Bi-fluorescent infection enables single-cell analysis of intracellular killing .

Techniques for studying the clearance of bacterial infections are critical for advances in understanding disease states, immune cell effector functions, and novel antimicrobial therapeutics. Intracellular killing of by neutrophils can be monitored using a strain stably expressing GFP, a fluorophore that is quenched when exposed to the reactive oxygen species (ROS) present in the phagolysosome. Here, we expand upon this method by developing a bi-fluorescent killing assay for use Conjugating with a stable secondary fluorescent marker enables the separation of infected cell samples into three populations: cells that have not engaged in phagocytosis, cells that have engulfed and killed , and cells that have viable internalized .

Identification and mechanistic basis of non-ACE2 blocking neutralizing antibodies from COVID-19 patients with deep RNA sequencing and molecular dynamics simulations.

Variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continue to cause disease and impair the effectiveness of treatments. The therapeutic potential of convergent neutralizing antibodies (NAbs) from fully recovered patients has been explored in several early stages of novel drugs. Here, we identified initially elicited NAbs (Ig Heavy, Ig lambda, Ig kappa) in response to COVID-19 infection in patients admitted to the intensive care unit at a single center with deep RNA sequencing (>100 million reads) of peripheral blood as a diagnostic tool for predicting the severity of the disease and as a means to pinpoint specific compensatory NAb treatments.

Natriuretic peptide receptor-C mediates the inhibitory effect of atrial natriuretic peptide on neutrophil recruitment to the lung during acute lung injury.

Atrial natriuretic peptide (ANP) protects against acute lung injury (ALI), but the receptor that mediates this effect is not known. Transgenic mice with 0 (knockout), 1 (heterozygote), or 2 (wild-type) functional copies of , the gene that encodes for natriuretic peptide receptor-C (NPR-C), were treated with intravenous infusion of ANP or saline vehicle before oropharyngeal aspiration of (PA103) or saline vehicle. Lung injury was assessed 4 h following aspiration by measurement of lung wet/dry (W/D) weight, whole lung leukocyte and cytokine levels, and protein, leukocyte, and cytokine concentration in bronchoalveolar lavage fluid (BALF).

HS1 deficiency protects against sepsis by attenuating neutrophil-inflicted lung damage.

Sepsis remains an important health problem worldwide due to inefficient treatments often resulting in multi-organ failure. Neutrophil recruitment is critical during sepsis. While neutrophils are required to combat invading bacteria, excessive neutrophil recruitment contributes to tissue damage due to their arsenal of molecular weapons that do not distinguish between host and pathogen.

Engraftment, Fate, and Function of HoxB8-Conditional Neutrophil Progenitors in the Unconditioned Murine Host.

The development and use of murine myeloid progenitor cell lines that are conditionally immortalized through expression of HoxB8 has provided a valuable tool for studies of neutrophil biology. Recent work has extended the utility of HoxB8-conditional progenitors to the setting via their transplantation into irradiated mice. Here, we describe the isolation of HoxB8-conditional progenitor cell lines that are unique in their ability to engraft in the naïve host in the absence of conditioning of the hematopoietic niche.

A Mouse Model of Pseudomonas aeruginosa Pneumonia.

The rapid innate immune response to respiratory infection is essential to prevent the systemic dissemination of pathogens. This chapter outlines an experimental mouse model of respiratory infection by gram-negative Pseudomonas aeruginosa and analyses of leukocyte trafficking in the lungs. The reader will learn two methods to induce respiratory infection in mice that differ in whether the initial bolus is targeted within a specific lobe of the lung.

Murine Myeloid Progenitors Attenuate Immune Dysfunction Induced by Hemorrhagic Shock.

Hemorrhagic shock induces an aberrant immune response characterized by simultaneous induction of a proinflammatory state and impaired host defenses. The objective of this study was to evaluate the impact of conditionally immortalized neutrophil progenitors (NPs) on this aberrant immune response. We employed a mouse model of hemorrhagic shock, followed by the adoptive transfer of NPs and subsequent inoculation of Staphylococcus aureus to induce pneumonia.

Distinct Contributions of CD18 Integrins for Binding and Phagocytic Internalization of Pseudomonas aeruginosa.

Phagocytosis is the key mechanism for host control of , a motile Gram-negative, opportunistic bacterial pathogen which frequently undergoes adaptation and selection for traits that are advantageous for survival. One such clinically relevant adaptation is the loss of bacterial motility, observed within chronic infections, that is associated with increased antibiotic tolerance and phagocytic resistance. Previous studies using phagocytes from a leukocyte adhesion deficiency type 1 (LAD-I) patient identified CD18 as a putative cell surface receptor for uptake of live However, how bacterial motility alters direct engagement with CD18-containing integrins remains unknown.

Context-Dependent Role of Vinculin in Neutrophil Adhesion, Motility and Trafficking.

Neutrophils are innate immune effector cells that traffic from the circulation to extravascular sites of inflammation. β2 integrins are important mediators of the processes involved in neutrophil recruitment. Although neutrophils express the cytoskeletal protein vinculin, they do not form mature focal adhesions.

Differential attenuation of β2 integrin-dependent and -independent neutrophil migration by Ly6G ligation.

Antibody ligation of the murine neutrophil surface protein Ly6G disrupts neutrophil migration in some contexts but not others. We tested whether this variability reflected divergent dependence of neutrophil migration on β2 integrins, adhesion molecules that interact with Ly6G at the neutrophil surface. In integrin-dependent murine arthritis, Ly6G ligation attenuated joint inflammation, even though mice lacking Ly6G altogether developed arthritis normally.

CD177 modulates human neutrophil migration through activation-mediated integrin and chemoreceptor regulation.

CD177 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed by a variable proportion of human neutrophils that mediates surface expression of the antineutrophil cytoplasmic antibody antigen proteinase 3. CD177 associates with β2 integrins and recognizes platelet endothelial cell adhesion molecule 1 (PECAM-1), suggesting a role in neutrophil migration. However, CD177 neutrophils exhibit no clear migratory advantage in vivo, despite interruption of in vitro transendothelial migration by CD177 ligation.

Activated β Integrins Restrict Neutrophil Recruitment during Murine Acute Pseudomonal Pneumonia.

Rapid neutrophil recruitment is critical for the efficient clearance of bacterial pathogens from the lungs. Although β integrins and their activation are required for neutrophil recruitment from postcapillary venules of the systemic circulation into inflamed tissues, the involvement of integrins in neutrophil recruitment in response to respiratory infection varies among bacterial pathogens. For stimuli eliciting β integrin-dependent neutrophil influx, including Pseudomonas aeruginosa, it remains unclear whether the activation of β integrins is an essential step in this process.

Integrin Cross-Talk Regulates the Human Neutrophil Response to Fungal β-Glucan in the Context of the Extracellular Matrix: A Prominent Role for VLA3 in the Antifungal Response.

Candida albicans infection produces elongated hyphae resistant to phagocytic clearance compelling alternative neutrophil effector mechanisms to destroy these physically large microbial structures. Additionally, all tissue-based neutrophilic responses to fungal infections necessitate contact with the extracellular matrix (ECM). Neutrophils undergo a rapid, ECM-dependent mechanism of homotypic aggregation and NETosis in response to C.

The mechanism of kindlin-mediated activation of integrin αIIbβ3.

Increased ligand binding to cellular integrins ("activation") plays important roles in processes such as development, cell migration, extracellular matrix assembly, tumor metastasis, hemostasis, and thrombosis. Integrin activation encompasses both increased integrin monomer affinity and increased receptor clustering and depends on integrin-talin interactions. Loss of kindlins results in reduced activation of integrins.

The PSGL-1-L-selectin signaling complex regulates neutrophil adhesion under flow.

Neutrophils are recruited from the blood to sites of inflammation, where they contribute to immune defense but may also cause tissue damage. During inflammation, neutrophils roll along the microvascular endothelium before arresting and transmigrating. Arrest requires conformational activation of the integrin lymphocyte function-associated antigen 1 (LFA-1), which can be induced by selectin engagement.

Neutrophil arrest by LFA-1 activation.

Lymphocyte function-associated antigen-1 (LFA-1) is a heterodimeric integrin consisting of α(L) (gene name, Itgal) and β(2) (gene name, Itgb2) subunits expressed in all leukocytes. LFA-1 is essential for neutrophil recruitment to inflamed tissue. Activation of LFA-1 by chemokines allows neutrophils and other leukocytes to undergo arrest, resulting in firm adhesion on endothelia expressing intercellular adhesion molecules (ICAMs).

Global metabolic inhibitors of sialyl- and fucosyltransferases remodel the glycome.

Despite the fundamental roles of sialyl- and fucosyltransferases in mammalian physiology, there are few pharmacological tools to manipulate their function in a cellular setting. Although fluorinated analogs of the donor substrates are well-established transition state inhibitors of these enzymes, they are not membrane permeable. By exploiting promiscuous monosaccharide salvage pathways, we show that fluorinated analogs of sialic acid and fucose can be taken up and metabolized to the desired donor substrate-based inhibitors inside the cell.

Distinct roles for talin-1 and kindlin-3 in LFA-1 extension and affinity regulation.

In inflammation, neutrophils and other leukocytes roll along the microvascular endothelium before arresting and transmigrating into inflamed tissues. Arrest requires conformational activation of the integrin lymphocyte function-associated antigen-1 (LFA-1). Mutations of the FERMT3 gene encoding kindlin-3 underlie the human immune deficiency known as leukocyte adhesion deficiency-III.

Monitoring integrin activation by fluorescence resonance energy transfer.

Aberrant integrin activation is associated with several immune pathologies. In leukocyte adhesion deficiency (LAD), the absence or inability of β(2) integrins to undergo affinity upregulation contributes to recurrent infectious episodes and impaired wound healing, while excessive integrin activity leads to an exaggerated inflammatory response with associated tissue damage. Therefore, integrin activation is an attractive target for immunotherapies, and monitoring the effect of agents on integrin activation is necessary during preclinical drug development.

Adam17-dependent shedding limits early neutrophil influx but does not alter early monocyte recruitment to inflammatory sites.

TNF-α-converting enzyme (TACE, herein denoted as Adam17) proteolytically sheds several cell-surface inflammatory proteins, but the physiologic importance of the cleavage of these substrates from leukocyte subsets during inflammation is incompletely understood. In this study, we show that Adam17-null neutrophils have a 2-fold advantage in their initial recruitment during thioglycollate-induced peritonitis, and they roll slower and adhere more readily in the cremaster model than wild-type neutrophils. Although CD44 and ICAM-1 are both in vitro substrates of Adam17, their surface levels are not altered on Adam17-null neutrophils.

N-cadherin cell-cell adhesion complexes are regulated by fibronectin matrix assembly.

Fibronectin is a principal component of the extracellular matrix. Soluble fibronectin molecules are assembled into the extracellular matrix as insoluble, fibrillar strands via a cell-dependent process. In turn, the interaction of cells with the extracellular matrix form of fibronectin stimulates cell functions critical for tissue repair.

Human T lymphocyte isolation, culture and analysis of migration in vitro.

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility.

Combinatorial libraries against libraries for selecting neoepitope activation-specific antibodies.

A systematic approach to the discovery of conformation-specific antibodies or those that recognize activation-induced neoepitopes in signaling molecules and enzymes will be a powerful tool in developing antibodies for basic science and therapy. Here, we report the isolation of antibody antagonists that preferentially bind activated integrin Mac-1 (alpha(M)beta(2)) and are potent in blocking neutrophil adhesion and migration. A novel strategy was developed for this task, consisting of yeast surface display of Mac-1 inserted (I) domain library, directed evolution to isolate active mutants of the I domain, and screening of phage display of human antibody library against the active I domain in yeast.

Outside-in signal transmission by conformational changes in integrin Mac-1.

Intracellular signals associated with or triggered by integrin ligation can control cell survival, differentiation, proliferation, and migration. Despite accumulating evidence that conformational changes regulate integrin affinity to its ligands, how integrin structure regulates signal transmission from the outside to the inside of the cell remains elusive. Using fluorescence resonance energy transfer, we addressed whether conformational changes in integrin Mac-1 are sufficient to transmit outside-in signals in human neutrophils.