Statistical method for testing the neutral mutation hypothesis by DNA polymorphism.

To get to the site of inflammation, leukocytes must first adhere to and traverse the blood-vessel wall, events that occur in a cascade-like manner. But what are the exact steps in this cascade and what molecules are involved?

Getting to the site of inflammation: the leukocyte adhesion cascade updated

Neutrophils have traditionally been thought of as simple foot soldiers of the innate immune system with a restricted set of pro-inflammatory functions. More recently, it has become apparent that neutrophils are, in fact, complex cells capable of a vast array of specialized functions. Although neutrophils are undoubtedly major effectors of acute inflammation, several lines of evidence indicate that they also contribute to chronic inflammatory conditions and adaptive immune responses. Here, we discuss the key features of the life of a neutrophil, from its release from bone marrow to its death. We discuss the possible existence of different neutrophil subsets and their putative anti-inflammatory roles. We focus on how neutrophils are recruited to infected or injured tissues and describe differences in neutrophil recruitment between different tissues. Finally, we explain the mechanisms that are used by neutrophils to promote protective or pathological immune responses at different sites.

Neutrophil recruitment and function in health and inflammation

Reactive oxygen species (ROS) are key signaling molecules that play an important role in the progression of inflammatory disorders. An enhanced ROS generation by polymorphonuclear neutrophils (PMNs) at the site of inflammation causes endothelial dysfunction and tissue injury. The vascular endothelium plays an important role in passage of macromolecules and inflammatory cells from the blood to tissue. Under the inflammatory conditions, oxidative stress produced by PMNs leads to the opening of inter-endothelial junctions and promotes the migration of inflammatory cells across the endothelial barrier. The migrated inflammatory cells not only help in the clearance of pathogens and foreign particles but also lead to tissue injury. The current review compiles the past and current research in the area of inflammation with particular emphasis on oxidative stress-mediated signaling mechanisms that are involved in inflammation and tissue injury.

Reactive Oxygen Species in Inflammation and Tissue Injury

Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

Integrative Genomics Viewer

The prevailing view is that the beta2-integrins Mac-1 (alphaMbeta2, CD11b/CD18) and LFA-1 (alphaLbeta2, CD11a/CD18) serve similar biological functions, namely adhesion, in the leukocyte recruitment cascade. Using real-time and time-lapse intravital video-microscopy and confocal microscopy within inflamed microvessels, we systematically evaluated the function of Mac-1 and LFA-1 in the recruitment paradigm. The chemokine macrophage inflammatory protein-2 induced equivalent amounts of adhesion in wild-type and Mac-1-/- mice but very little adhesion in LFA-1-/- mice. Time-lapse video-microscopy within the postcapillary venules revealed that immediately upon adhesion, there is significant intraluminal crawling of all neutrophils to distant emigration sites in wild-type mice. In dramatic contrast, very few Mac-1-/- neutrophils crawled with a 10-fold decrease in displacement and a 95% reduction in velocity. Therefore, Mac-1-/- neutrophils initiated transmigration closer to the initial site of adhesion, which in turn led to delayed transmigration due to movement through nonoptimal emigration sites. Interestingly, the few LFA-1-/- cells that did adhere crawled similarly to wild-type neutrophils. Intercellular adhesion molecule-1 but not intercellular adhesion molecule-2 mediated the Mac-1-dependent crawling. These in vivo results clearly delineate two fundamentally different molecular mechanisms for LFA-1 and Mac-1 in vivo, i.e., LFA-1-dependent adhesion followed by Mac-1-dependent crawling, and both steps ultimately contribute to efficient emigration out of the vasculature.

/pdf/intraluminal-crawling-of-neutrophils-to-emigration-sites-a-50l0043bfx.pdf

Intraluminal crawling of neutrophils to emigration sites: a molecularly distinct process from adhesion in the recruitment cascade

Neutrophils must follow both endogenous and bacterial chemoattractant signals out of the vasculature and through the interstitium to arrive at a site of infection. By necessity, in the setting of multiple chemoattractants, the neutrophils must prioritize, favoring end target chemoattractants (e.g., fMLP and C5a) emanating from the site of infection over intermediary endogenous chemoattractants (e.g., IL-8 and LTB4) encountered en route to sites of infection. In this study, we propose a hierarchical model of two signaling pathways mediating the decision-making process of the neutrophils, which allows end target molecules to dominate over intermediary chemoattractants. In an under agarose assay, neutrophils predominantly migrated toward end target chemoattractants via p38 MAPK, whereas intermediary chemoattractant-induced migration was phosphoinositide 3-kinase (PI3K)/Akt dependent. When faced with competing gradients of end target and intermediary chemoattractants, Akt activation was significantly reduced within neutrophils, and the cells migrated preferentially toward end target chemoattractants even at 1/1,000th that of intermediary chemoattractants. End target molecules did not require chemotactic properties, since the p38 MAPK activator, LPS, also inhibited Akt and prevented migration to intermediary chemoattractants. p38 MAPK inhibitors not only reversed this hierarchy, such that neutrophils migrated preferentially toward intermediary chemoattractants, but also allowed neutrophils to be drawn out of a local end target chemoattractant environment and toward intermediary chemoattractants unexpectedly in an exaggerated (two- to fivefold) fashion. This was entirely related to significantly increased magnitude and duration of Akt activation. Finally, end target chemoattractant responses were predominantly Mac-1 dependent, whereas nondominant chemoattractants used primarily LFA-1. These data provide support for a two pathway signaling model wherein the end target chemoattractants activate p38 MAPK, which inhibits intermediary chemoattractant-induced PI3K/Akt pathway, establishing an intracellular signaling hierarchy.

/pdf/an-intracellular-signaling-hierarchy-determines-direction-of-1ryvdyz58s.pdf

An intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients

Neutrophils encounter and 'prioritize' many chemoattractants in their pursuit of bacteria. Here we tested the possibility that the phosphatase PTEN is responsible for the prioritization of chemoattractants. Neutrophils induced chemotaxis by two separate pathways, the phosphatidylinositol-3-OH kinase (PI(3)K) phosphatase and tensin homolog (PTEN) pathway, and the p38 mitogen-activated protein kinase pathway, with the p38 pathway dominating over the PI(3)K pathway. Pten(-/-) neutrophils could not prioritize chemoattractants and were 'distracted' by chemokines when moving toward bacterial chemoattractants. In opposing gradients, PTEN became distributed throughout the cell circumference, which inhibited all PI(3)K activity, thus permitting 'preferential' migration toward bacterial products via phospholipase A(2) and p38. Such prioritization was defective in Pten(-/-) neutrophils, which resulted in defective bacterial clearance in vivo. Our data identify a PTEN-dependent mechanism in neutrophils to prioritize, 'triage' and integrate responses to multiple chemotactic cues.

PTEN functions to 'prioritize' chemotactic cues and prevent 'distraction' in migrating neutrophils.

Lymphocyte CD44 interactions with hyaluronan localized on the endothelium have been demonstrated to mediate rolling and regulate lymphocyte entry into sites of chronic inflammation. Because neutrophils also express CD44, we investigated the role of CD44 and hyaluronan in the multistep process of neutrophil recruitment. CD44(-/-) and wild-type control mice were intrascrotally injected with the neutrophil-activating chemokine, MIP-2, and leukocyte kinetics in the cremasteric microcirculation were investigated 4 h subsequently using intravital microscopy. Neither the rolling flux nor the rolling velocities were decreased in CD44(-/-) mice relative to wild-type mice. In vitro, neutrophils did not roll on the CD44 ligand hyaluronan, consistent with the in vivo data that CD44/hyaluronan did not mediate rolling. However, the number of adherent leukocytes in the venule was decreased by 65% in CD44(-/-) mice compared with wild-type mice. Leukocyte emigration was also greatly decreased in the CD44(-/-) mice. The same decrease in adhesion and emigration was observed in the wild-type mice given hyaluronidase. Histology revealed neutrophils as being the dominant infiltrating population. We generated chimeric mice that express CD44 either on their leukocytes or on their endothelium and found that CD44 on both the endothelium and neutrophils was important for optimal leukocyte recruitment into tissues. Of those neutrophils that emigrated in wild-type and CD44(-/-) mice, there was no impairment in migration through the interstitium. This study suggests that CD44 can mediate some neutrophil adhesion and emigration, but does not appear to affect subsequent migration within tissues.

Role of CD44 and Hyaluronan in Neutrophil Recruitment

The success of Staphylococcus aureus as a pathogen is partly attributable to its ability to thwart host innate immune responses, which includes resisting the antimicrobial functions of phagocytes. Here, we have studied the interaction of methicillin-resistant S. aureus (MRSA) strain USA300 with murine RAW 264.7 and primary human macrophages using molecular imaging and single cell analysis to obtain an unprecedented understanding of the interaction between the macrophage and MRSA. Herein we demonstrate that macrophages fail to control intracellular infection by MRSA USA300 despite trafficking the bacteria into mature phagolysosomes. Using fluorescence-based proliferation assays we also show that intracellular staphylococci proliferate and that replication commences while the bacteria are residing in mature phagolysosomes hours after initial phagocytosis. Finally, live-cell fluorescence video microscopy allowed for unprecedented visual insight into the escape of MRSA from macrophages, demonstrating that the macrophages die through a pathway characterized by membrane blebbing and activation of caspase-3 followed by acquisition of the vital dye propidium iodide. Moreover, cell death precedes the emergence of MRSA from infected macrophages, and these events can be ablated by prolonged exposure of infected phagocytes to gentamicin.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1111/cmi.12527

Intracellular replication of Staphylococcus aureus in mature phagolysosomes in macrophages precedes host cell death, and bacterial escape and dissemination.

Bryan Heit

Papers

Intraluminal crawling of neutrophils to emigration sites: a molecularly distinct process from adhesion in the recruitment cascade

An intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients

PTEN functions to 'prioritize' chemotactic cues and prevent 'distraction' in migrating neutrophils.

Role of CD44 and Hyaluronan in Neutrophil Recruitment

Intracellular replication of Staphylococcus aureus in mature phagolysosomes in macrophages precedes host cell death, and bacterial escape and dissemination.