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.

PTEN, a tumor suppressor protein that dephosphorylates phosphoinositides at the 3-position of the inositol ring, is a cytosolic protein that needs to associate with the plasma membrane or other subcellular membranes to exert its lipid phosphatase function. Upon membrane association PTEN interacts with at least three different lipid entities: An anionic lipid that is present in sufficiently high concentration to create a negative potential that allows PTEN to interact electrostatically with the membrane, phosphatidylinositol-4,5-bisphosphate, which interacts with PTEN's N-terminal end and the substrate, usually phosphatidylinositol-3,4,5-trisphosphate. Many parameters influence PTEN's interaction with the lipid bilayer, for example, the lateral organization of the lipids or the presence of other chemical species like cholesterol or other lipids. To investigate systematically the different steps of PTEN's complex binding mechanism and to explore its dynamic behavior in the membrane bound state, in vitro methods need to be employed that allow for a systematic variation of the experimental conditions. In this review we survey a variety of methods that can be used to assess PTEN lipid binding affinity, the dynamics of its membrane association as well as its dynamic behavior in the membrane bound state.

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 in vivo 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. Our results indicate that HoxB8-conditional progenitors engraft in a β1 integrin-dependent manner and transiently generate donor-derived mature neutrophils. Furthermore, we show that neutrophils derived in vivo from transplanted HoxB8-conditional progenitors are mobilized to the periphery and recruited to sites of inflammation in a manner that depends on the C-X-C chemokine receptor 2 and β2 integrins, the same mechanisms that have been described for recruitment of endogenous primary neutrophils. Together, our studies advance the understanding of HoxB8-conditional neutrophil progenitors and describe an innovative tool that, by virtue of its ability to engraft in the naïve host, will facilitate mechanistic in vivo experimentation on neutrophils.

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 in vivo 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. Our results indicate that HoxB8-conditional progenitors engraft in a β1 integrin-dependent manner and transiently generate donor-derived mature neutrophils. Furthermore, we show that neutrophils derived in vivo from transplanted HoxB8-conditional progenitors are mobilized to the periphery and recruited to sites of inflammation in a manner that depends on the C-X-C chemokine receptor 2 and β2 integrins, the same mechanisms that have been described for recruitment of endogenous primary neutrophils. Together, our studies advance the understanding of HoxB8-conditional neutrophil progenitors and describe an innovative tool that, by virtue of its ability to engraft in the naïve host, will facilitate mechanistic in vivo experimentation on neutrophils.

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.

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