Neutrophils are innate immune effector cells that migrate from the blood to resolve bacterial and fungal infections. Understanding how neutrophils migrate is critical for regulating excessive inflammation and subsequent collateral injury. β2 integrins are essential to classical neutrophil recruitment from the blood, and the activation of β2 integrins has been well defined in previous studies. Adhesion stabilization of neutrophils on the endothelial surface as they crawl into a favorable position for transmigration is not as well defined. Neutrophils do not make mature focal adhesions, but do express the focal adhesion protein vinculin. Vinculin associates with integrins by binding to talin‐1 and stabilizes integrin adhesions by recruiting various actin‐associated proteins or by associating with actin directly. This study characterizes the role of vinculin in neutrophil β2 integrin‐dependent adhesion, motility and anti‐bacterial function. Intrinsic activation of β2 integrins is unaffected by vinculin knockout after CXCL1 activation. Vinculin knockout attenuates neutrophil adhesion, spreading, and motility on glass coated with β2 integrin ligand, ICAM‐1, and activating CXCL1. Vinculin knockout also reduces neutrophil spreading in response to ICAM‐1/CXCL1 on polyacrylamide gels of high stiffness but not lower stiffness. Vinculin knockout reduces traction stresses of neutrophils and the actin stiffening response after stimulation. Unlike static conditions, vinculin knockout does not affect neutrophil motility under flow conditions. Vinculin knockout attenuates respiratory burst, but does not affect phagocytosis. In mixed chimeric mice given intraperitoneal thioglycollate, we find comparable migration of vinculin‐knockout and vinculin‐sufficient neutrophils into the peritoneum. Altogether, while vinculin enhances neutrophil β2 integrin adhesion strength, vinculin knockout does not affect neutrophil motility and trafficking under physiological conditions. Support or Funding Information American Heart Association (12SDG12080281), Scientist Development Grant CL Department of Surgery, Rhode Island Hospital This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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 extracellular matrix (ECM). Neutrophils undergo a rapid, ECM-dependent mechanism of homotypic aggregation and NETosis in response to C. albicans mediated by the β2 integrin, Complement Receptor 3 (CR3, CD11b/CD18, αMβ2). Neither homotypic aggregation nor NETosis occurs when human neutrophils are exposed either to immobilized fungal β-glucan or to C. albicans hyphae without ECM. The current study provides a mechanistic basis to explain how matrix controls the anti-fungal effector functions of neutrophils under conditions that preclude phagocytosis. We show that CR3 ligation initiates a complex mechanism of integrin cross-talk resulting in differential regulation of the β1 integrins VLA3 (α3β1) and VLA5 (α5β1). These β1 integrins control distinct anti-fungal effector functions in response to either fungal β-glucan or C. albicans hyphae and fibronectin (Fn), with VLA3 inducing homotypic aggregation and VLA5 regulating NETosis. These integrin-dependent effector functions are controlled temporally whereby VLA5 and CR3 induce rapid, focal NETosis early after binding Fn and β-glucan. Within minutes, CR3 undergoes inside-out auto-activation that drives the down-regulation of VLA5 and the up-regulation of VLA3 to support neutrophil swarming and aggregation. Forcing VLA5 to remain in the activated state permits NETosis but prevents homotypic aggregation. Therefore, CR3 serves as a master regulator during the antifungal neutrophil response, controlling the affinity states of two different β1 integrins which in turn elicit distinct effector functions.

We introduce a novel method to compute three-dimensional (3D) displacements and both in-plane and out-of-plane tractions on nominally planar transparent materials using standard epifluorescence microscopy. Despite the importance of out-of-plane components to fully understanding cell behavior, epifluorescence images are generally not used for 3D traction force microscopy (TFM) experiments due to limitations in spatial resolution and measuring out-of-plane motion. To extend an epifluorescence-based technique to 3D, we employ a topology-based single particle tracking algorithm to reconstruct high spatial-frequency 3D motion fields from densely seeded single-particle layer images. Using an open-source finite element (FE) based solver, we then compute the 3D full-field stress and strain and surface traction fields. We demonstrate this technique by measuring tractions generated by both single human neutrophils and multicellular monolayers of Madin–Darby canine kidney cells, highlighting its acuity in reconstructing both individual and collective cellular tractions. In summary, this represents a new, easily accessible method for calculating fully three-dimensional displacement and 3D surface tractions at high spatial frequency from epifluorescence images. We released and support the complete technique as a free and open-source code package.

In vitro and in vivo experiments were conducted to determine whether intraluminal saline in breast implants can support the growth of common wound-infecting microorganisms over a prolonged period of time. The bacteria tested were Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Corynebacterium jeikeium, Enterobacter cloacae, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Three fungal species also were tested: Aspergillus fumigatus, Paecilomyces variotii, and Candida albicans. In the in vitro study, four organisms survived in flasks of sterile saline for the 2 weeks in which serial cultures were performed: K. pneumoniae, C. albicans, A. fumigatus, and P. variotii. In the in vivo study, 61 white rabbits (122 implants) received both an experimental implant inoculated with one of the test organisms and a control implant containing only sterile saline. They were sacrificed at 1-, 3-, or 6-month scheduled endpoints. None of the control implants containing sterile saline had positive cultures. In contrast, the intraluminal saline was culture positive for 7 of the 10 inoculated organisms after varying lengths of time: S. epidermidis, E. coli, E. cloacae, K. pneumoniae, P. aeruginosa, A. fumigatus, and P. variotii. Samples of capsular tissue also were cultured. Of the 122 capsular tissue specimens, 21 (17 percent) had positive cultures and surrounded both inoculated and sterile implants. In most instances, capsules that were culture positive contained an organism different from the one that had been inoculated in the group. In only 3 cases was the same organism cultured from both the periprosthetic tissue and the intraluminal saline, and these may represent instances of the inoculated organism migrating through the implants filler valves. The data show that several types of bacteria (particularly gram-negative species) and fungi can grow and reproduce in a restricted saline environment for extended periods of time.

The generation of traction forces by neutrophils regulates many crucial effector functions responsible for host defense, such as attachment, spreading, migration, phagocytosis, and NETosis. The activation state of the cell is a strong determinant of the functional efficacy of the neutrophil; however, the effect of activation on traction force production has not yet been determined experimentally. Previously, the mapping of cellular-generated forces produced by human neutrophils via a Traction Force Microscopy (TFM) method has required a three-dimensional imaging modality to capture out-of-plane forces, such as confocal or multiphoton techniques. A method newly developed in our laboratories can capture out-of-plane forces using only a two-dimensional imaging modality. This novel technique—combined with a topology-based single particle tracking algorithm and finite element method calculations—can construct high spatial frequency three-dimensional traction fields, allowing for traction forces in-plane and out-of-plane to the substrate to now be differentially visualized and quantified with a standard epifluorescence microscope. Here we apply this technology to determine the effect of neutrophil activation on force generation. Sepsis is a systemic inflammatory response that causes dysregulated neutrophil activation in vivo. We found that neutrophils from septic patients produced greater total forces than neutrophils from healthy donors and that the majority of this dysregulation occurred in-plane to the substrate. Ex vivo activation of neutrophils from healthy donors showed differential consequences depending on activation stimuli with mechanosensitive force decreases observed in some cases. These findings demonstrate the feasibility of epifluorescence-based microscopy in mapping traction forces to ask biologically significant questions regarding neutrophil function.

No abstract

The generation of traction forces by neutrophils regulates many crucial effector functions responsible for host defense, such as attachment, spreading, migration, phagocytosis, and NETosis. The activation state of the cell is a strong determinant of the functional efficacy of the neutrophil; however, the effect of activation on traction force production has not yet been determined experimentally. Previously, the mapping of cellular-generated forces produced by human neutrophils via a Traction Force Microscopy (TFM) method has required a three-dimensional imaging modality to capture out-of-plane forces, such as confocal or multiphoton techniques. A method newly developed in our laboratories can capture out-of-plane forces using only a two-dimensional imaging modality. This novel technique—combined with a topology-based single particle tracking algorithm and finite element method calculations—can construct high spatial frequency three-dimensional traction fields, allowing for traction forces in-plane and out-of-plane to the substrate to now be differentially visualized and quantified with a standard epifluorescence microscope. Here we apply this technology to determine the effect of neutrophil activation on force generation. Sepsis is a systemic inflammatory response that causes dysregulated neutrophil activation in vivo. We found that neutrophils from septic patients produced greater total forces than neutrophils from healthy donors and that the majority of this dysregulation occurred in-plane to the substrate. Ex vivo activation of neutrophils from healthy donors showed differential consequences depending on activation stimuli with mechanosensitive force decreases observed in some cases. These findings demonstrate the feasibility of epifluorescence-based microscopy in mapping traction forces to ask biologically significant questions regarding neutrophil function.

Neutrophils are innate immune effector cells that traffic from the peripheral blood to extravascular sites of inflammation. β2 integrins are involved during multiple phases of neutrophil recruitment, including the transition from rolling to arrest, firm attachment and motility within the vasculature. Following neutrophil arrest, adhesion stabilization occurs as the neutrophil interacts with the endothelial surface and crawls into a favorable position for extravasation. The cytoskeletal protein vinculin has been implicated in other cell types as a regulator of adhesion strength by promoting focal adhesion maturation and as a sensor of the mechanical properties of the microenvironment. Neutrophils express vinculin but do not form mature focal adhesions. Here, we characterize the role of vinculin in β2 integrin-dependent neutrophil adhesion, motility, mechanosensing, and recruitment. We observe that knockout of vinculin attenuates, but does not completely abrogate, neutrophil adhesion, spreading, and crawling under static conditions. In the presence of forces from fluid flow, vinculin was not required for neutrophil adhesion or migration. Vinculin deficiency only mildly attenuated neutrophil traction stresses and spreading on stiff, but not soft, polyacrylamide gels indicating a minor role for vinculin in the mechanosensing of the neutrophil as compared to slower moving mesenchymal cells that form mature focal adhesions. Consistent with these findings, we observe in vivo neutrophil recruitment into the inflamed peritoneum of mice remains intact in the absence of vinculin. Together, these data suggest that while vinculin regulates some aspects of neutrophil adhesion and spreading, it may be dispensable for neutrophil recruitment and motility in vivo.