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.

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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.

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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.

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 InformationAmerican Heart Association (12SDG12080281), Scientist Development Grant CLDepartment of Surgery, Rhode Island HospitalThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mapping of tractional forces produced by human neutrophils enables a precise understanding of mechanisms responsible for well‐regulated cell motility. In collaboration with the Henann Lab at Brown University's Department of Solid Mechanics and the Franck Lab at University of Wisconsin's College of Engineering, we have developed a novel method of quantifying cellular force generation. Previously, Traction Force Microscopy (TFM) methods have required a 3D imaging modality to capture 3D forces. Here, we demonstrate a new method that can capture 3D forces using only a 2D imaging modality. Therefore, both forces in‐plane with the substrate as well as forces out‐of‐plane to the substrate can now be seen with a standard epifluorescent microscope. Our technique involves embedding a single layer of fiducial markers under the surface of a soft fibronectin‐coated polyacrylamide gel, followed by our previously published single particle tracking algorithm (T‐PT) and a novel finite element method to quantify forces from substrate displacement. To validate this system, PMNs were obtained from healthy donors with ex vivo activation and from septic donors. Sepsis is a potentially fatal systemic inflammatory response to infection that can progress to multiorgan system failure. We hypothesize that ex vivo activation of PMNs from healthy donors and different substrate stiffnesses will lead to differential force production. On soft substrates (1kPa), when comparing PMN force generation from healthy and septic donors, we found that neutrophils from septic donors produce greater forces. However, ex vivo activation of PMNs from healthy donors with Lipopolysaccharide (LPS) had no effect, while a stimulant cocktail of LPS, Interferon Gamma (IFN‐g), and Granulocyte‐Macrophage Colony‐Stimulating Factor (GM‐CSF) decreased force generation. Further, ex vivo activation with Phorbol Myristate Acetate (PMA) and N‐Formylmethionine‐leucyl‐phenylalanine (fMLP) increased force generation. However, on stiff substrates (10kPa) LPS, PMA, and fMLP all had no effect on force generation, while the stimulant cocktail decreased total forces. Taken together with our novel TFM technique, these findings indicate that PMNs from septic patients are abnormal in their spatiotemporal generation of 3D forces. Further, secondary stimulation leads to differential dysregulation of force generation that is also mechanosensitive to substrate stiffness. Because there is still the need to further elucidate the deleterious molecular mechanisms during PMN‐mediated sepsis, our methods will help bridge the divide between understanding dysregulation both in its biochemical and its mechanical interactions with the microenvironment.

Approximately 55,500 proximal humeral fractures require surgical fixation annually. The current standard for internal humeral fracture fixation involves implantation of rigid metallic devices to prevent dislocation of bone fragments. However, these devices have high stiffness characteristics which can cause stress shielding in bone. A second method of fixation, called biological fixation, decreases stiffness which reduces stress shielding by utilizing more flexible devices. This approach tends leads to increased incidences of delayed healing and nonunion of fracture fragments. Therefore, this device design implements two bioabsorbable polymers in two distinct layers that degrade at different rates. The purpose of this design is to provide rigid fixation during the initial fracture healing phase followed by a period of biological fixation, allowing for functional healing along with a reduction in stress shielding over time compared to current devices. The bioabsorbable property permits the device to remain in situ, thus eliminating the need for removal surgery and reducing the risk of surgical site infection. Using finite element analysis, the design has been demonstrated to exhibit varying axial, torsional, and flexural stiffness over time. The final device was fabricated by injection molding, and tested for flexural stiffness. In addition, the polymers were tested for stiffness at specific time intervals over the course of the degradation period. All stiffness tests were performed under simple three point loads. A Nikon 3200 camera (Nikon Inc., Melville, NY) was used to sequentially image the material samples and plate throughout each load application. The flexural stiffness of the device was determined by utilizing Digital Image Correlation analysis in Matlab (MathWorks, Inc.) to analyze surface displacements between image frames. The success of the device was determined by comparing the observed difference in stiffness to standard stiffness values for humeral fixation devices currently available on the market. A substantial decrease in stiffness combines the benefits of rigid and biological fixation devices as well as eliminates the complications associated with each, providing an improved solution for proximal humeral fractures.