferative neoplasms.

V617F driver mutation of JAK2 is the leading cause of the Philadelphia-chromosome-negative myeloproliferative neoplasms (MPNs). Although thrombosis is a leading cause of mortality and morbidity in MPNs, the mechanisms underlying their pathogenesis are unclear. Here, we identified pleckstrin-2 (Plek2) as a downstream target of the JAK2/STAT5 pathway in erythroid and myeloid cells, and showed that it is upregulated in a JAK2V617F-positive MPN mouse model and in patients with MPNs. Loss of Plek2 ameliorated JAK2V617F-induced myeloproliferative phenotypes including erythrocytosis, neutrophilia, thrombocytosis, and splenomegaly, thereby reverting the widespread vascular occlusions and lethality in JAK2V617F-knockin mice. Additionally, we demonstrated that a reduction in red blood cell mass was the main contributing factor in the reversion of vascular occlusions. Thus, our study identifies Plek2 as an effector of the JAK2/STAT5 pathway and a key factor in the pathogenesis of JAK2V617F-induced MPNs, pointing to Plek2 as a viable target for the treatment of MPNs.

Expression of Lewis-a glycans on polymorphonuclear leukocytes augments function by increasing transmigration.

PMN-expressed fucosylated glycans from the Lewis glycan family, including Lewis-x (Lex) and sialyl Lewis-x (sLex), have previously been implicated in the regulation of important PMN functions, including selectin-mediated trafficking across vascular endothelium. Although glycans, such as Lex and sLex, which are based on the type 2 sequence (Galß1-4GlcNAc-R), are abundant on PMNs, the presence of type 1 Galß1-3GlcNAc-R glycans required for PMN expression of the closely related stereoisomer of Lex, termed Lewis-A (Lea), has not, to our knowledge, been reported. Here, we show that Lea is abundantly expressed by human PMNs and functionally regulates PMN migration. Using mAbs whose precise epitopes were determined using glycan array technology, Lea function was probed using Lea-selective mAbs and lectins, revealing increased PMN transmigration across model intestinal epithelia, which was independent of epithelial-expressed Lea Analyses of glycan synthetic machinery in PMNs revealed expression of ß1-3 galactosyltransferase and α1-4 fucosyltransferase, which are required for Lea synthesis. Specificity of functional effects observed after ligation of Lea was confirmed by failure of anti-Lea mAbs to enhance migration using PMNs from individuals deficient in α1-4 fucosylation. These results demonstrate that Lea is expressed on human PMNs, and its specific engagement enhances PMN migration responses. We propose that PMN Lea represents a new target for modulating inflammation and regulating intestinal, innate immunity.