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Neutrophil extravasation is a critical event during immune responses to infection or injury that ensures survival. However, given the destructive potential of neutrophil effector molecules such as proteases and reactive oxygen species, strict control of neutrophil recruitment is required to avoid tissue damage. Neutrophil extravasation is a multistep process of adhesive interactions between neutrophils and components of the venular wall, that is, endothelium, basement membrane and pericytes. A plethora of proteins have been identified as critical regulators of each step that provide for compensatory mechanisms thus ensuring that extravasation can happen even if one mechanism is defective. Here, we discuss recent discoveries of how extravasation is regulated with emphasis on neutrophil-specific mechanisms; and discuss potential implications for future research.

Here we investigated the role of murine mast cell protease 4 (MCPT4), the functional counterpart of human mast cell chymase, in an experimental model of renal ischemia reperfusion injury, a major cause of acute kidney injury. MCPT4-deficient mice had worsened kidney function compared to wildtype mice. MCPT4 absence exacerbated pathologic neutrophil infiltration in the kidney and increased kidney myeloperoxidase expression, cell death and necrosis. In kidneys with ischemia reperfusion injury, when compared to wildtype mice, MCPT4-deficient mice showed increased surface expression of adhesion molecules necessary for leukocyte extravasation including neutrophil CD162 and endothelial cell CD54. In vitro, human chymase mediated the cleavage of neutrophil expressed CD162 and also CD54, P- and E-Selectin expressed on human glomerular endothelial cells. MCPT4 also dampened systemic neutrophil activation after renal ischemia reperfusion injury as neutrophils expressed more CD11b integrin and produced more reactive oxygen species in MCPT4-deficient mice. Accordingly, after renal injury, neutrophil migration to an inflammatory site distal from the kidney was increased in MCPT4-deficient versus wildtype mice. Thus, contrary to the described overall aggravating role of mast cells, one granule-released mediator, the MCPT4 chymase, exhibits a potent anti-inflammatory function in renal ischemia reperfusion injury by controlling neutrophil extravasation and activation thereby limiting associated damage.

Aims Neutrophil trafficking within the vasculature strongly relies on intracellular calcium signaling. Sustained Ca2+ influx into the cell requires a compensatory efflux of potassium to maintain membrane potential. Here, we aimed to investigate whether the voltage-gated potassium channel KV1.3 regulates neutrophil function during the acute inflammatory process by affecting sustained Ca2+ signaling. Methods and results Using in vitro assays and electrophysiological techniques, we show that KV1.3 is functionally expressed in human neutrophils regulating sustained store operated Ca2+ entry (SOCE) through membrane potential stabilizing K+ efflux. Inhibition of KV1.3 on neutrophils by the specific inhibitor 5-(4-Phenoxybutoxy)psoralen (PAP-1) impaired intracellular Ca2+ signaling, thereby preventing cellular spreading, adhesion strengthening and appropriate crawling under flow conditions in vitro. Using intravital microscopy, we show that pharmacological blockade or genetic deletion of KV1.3 in mice decreased neutrophil adhesion in a blood flow dependent fashion in inflamed cremaster muscle venules. Furthermore, we identified KV1.3 as a critical component for neutrophil extravasation into the inflamed peritoneal cavity. Finally, we also revealed impaired phagocytosis of E.coli particles by neutrophils in the absence of KV1.3. Conclusion We show that the voltage gated potassium channel KV1.3 is critical for Ca2+ signaling and neutrophil trafficking during acute inflammatory processes. Our findings do not only provide evidence for a role of KV1.3 for sustained calcium signaling in neutrophils affecting key functions of these cells, they also open up new therapeutic approaches to treat inflammatory disorders characterized by overwhelming neutrophil infiltration. Translational perspective Neutrophils exert important immune functions during tissue injury or bacterial infection through leaving the vasculature and extravasate into affected tissues. Conversely, neutrophils trigger the pathogenesis of acute and chronic inflammatory disorders and are involved in the development and maintenance of various autoimmune diseases. Within this study, we show that the voltage-gated potassium channel KV1.3 is functionally expressed on neutrophils and affects calcium signaling thereby regulating neutrophil effector functions during immune responses. Hence, KV1.3 represents an interesting potential new target to treat unwanted excessive neutrophil invasion in various disorders ranging from autoinflammatory disorders to ischemic tissue injury.

Precise spatiotemporal regulation of leukocyte extravasation is key for generating an efficient immune response to injury or infection. The integrins LFA-1(CD11a/CD18) and Mac-1(CD11b/CD18) play overlapping roles in neutrophil migration because they bind the same as well as different ligands in response to extracellular signaling. Using two-photon intravital imaging and transmission electron microscopy, we observed the existence of preferred sites for neutrophil entrance into the endothelial cell monolayer and exit from the basement membrane and pericyte sheath during neutrophil extravasation, namely, hotspots I and II, by elucidating distinctive roles of LFA-1 and Mac-1. To penetrate the vascular endothelium, neutrophils must first penetrate the endothelial cell layer through hotspot I (i.e., the point of entry into the endothelium). Neutrophils frequently remain in the space between the endothelial cell layer and the basement membrane for a prolonged period (>20 min). Subsequently, neutrophils penetrate the basement membrane and pericyte sheath at hotspot II, which is the final stage of exiting the vascular endothelium. To further investigate the roles of LFA-1 and Mac-1, we newly generated LFA-1 FRET (CD11a-YFP/CD18-CFP) mice and Mac-1 FRET (CD11b-YFP/CD18-CFP) mice. Using both FRET mice, we were able to determine that LFA-1 and Mac-1 distinctly regulate the neutrophil extravasation cascade. Our data suggest that the vascular endothelium functions as a double-layered barrier in the steps of neutrophil extravasation. We propose that the harmonized regulation of neutrophil penetration through the endothelium via hotspots I and II may be critical for vascular homeostasis during inflammation., Immune response: White blood cells follow the leader In response to infection or tissue damage, white blood cells known as leukocytes exit blood vessels at specific sites, “hotspots.” Many leukocytes follow each other out through the same hotspot. As part of the body’s initial response to infection or tissue damage, leukocytes are carried to infected or damaged tissues by blood vessels. To fight infection, they must exit the vessels, but how they exited was poorly understood. Young-Min Hyun at the Yonsei University College of Medicine in Seoul, South Korea and co-workers used advanced imaging techniques to visualize leukocyte delivery. They found that many leukocytes use a single hotspot to enter the blood vessel wall, travel through the wall’s interior, and then exit the wall at another hotspot. Using hotspots is thought to minimize the number of perforations in the vessel wall, maintaining vessel integrity. These results illuminate a key aspect of how efficiently the body fights infection from the viewpoint of leukocyte migration.

Primary graft dysfunction (PGD) is the predominant cause of early graft loss following lung transplantation. We recently demonstrated that donor pulmonary intravascular nonclassical monocytes (NCM) initiate neutrophil recruitment. Simultaneously, host-origin classical monocytes (CM) permeabilize the vascular endothelium to allow neutrophil extravasation necessary for PGD. Here, we show that a CCL2-CCR2 axis is necessary for CM recruitment. Surprisingly, although intravital imaging and multichannel flow cytometry revealed that depletion of donor NCM abrogated CM recruitment, single cell RNA sequencing identified donor alveolar macrophages (AM) as predominant CCL2 secretors. Unbiased transcriptomic analysis of murine tissues combined with murine KOs and chimeras indicated that IL-1β production by donor NCM was responsible for the early activation of AM and CCL2 release. IL-1β production by NCM was NLRP3 inflammasome dependent and inhibited by treatment with a clinically approved sulphonylurea. Production of CCL2 in the donor AM occurred through IL-1R-dependent activation of the PKC and NF-κB pathway. Accordingly, we show that IL-1β-dependent paracrine interaction between donor NCM and AM leads to recruitment of recipient CM necessary for PGD. Since depletion of donor NCM, IL-1β, or IL-1R antagonism and inflammasome inhibition abrogated recruitment of CM and PGD and are feasible using FDA-approved compounds, our findings may have potential for clinical translation.

Neutrophils are key first-responders in the innate immune response to C. difficile infection (CDI) and play a central role in disease pathogenesis. Studies have clearly shown that tissue neutrophil numbers need to be tightly regulated for optimal CDI outcomes: while excessive colonic neutrophilia is associated with severe CDI, neutrophil depletion also results in worse outcomes. However, the biological mechanisms that control CDI-induced neutrophilia remain poorly defined. C-X-C chemokine receptor 2 (CXCR2) is a chemotactic receptor that is critical in neutrophil mobilization from bone marrow to blood and tissue sites. We have previously reported that a single nucleotide polymorphism (SNP) in leptin receptor (LEPR), present in up to 50% of people, influenced CDI-induced neutrophil CXCR2 expression and tissue neutrophilia. Homozygosity for mutant LEPR (i.e. RR genotype) was associated with higher CXCR2 expression and more tissue neutrophils. Here, we investigated the biological mechanisms that regulate neutrophil CXCR2 expression after CDI, and the influence of host genetics on this process. Our data reveal that: a) CXCR2 plays a key role in CDI-induced neutrophil extravasation from blood to colonic tissue; b) plasma from C. difficile-infected mice upregulated CXCR2 on bone marrow neutrophils; c) plasma from C. difficile-infected RR mice induced a higher magnitude of CXCR2 upregulation and had more IL-1β; and d) IL-1β neutralization reduced CXCR2 expression on bone marrow and blood neutrophils and their subsequent accrual to colonic tissue. In sum, our data indicate that IL-1β is a key molecular mediator that communicates between gastro-intestinal tract (i.e. site of CDI) and bone marrow (i.e. primary neutrophil reservoir) and regulates the intensity of CDI-induced tissue neutrophilia by modulating CXCR2 expression. Further, our studies highlight the importance of host genetics in affecting these innate immune responses and provide novel insights into the mechanisms by which a common SNP influences CDI-induced neutrophilia.

Author(s): Dehghani, Tima; Thai, Phung N; Sodhi, Harkanwalpreet; Ren, Lu; Sirish, Padmini; Nader, Carol E; Timofeyev, Valeriy; Overton, James L; Li, Xiaocen; Lam, Kit S; Chiamvimonvat, Nipavan; Panitch, Alyssa | Abstract: AimsOne of the hallmarks of myocardial infarction (MI) is excessive inflammation. During an inflammatory insult, damaged endothelial cells shed their glycocalyx, a carbohydrate-rich layer on the cell surface which provides a regulatory interface to immune cell adhesion. Selectin-mediated neutrophilia occurs as a result of endothelial injury and inflammation. We recently designed a novel selectin-targeting glycocalyx mimetic (termed DS-IkL) capable of binding inflamed endothelial cells. This study examines the capacity of DS-IkL to limit neutrophil binding and platelet activation on inflamed endothelial cells, as well as the cardio-protective effects of DS-IkL after acute myocardial infarction.Methods and resultsIn vitro, DS-IkL diminished neutrophil interactions with both recombinant selectin and inflamed endothelial cells, and limited platelet activation on inflamed endothelial cells. Our data demonstrated that DS-IkL localized to regions of vascular inflammation in vivo after 45 minutes of left anterior descending coronary artery ligation induced MI. Further, findings from this study show DS-IkL treatment had short- and long-term cardioprotective effects after ischemia/reperfusion at the left anterior descending coronary artery. Mice treated with DS-IkL immediately after ischemia/reperfusion and 24 hours later exhibited reduced neutrophil extravasation, macrophage accumulation, fibroblast and endothelial cell proliferation, and fibrosis compared to saline controls.ConclusionsOur findings suggest that DS-IkL has great therapeutic potential after MI by limiting reperfusion injury induced by the immune response.Translational perspectiveCardiovascular disease remains the leading cause of mortality worldwide. Acute inflammation from myocardial infarction (MI) results in damaged endothelial cells, leading to the loss of glycocalyx. Here, we designed a novel selectin-targeting glycocalyx mimetic (termed DS-IkL) capable of binding inflamed endothelial cells. DS-IkL limits neutrophil binding and platelet activation on inflamed endothelial cells. Treatment with DS-IkL in a preclinical model of MI reduces infarct size by preventing neutrophil extravasation, macrophage accumulation, fibroblast and endothelial cell proliferation, and fibrosis. Our findings suggest that DS-IkL has great therapeutic potential after MI by limiting reperfusion injury induced by the immune response.

We have previously shown that multimers of plasma pentraxin-3 (PTX3) were predictive of survival in patients with sepsis. To characterize the release kinetics and cellular source of plasma protein changes in sepsis, serial samples were obtained from healthy volunteers (n = 10; three time points) injected with low-dose endotoxin (lipopolysaccharide [LPS]) and analyzed using data-independent acquisition MS. The human plasma proteome response was compared with an LPS-induced endotoxemia model in mice. Proteomic analysis of human plasma revealed a rapid neutrophil degranulation signature, followed by a rise in acute phase proteins. Changes in circulating PTX3 correlated with increases in neutrophil-derived proteins following LPS injection. Time course analysis of the plasma proteome in mice showed a time-dependent increase in multimeric PTX3, alongside increases in neutrophil-derived myeloperoxidase (MPO) upon LPS treatment. The mechanisms of oxidation-induced multimerization of PTX3 were explored in two genetic mouse models: MPO global knock-out (KO) mice and LysM Cre Nox2 KO mice, in which NADPH oxidase 2 (Nox2) is only deficient in myeloid cells. Nox2 is the enzyme responsible for the oxidative burst in neutrophils. Increases in plasma multimeric PTX3 were not significantly different between wildtype and MPO or LysM Cre Nox2 KO mice. Thus, PTX3 may already be stored and released in a multimeric form. Through in vivo neutrophil depletion and multiplexed vascular proteomics, PTX3 multimer deposition within the aorta was confirmed to be neutrophil dependent. Proteomic analysis of aortas from LPS-injected mice returned PTX3 as the most upregulated protein, where multimeric PTX3 was deposited as early as 2 h post-LPS along with other neutrophil-derived proteins. In conclusion, the rise in multimeric PTX3 upon LPS injection correlates with neutrophil-related protein changes in plasma and aortas. MPO and myeloid Nox2 are not required for the multimerization of PTX3; instead, neutrophil extravasation is responsible for the LPS-induced deposition of multimeric PTX3 in the aorta., Graphical Abstract, Highlights • Multimer formation of pentraxin-3 (PTX3) has been linked to outcome in sepsis. • PTX3 plasma levels have been correlated to increases in neutrophil-derived proteins. • Neutrophil-derived myeloperoxidase and NADPH oxidase 2 are not responsible for PTX3 oxidation. • PTX3 multimer deposition within the aorta is neutrophil dependent., In Brief Circulating proteome alterations, in both humans and mice, in response to endotoxin were mapped utilizing proteomic approaches. Multimeric pentraxin-3 (PTX3) plasma and aortic levels mimicked changes observed in proteins of known neutrophil origin in response to endotoxin. Genetic mouse models ruled out myeloperoxidase and NADPH oxidase 2 as drivers of PTX3 oxidation; however, neutrophil extravasation promotes PTX3 deposition within the vasculature. The results reveal a link between the systemic inflammatory response and the neutrophil-dependent vascular deposition of multimeric PTX3.

Neutrophils arrest and extravasate from the blood vessels in response to infection and injury. Using intravital subcellular microscopy, Subramanian et al. identify a role for extracellular vesicle–based autocrine/paracrine LTB4/BLT1 signaling in promoting the rearrangement of the actomyosin cytoskeleton and β2-integrin during neutrophil extravasation in live animals., The eicosanoid leukotriene B4 (LTB4) relays chemotactic signals to direct neutrophil migration to inflamed sites through its receptor BLT1. However, the mechanisms by which the LTB4–BLT1 axis relays chemotactic signals during intravascular neutrophil response to inflammation remain unclear. Here, we report that LTB4 produced by neutrophils acts as an autocrine/paracrine signal to direct the vascular recruitment, arrest, and extravasation of neutrophils in a sterile inflammation model in the mouse footpad. Using intravital subcellular microscopy, we reveal that LTB4 elicits sustained cell polarization and adhesion responses during neutrophil arrest in vivo. Specifically, LTB4 signaling coordinates the dynamic redistribution of non-muscle myosin IIA and β2-integrin, which facilitate neutrophil arrest and extravasation. Notably, we also found that neutrophils shed extracellular vesicles in the vascular lumen and that inhibition of extracellular vesicle release blocks LTB4-mediated autocrine/paracrine signaling required for neutrophil arrest and extravasation. Overall, we uncover a novel complementary mechanism by which LTB4 relays extravasation signals in neutrophils during early inflammation response.

Uromodulin (UMOD) is produced and secreted by tubular epithelial cells. Secreted UMOD polymerizes (pUMOD) within the lumen, where it regulates salt transport and protects the kidney from bacteria and stone formation. Under various pathological conditions, pUMOD accumulates within the tubular lumen and reaches extratubular sites where it may interact with renal interstitial cells. Here, we investigated the potential of extratubular pUMOD to act as a damage associated molecular pattern (DAMP) molecule thereby creating local inflammation. We found that intrascrotal and intraperitoneal injection of pUMOD induced leukocyte recruitment in vivo and led to TNF-α secretion by F4/80 positive macrophages. Additionally, pUMOD directly affected vascular permeability and increased neutrophil extravasation independent of macrophage-released TNF-α. Interestingly, pUMOD did not directly upregulate adhesion molecules on endothelial cells and did not directly activate β2 integrins on neutrophils. In obstructed neonatal murine kidneys, we observed extratubular UMOD accumulation with tubular atrophy and leukocyte infiltrates. Finally, we found extratubular UMOD deposits associated with peritubular leukocyte infiltration in kidneys from patients with inflammatory kidney diseases. Taken together, we identified extratubular pUMOD as a strong inducer of leukocyte recruitment, underlining its critical role in mounting an inflammatory response in various kidneys pathologies.

Tissue resident mast cells (MCs) rapidly initiate neutrophil infiltration upon inflammatory insult, yet the molecular mechanism is still unknown. Here, we demonstrated that MC-derived tumor necrosis factor (TNF) was crucial for neutrophil extravasation to sites of contact hypersensitivity-induced skin inflammation by promoting intraluminal crawling. MC-derived TNF directly primed circulating neutrophils via TNF receptor-1 (TNFR1) while being dispensable for endothelial cell activation. The MC-derived TNF was infused into the bloodstream by directional degranulation of perivascular MCs that were part of the vascular unit with access to the vessel lumen. Consistently, intravenous administration of MC granules boosted neutrophil extravasation. Pronounced and rapid intravascular MC degranulation was also observed upon IgE crosslinking or LPs challenge indicating a universal MC potential. Consequently, the directional MC degranulation of pro-inflammatory mediators into the bloodstream may represent an important target for therapeutic approaches aimed at dampening cytokine storm syndromes or shock symptoms, or intentionally pushing immune defense.

RationalePulmonary endothelial permeability contributes to the high-permeability pulmonary edema that characterizes acute respiratory distress syndrome (ARDS), which carries a high mortality. Circulating bone morphogeneic protein 9 (BMP9) is emerging as an important regulator of pulmonary vascular homeostasis.ObjectiveTo determine whether endogenous BMP9 plays a role in preserving pulmonary endothelial integrity, and whether loss of endogenous BMP9 occurs during lipopolysacharride (LPS)-induced lung inflammation and permeability.MethodsA BMP9-neutralizing antibody was administrated to healthy adult mice and lung vasculature was examined. Potential mechanisms were delineated by transcript analysis in human primary lung endothelial cells. Impact of BMP9 was evaluated in a murine acute lung injury model induced by inhaled LPS. Levels of BMP9 were measured in plasma from patients with sepsis and endotoxemic mice.Main ResultsSubacute neutralization of endogenous BMP9 in mice resulted in increased lung vascular permeability, interstitial edema and neutrophil extravasation. In lung endothelial cells, BMP9 regulated a programme of gene expression and pathways controlling vascular permeability and cell membrane integrity. Augmentation of BMP9 signalling in mice with exogenous BMP9 prevented inhaled LPS-caused lung injury and edema. In endotoxemic mice, endogenous BMP9 levels were markedly reduced, due to a transient reduction in hepatic BMP9 mRNA expression and increased elastase activity in plasma. In human sepsis patients, circulating levels of BMP9 were also markedly reduced.ConclusionsEndogenous circulating BMP9 is a pulmonary endothelial protective factor, down-regulated during inflammation. Supplementation with exogenous BMP9 offers a potential therapy to prevent increased pulmonary endothelial permeability in the setting of lung injury.Short summaryScientific Knowledge on the SubjectIncreased pulmonary endothelial permeability is a major factor in the development of acute respiratory distress syndrome (ARDS). Evidence is emerging that circulating BMP9, secreted from the liver, might protect the pulmonary endothelium from injury. For example, loss of BMP9 levels or signalling receptor contributes to the development of pulmonary arterial hypertension. The role of endogenous BMP9 in endothelial permeability remains unclear.What This Study Adds to the FieldHere we show that subacute neutralization of endogenous BMP9 leads to lung vascular injury, including enhanced permeability and neutrophil extravasation. BMP9 levels were markedly reduced in the setting of inflammation in mice and humans. Conversely, exogenous supplementation of BMP9 protected the lung from LPS-induced injury. This study suggests that exogenous BMP9 could offer a novel approach to prevent increased pulmonary endothelial permeability in the setting of lung injury and ARDS.

Acute lung inflammation has severe morbidity, as seen in COVID-19 patients. Lung inflammation is accompanied or led by massive accumulation of neutrophils in pulmonary capillaries (“margination”). We sought to identify nanostructural properties that predispose nanoparticles to accumulate in pulmonary marginated neutrophils, and therefore to target severely inflamed lungs. We designed a library of nanoparticles and conducted an in vivo screen of biodistributions in naive mice and mice treated with lipopolysaccharides. We found that supramolecular organization of protein in nanoparticles predicts uptake in inflamed lungs. Specifically, nanoparticles with agglutinated protein (NAPs) efficiently home to pulmonary neutrophils, while protein nanoparticles with symmetric structure (e.g. viral capsids) are ignored by pulmonary neutrophils. We validated this finding by engineering protein-conjugated liposomes that recapitulate NAP targeting to neutrophils in inflamed lungs. We show that NAPs can diagnose acute lung injury in SPECT imaging and that NAP-like liposomes can mitigate neutrophil extravasation and pulmonary edema arising in lung inflammation. Finally, we demonstrate that ischemic ex vivo human lungs selectively take up NAPs, illustrating translational potential. This work demonstrates that structure-dependent interactions with neutrophils can dramatically alter the biodistribution of nanoparticles, and NAPs have significant potential in detecting and treating respiratory conditions arising from injury or infections.

Luminol-based bioluminescence imaging allows noninvasive tracking of oxidatively active cells such as neutrophils. Luminol is given intravenously or intraperitoneally, followed by bioluminescence imaging at 425 nm. Here we describe a method for tracking neutrophil extravasation into an inflammatory site, especially focusing on mammary carcinoma.

Tissue resident mast cells (MCs) rapidly initiate neutrophil infiltration upon inflammatory insult, yet the molecular mechanism is still unknown. Here, we demonstrate that MC-derived TNF is crucial for neutrophil extravasation to sites of sterile skin inflammation by promoting intraluminal adhesion and crawling. Strikingly, MC-TNF directly primes circulating neutrophils via TNFR1, while being dispensable for chemokine production and endothelial cell activation. The MC-TNF is fused into the bloodstream by directional degranulation of perivascular MCs that are part of the vascular unit with access to the vessel lumen. Consistently, intravenous administration of MC granules can boost neutrophil extravasation. Consequently, the directional MC degranulation of pro-inflammatory mediators into the bloodstream may represent an important target for therapeutic approaches aimed at dampening cytokine storm syndromes or shock symptoms, or intentionally pushing immune defense.

Dysregulated neutrophil extravasation contributes to the pathogenesis of many inflammatory disorders. Pericytes (PCs) have been implicated in the regulation of neutrophil transmigration, and previous work demonstrates that endothelial cell (EC)-derived signals reduce PC barrier function; however, the signaling mechanisms are unknown. Here, we demonstrate a novel role for EC-derived macrophage migration inhibitory factor (MIF) in inhibiting PC contractility and facilitating neutrophil transmigration. With the use of micro-ELISAs, RNA sequencing, quantitative PCR, and flow cytometry, we found that ECs secrete MIF, and PCs upregulate CD74 in response to TNF-α. We demonstrate that EC-derived MIF decreases PC contractility on 2-dimensional silicone substrates via reduction of phosphorylated myosin light chain. With the use of an in vitro microvascular model of the human EC–PC barrier, we demonstrate that MIF decreases the PC barrier to human neutrophil transmigration by increasing intercellular PC gap formation. For the first time, an EC-specific MIF knockout mouse was used to investigate the effects of selective deletion of EC MIF. In a model of acute lung injury, selective deletion of EC MIF decreases neutrophil infiltration to the bronchoalveolar lavage and tissue and simultaneously decreases PC relaxation by increasing myosin light-chain phosphorylation. We conclude that paracrine signals from EC via MIF decrease PC contraction and enhance PC-regulated neutrophil transmigration.—Pellowe, A. S., Sauler, M., Hou, Y., Merola, J., Liu, R., Calderon, B., Lauridsen, H. M., Harris, M. R., Leng, L., Zhang, Y., Tilstam, P. V., Pober, J. S., Bucala, R., Lee, P. J., Gonzalez, A. L. Endothelial cell-secreted MIF reduces pericyte contractility and enhances neutrophil extravasation.

Neutrophils are the first leukocytes to arrive at sites of injury during the acute inflammatory response. To maintain the polarized morphology during migration, nonmuscle myosins class II are essential, but studies using genetic models to investigate the role of Myh9 for neutrophil migration were missing. In this study, we analyzed the functional role of Myh9 on neutrophil trafficking using genetic downregulation of Myh9 in Vav-iCre+/Myh9wt/fl mice because the complete knockout of Myh9 in the hematopoietic system was lethal. Migration velocity and Euclidean distance were significantly diminished during mechanotactic migration of Vav-iCre+/Myh9wt/fl neutrophils compared with Vav-iCre−/Myh9wt/fl control neutrophils. Similar results were obtained for transmigration and migration in confined three-dimensional environments. Stimulated emission depletion nanoscopy revealed that a certain threshold of Myh9 was required to maintain proper F-actin dynamics in the front of the migrating cell. In laser-induced skin injury and in acute peritonitis, reduced Myh9 expression in the hematopoietic system resulted in significantly diminished neutrophil extravasation. Investigation of bone marrow chimeric mice in the peritonitis model revealed that the migration defect was cell intrinsic. Expression of Myh9-EGFP rescued the Myh9-related defects in two-dimensional and three-dimensional migration of Hoxb8-SCF cell–derived neutrophils generated from fetal liver cells with a Myh9 knockdown. Live cell imaging provided evidence that Myh9 was localized in branching lamellipodia and in the uropod where it may enable fast neutrophil migration. In summary, the severe migration defects indicate an essential and fundamental role of Myh9 for neutrophil trafficking in innate immunity.

Spinal cord injury (SCI) is a serious devastating condition and it has a high mortality rate and morbidity rate. The early pathological changes in the immediate phase of SCI may play a major part in the development of secondary injury. Alterations in the expression of many long noncoding RNAs (lncRNAs) have been shown to play fundamental roles in the diseases of the central nervous system. However, the roles of lncRNAs and messenger RNAs (mRNAs) in the immediate phase of SCI are not clear. We examined the expression of mRNAs and lncRNAs in a rat model at 2 h after SCI and identified the differentially expressed lncRNAs (DE lncRNAs) and differentially expressed mRNAs (DE mRNAs) using microarray analysis. 772 DE lncRNAs and 992 DE mRNAs were identified in spinal cord samples in the immediate phase following SCI compared with control samples. Moreover, Gene Ontology (GO) term annotation results showed that CXCR chemokine receptor binding, neutrophil apoptotic process, neutrophil migration, neutrophil extravasation, macrophage differentiation, monocyte chemotaxis and cellular response to interleukin-1 (IL-1) were the main significantly enriched GO terms. The results of Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the DEGs were enriched in toll−like receptor signaling pathway, p53 signaling pathway, MAPK signaling pathway and Jak–STAT signaling pathway. IL6, MBOAT4, FOS, TNF, JUN, STAT3, CSF2, MYC, CCL2 and FGF2 were the top 10 high-degree hub nodes and may be important targets in the immediate phase of SCI. The current study on provides novel insights into how lncRNAs and mRNAs regulate the pathogenesis of the immediate phase after SCI.

Inflammation is a complex biological response that serves to protect the body's tissues following harmful stimuli such as infection, irritation or injury and initiates tissue repair. At the start of an inflammatory response, pro-inflammatory mediators induce changes in the endothelial lining of the blood vessels and in leukocytes. This results in increased vascular permeability and increased expression of adhesion proteins, and promotes adhesion of leukocytes, especially neutrophils to the endothelium. Adhesion is a prerequisite for neutrophil extravasation and chemoattractant-stimulated recruitment to inflammatory sites, where neutrophils phagocytose and kill microbes, release inflammatory mediators and cross-talk with other immune cells to co-ordinate the immune response in preparation for tissue repair. Many signalling proteins are critically involved in the complex signalling processes that underpin the inflammatory response and cross-talk between endothelium and leukocytes. As key regulators of cell–cell and cell–substratum adhesion, small GTPases (guanosine triphosphatases) act as important controls of neutrophil-endothelial cell interactions as well as neutrophil recruitment to sites of inflammation. Here, we summarise key processes that are dependent upon small GTPases in leukocytes during these early inflammatory events. We place a particular focus on the regulation of integrin-dependent events and their control by Rho and Rap family GTPases as well as their regulators during neutrophil adhesion, chemotaxis and recruitment.

Neutrophil extravasation and interstitial migration are important steps during the recruitment of neutrophils to sites of inflammation. In the present study, we addressed the functional importance of the unconventional class I myosin 1f (Myo1f) for neutrophil trafficking during acute inflammation. In contrast to leukocyte rolling and adhesion, the genetic absence of Myo1f severely compromised neutrophil extravasation into the inflamed mouse cremaster tissue when compared with Myo1f+/+ mice as studied by intravital microscopy. Similar results were obtained in experimental models of acute peritonitis and acute lung injury. In contrast to 2-dimensional migration, which occurred independently of Myo1f, Myo1f was indispensable for neutrophil migration in 3-dimensional (3D) environments, that is, transmigration and migration in collagen networks as it regulated squeezing and dynamic deformation of the neutrophil nucleus during migration through physical barriers. Thus, we provide evidence for an important role of Myo1f in neutrophil trafficking during inflammation by specifically regulating neutrophil extravasation and migration in 3D environments.

Intercellular adhesion molecule-1 (ICAM-1) mediates the firm adhesion of leukocytes to endothelial cells and initiates subsequent signaling that promotes their transendothelial migration (TEM). Vascular endothelial (VE)-cadherin plays a critical role in endothelial cell–cell adhesion, thereby controlling endothelial permeability and leukocyte transmigration. This study aimed to determine the molecular signaling events that originate from the ICAM-1–mediated firm adhesion of neutrophils that regulate VE-cadherin’s role as a negative regulator of leukocyte transmigration. We observed that ICAM-1 interacts with Src homology domain 2–containing phosphatase-2 (SHP-2), and SHP-2 down-regulation via silencing of small interfering RNA in endothelial cells enhanced neutrophil adhesion to endothelial cells but inhibited neutrophil transmigration. We also found that VE-cadherin associated with the ICAM-1–SHP-2 complex. Moreover, whereas the activation of ICAM-1 leads to VE-cadherin dissociation from ICAM-1 and VE-cadherin association with actin, SHP-2 down-regulation prevented ICAM-1–VE-cadherin association and promoted VE-cadherin–actin association. Furthermore, SHP-2 down-regulation in vivo promoted LPS-induced neutrophil recruitment in mouse lung but delayed neutrophil extravasation. These results suggest that SHP-2—via association with ICAM-1—mediates ICAM-1–induced Src activation and modulates VE-cadherin switching association with ICAM-1 or actin, thereby negatively regulating neutrophil adhesion to endothelial cells and enhancing their TEM.—Yan, M., Zhang, X., Chen, A., Gu, W., Liu, J., Ren, X., Zhang, J., Wu, X., Place, A. T., Minshall, R. D., Liu, G. Endothelial cell SHP-2 negatively regulates neutrophil adhesion and promotes transmigration by enhancing ICAM-1–VE-cadherin interaction.

Background and Purpose Neuroinflammation is initiated by a variety of stimuli including infections, sepsis, neurodegenerative diseases or traumatic brain injury and, if not adequately controlled, can lead to various degrees of neuronal damage and behavioural impairment. A critical event in the initial steps of inflammation is neutrophil extravasation. Semicarbazide-sensitive amine oxidase (SSAO, also known as vascular adhesion protein 1 or VAP-1) regulates neutrophil adhesion and extravasation. Here, we elucidate the role of SSAO/VAP-1 in the early stage inflammatory response after LPS insult in the brain. Experimental Approach PXS-4681A, a selective and irreversible SSAO/VAP-1 inhibitor, was tested in two rat models of neuroinflammation, following systemic or i.c.v. LPS. Immunohistochemical and immunofluorescence techniques were used to measure neutrophils and microglia. VAP-1 was quantitated by Western blotting. Key Results Both systemic and i.c.v. administration of LPS induced an increase in neutrophil recruitment and microglial response in various brain areas including the substantia nigra and striatum. PXS-4681A produced a significant inhibition of neutrophil recruitment and extravasation after i.c.v. LPS injection and also reversed microglial cell recruitment and morphological changes to the level of the sham controls in both LPS models. Conclusions and Implications PXS-4681A acted as an effective anti-inflammatory agent after both systemic and i.c.v. LPS injections suggesting that SSAO/VAP-1 inhibition could be beneficial in the treatment of brain inflammation.

Background and purpose In recent years, studies have focused on the resolution of inflammation, which can be achieved by endogenous anti-inflammatory agonists such as Annexin A1 (AnxA1). Here, we investigated the effects of mast cells (MCs) on early lipopolysaccharide (LPS)-induced neutrophil recruitment and the involvement of the AnxA1-Formyl peptide receptor 2/ALX (Fpr2/ALX or lipoxin A4 receptor) pathway. Experimental approach Intravital microscopy (IVM) was used to visualize and quantify the effects of LPS (10 µg per mouse i.p.) on murine mesenteric cellular interactions. Furthermore, the role MCs play in these inflammatory responses was determined in vivo and in vitro and effects of AnxA1 mimetic peptide Ac2-26 were assessed. Key Results LPS increased both neutrophil endothelial cell interactions within the mesenteric microcirculation and MC activation (determined by IVM and ruthenium red dye uptake) which in turn lead to the early stages of neutrophil recruitment. MC recruitment of neutrophils could be blocked by preventing the pro-inflammatory activation (using cromolyn sodium) or enhancing an anti-inflammatory phenotype (using Ac2-26) in MCs. Furthermore, MCs induced neutrophil migration in vitro and MC stabilization enhanced the release of AnxA1 from neutrophils. Pharmacological approaches (such as the administration of Fpr pan-antagonist Boc2, or the Fpr2/ALX antagonist WRW4) revealed neutrophil Fpr2/ALX to be important in this process. Conclusions and Implications Data presented here provides evidence for a role of MCs, which are ideally positioned in close proximity to the vasculature, to act as sentinel cells in neutrophil extravasation and resolution of inflammation via the AnxA1-Fpr2/ALX pathway.

Atherosclerosis is characterized by the accumulation of cholesterol esters, macrophages and fibrous elements on the inner artery wall. This process begins with accumulation of plasma lipoproteins on the inner wall of the artery, which leads to changes in the passage and elasticity of the blood vessels. Monocytes penetrating the arterial wall transforms into macrophages which digest cholesterol and form foam cells which is one of the first steps in atherosclerotic process. Activation of macrophages is affected by galectin-3, a β- galactoside-binding lectin which is also involved in cardiac remodeling. Cardiac matrix remodeling is the ultimate result of macrophages proliferation and chemotaxis, neutrophil extravasation, oxidative stress, apoptosis, angiogenesis, fibroblast proliferation and deposition of collagen. Studies show that elevated levels of galectin 3 within atherosclerotic lesions in humans are closely related to the development of a disease itself. With this review, we want to demonstrate the correlation between galectin-3 which is precipitated in atherosclerotic plaque and has an influence on the development of cardiovascular diseases and its role in the prognosis of recovery in cardiac patients.

Endothelial basement membranes constitute barriers to extravasating leukocytes during inflammation, a process where laminin isoforms define sites of leukocyte exit; however, how this occurs is poorly understood. In addition to a direct effect on leukocyte transmigration, we show that laminin 511 affects endothelial barrier function by stabilizing VE-cadherin at junctions and downregulating expression of CD99L2, correlating with reduced neutrophil extravasation. Binding of endothelial cells to laminin 511, but not laminin 411 or non-endothelial laminin 111, enhanced transendothelial cell electrical resistance (TEER) and inhibited neutrophil transmigration. Data suggest that endothelial adhesion to laminin 511 via β1 and β3 integrins mediates RhoA-induced VE-cadherin localization to cell-cell borders, and while CD99L2 downregulation requires integrin β1, it is RhoA-independent. Our data demonstrate that molecular information provided by basement membrane laminin 511 affects leukocyte extravasation both directly and indirectly by modulating endothelial barrier properties.

We thank Bhogal and colleagues for their positive comments on our manuscript and their perspective on VWF being a potential driver of COVID-19 microvascular thrombosis rather than a mere biomarker of endotheliopathy. It is well-known that VWF promotes neutrophil recruitment to endothelial surfaces under low-shear conditions and supports neutrophil extravasation towards inflamed tissues1 . This pro-inflammatory role of VWF involves two distinct pathways: (1) a direct interaction of neutrophil receptors (PSGL1 and s2-integrins) with VWF, and (2) an indirect interaction in which neutrophils adhere to VWF-bound platelets.

Background & Aim Recently, we reported that local infusion of human umbilical cord-derived mesenchymal stem cells (MSCs) during donor lung ablation may increase lung availability by improving organ preservation. We found that MSCs protect donor lungs against ischemic injury by reducing oxidative damage and neutrophil extravasation. Pre-conditioning has emerged as a strategy to improve MSC-based therapies. Here, we studied whether the preservation capacity of MSCs may be enhanced by their pre-activation into an anti-inflammatory phenotype with TNF-alpha (TNF-a). Methods, Results & Conclusion We settled a lung preservation model in rats that mimics the different phases by which donor organs must undergo before implantation which consist on: cardiac arrest, warm ischemia (2h at room temperature), cold ischemia (1.5h at 4°C, with Perfadex), and normothermic lung perfusion with ventilation (Steen solution, 1h). After 1h of warm ischemia, 1 × 10^6 cells (MSCs, or TNF-a treated cells) or vehicle were infused via the pulmonary artery. Physiologic data (pressure-volume curves) were acquired right after the cardiac arrest and at the end of the perfusion. Lung biopsies were taken at the end of the perfusion to study edema formation, neutrophil infiltration, redox profile, etc. Interestingly, although the TNF-a treated cells showed an anti-inflammatory phenotype in vitro, we did not register a significant change in their preservation capacity. In this line, lung compliance decreased 39% and 35% in the MSCs- and TNF-a-treated groups, respectively, while the vehicle group showed a stronger reduction (72%, p These results suggest that, in this therapeutic conditions, the anti-inflammatory effect of non-activated MSCs is strong enough to conserve the functionality of the lung during ischemia, and reaffirm the notion that cell therapy is a novel tool for organ preservation.

Summary Neutrophils require directional cues to navigate through the complex structure of venular walls and into inflamed tissues. Here we applied confocal intravital microscopy to analyze neutrophil emigration in cytokine-stimulated mouse cremaster muscles. We identified differential and non-redundant roles for the chemokines CXCL1 and CXCL2, governed by their distinct cellular sources. CXCL1 was produced mainly by TNF-stimulated endothelial cells (ECs) and pericytes and supported luminal and sub-EC neutrophil crawling. Conversely, neutrophils were the main producers of CXCL2, and this chemokine was critical for correct breaching of endothelial junctions. This pro-migratory activity of CXCL2 depended on the atypical chemokine receptor 1 (ACKR1), which is enriched within endothelial junctions. Transmigrating neutrophils promoted a self-guided migration response through EC junctions, creating a junctional chemokine “depot” in the form of ACKR1-presented CXCL2 that enabled efficient unidirectional luminal-to-abluminal migration. Thus, CXCL1 and CXCL2 act in a sequential manner to guide neutrophils through venular walls as governed by their distinct cellular sources., Graphical Abstract, Highlights • The CXCR2 ligands CXCL1 and CXCL2 play distinct roles in neutrophil diapedesis • Endothelial cell (EC)- and pericyte-derived CXCL1 mediate neutrophil crawling • CXCL2 is neutrophil derived and is retained at EC junctions by ACKR1 • ACKR1-CXCL2 axis is critical for unidirectional paracellular neutrophil TEM in vivo, Girbl et al. find that sequential interactions with the chemokines CXCL1 and CXCL2 guide neutrophil crawling and subsequent migration through venular walls during inflammation. Transmigrating neutrophils promoted unidirectional luminal-to-abluminal migration by depositing CXCL2 on the chemokine receptor ACKR1 at endothelial junctions, providing a paradigm of self-guided migration.

Neutrophil extravasation is a migratory event in response to inflammation that depends on cytoskeletal dynamics regulated by myosins. Myosin-1e (Myo1e) is a long-tailed class-I myosin that has not yet been studied in the context of neutrophil-endothelial interactions and neutrophil extravasation. Intravital microscopy of TNFα-inflamed cremaster muscles in Myo1e-deficient mice revealed that Myo1e is required for efficient neutrophil extravasation. Specifically, Myo1e deficiency caused increased rolling velocity, decreased firm adhesion, aberrant crawling, and strongly reduced transmigration. Interestingly, we observed a striking discontinuous rolling behavior termed "intermittent rolling," during which Myo1e-deficient neutrophils showed alternating rolling and jumping movements. Surprisingly, chimeric mice revealed that these effects were due to Myo1e deficiency in leukocytes. Vascular permeability was not significantly altered in Myo1e KO mice. Myo1e-deficient neutrophils showed diminished arrest, spreading, uropod formation, and chemotaxis due to defective actin polymerization and integrin activation. In conclusion, Myo1e critically regulates adhesive interactions of neutrophils with the vascular endothelium and neutrophil extravasation. Myo1e may therefore be an interesting target in chronic inflammatory diseases characterized by excessive neutrophil recruitment.

Neutrophil extravasation requires opening of the endothelial barrier but does not necessarily cause plasma leakage. Leaks are prevented by contractile actin filaments surrounding the diapedesis pore, keeping this opening tightly closed around the transmigrating neutrophils. We have identified the receptor system that is responsible for this. We show that silencing, or gene inactivation, of endothelial Tie-2 results in leak formation in postcapillary venules of the inflamed cremaster muscle at sites of neutrophil extravasation, as visualized by fluorescent microspheres. Leakage was dependent on neutrophil extravasation, because it was absent upon neutrophil depletion. We identified the Cdc42 GTPase exchange factor FGD5 as a downstream target of Tie-2 that is essential for leakage prevention during neutrophil extravasation. Looking for the Tie-2 agonist and its source, we found that platelet-derived angiopoietin-1 (Angpt1) was required to prevent neutrophil-induced leaks. Intriguingly, blocking von Willebrand factor (VWF) resulted in vascular leaks during transmigration, indicating that platelets interacting with endothelial VWF activate Tie-2 by secreting Angpt1, thereby preventing diapedesis-induced leakiness.

Neutrophils are essential to the pathogenesis of many inflammatory diseases. In the autoantibody-mediated K/BxN model of inflammatory arthritis, the alternative pathway (AP) of complement and Fc gamma receptors (FcγRs) are required for disease development while the classical pathway is dispensable. The reason for this differential requirement is unknown. We show that within minutes of K/BxN serum injection complement activation (CA) is detected on circulating neutrophils, as evidenced by cell surface C3 fragment deposition. CA requires the AP factor B and FcγRs but not C4, implying that engagement of FcγRs by autoantibody or immune complexes directly triggers AP C3 convertase assembly. The absence of C5 does not prevent CA on neutrophils but diminishes the upregulation of adhesion molecules. In vivo two-photon microscopy reveals that CA on neutrophils is critical for neutrophil extravasation and generation of C5a at the site of inflammation. C5a stimulates the release of neutrophil proteases, which contribute to the degradation of VE-cadherin, an adherens junction protein that regulates endothelial barrier integrity. C5a receptor antagonism blocks the extracellular release of neutrophil proteases, suppressing VE-cadherin degradation and neutrophil transendothelial migration in vivo. These results elucidate the AP-dependent intravascular neutrophil-endothelial interactions that initiate the inflammatory cascade in this disease model but may be generalizable to neutrophil extravasation in other inflammatory processes.

The factors that regulate the passage of bacteria between different anatomical compartments are unclear. We have used an experimental model of infection with Streptococcus pneumoniae to examine the host and bacterial factors involved in the passage of bacteria from the lung to the pleural space. The transcriptional profile of host and bacterial cells within the pleural space and lung was analyzed using deep sequencing of the entire transcriptome using the technique of dual RNA-seq. We found significant differences in the host and bacterial RNA profiles in infection, which shed light on the key factors that allow passage of this bacterium into the pleural space., Streptococcus pneumoniae is the dominant cause of community-acquired pneumonia worldwide. Invasion of the pleural space is common and results in increased mortality. We set out to determine the bacterial and host factors that influence invasion of the pleural space. In a murine model of pneumococcal infection, we isolated neutrophil-dominated samples of bronchoalveolar and pleural fluid containing bacteria 48 hours after infection. Using dual RNA sequencing (RNA-seq), we characterized bacterial and host transcripts that were differentially regulated between these compartments and bacteria in broth and resting neutrophils, respectively. Pleural and lung samples showed upregulation of genes involved in the positive regulation of neutrophil extravasation but downregulation of genes mediating bacterial killing. Compared to the lung samples, cells within the pleural space showed marked upregulation of many genes induced by type I interferons, which are cytokines implicated in preventing bacterial transmigration across epithelial barriers. Differences in the bacterial transcripts between the infected samples and bacteria grown in broth showed the upregulation of genes in the bacteriocin locus, the pneumococcal surface adhesin PsaA, and the glycopeptide resistance gene vanZ; the gene encoding the ClpP protease was downregulated in infection. One hundred sixty-nine intergenic putative small bacterial RNAs were also identified, of which 43 (25.4%) small RNAs had been previously described. Forty-two of the small RNAs were upregulated in pleura compared to broth, including many previously identified as being important in virulence. Our results have identified key host and bacterial responses to invasion of the pleural space that can be potentially exploited to develop alternative antimicrobial strategies for the prevention and treatment of pneumococcal pleural disease. IMPORTANCE The factors that regulate the passage of bacteria between different anatomical compartments are unclear. We have used an experimental model of infection with Streptococcus pneumoniae to examine the host and bacterial factors involved in the passage of bacteria from the lung to the pleural space. The transcriptional profile of host and bacterial cells within the pleural space and lung was analyzed using deep sequencing of the entire transcriptome using the technique of dual RNA-seq. We found significant differences in the host and bacterial RNA profiles in infection, which shed light on the key factors that allow passage of this bacterium into the pleural space.

Neutrophils are key players of the innate immune system, that are involved in coordinating the initiation, propagation and resolution of inflammation. Accurate neutrophil migration (chemotaxis) to sites of inflammation in response to gradients of chemoattractants is pivotal to these roles. Binding of chemoattractants to dedicated G-protein-coupled receptors (GPCRs) initiates downstream signalling events that promote neutrophil polarisation, a prerequisite for directional migration. We provide a brief summary of some of the recent insights into signalling events and feedback loops that serve to initiate and maintain neutrophil polarisation. This is followed by a discussion of recent developments in the understanding of in vivo neutrophil chemotaxis, a process that is frequently referred to as ‘recruitment’ or ‘trafficking’. Here, we summarise neutrophil mobilisation from and homing to the bone marrow, and briefly discuss the role of glucosaminoglycan-immobilised chemoattractants and their corresponding receptors in the regulation of neutrophil extravasation and neutrophil swarming. We furthermore touch on some of the most recent insights into the roles of atypical chemokine receptors (ACKRs) in neutrophil recruitment, and discuss neutrophil reverse (transendothelial) migration together with potential function(s) in the dissemination and/or resolution of inflammation.

Staphylococcus aureus has become a serious threat to human health. In addition to having increased antibiotic resistance, the bacterium is a master at adapting to its host by evading almost every facet of the immune system, the so-called immune evasion proteins. Many of these immune evasion proteins target neutrophils, the most important immune cells in clearing S. aureus infections. The neutrophil attacks pathogens via a plethora of strategies. Therefore, it is no surprise that S. aureus has evolved numerous immune evasion strategies at almost every level imaginable. In this review we discuss step by step the aspects of neutrophil-mediated killing of S. aureus , such as neutrophil activation, migration to the site of infection, bacterial opsonization, phagocytosis, and subsequent neutrophil-mediated killing. After each section we discuss how S. aureus evasion molecules are able to resist the neutrophil attack of these different steps. To date, around 40 immune evasion molecules of S. aureus are known, but its repertoire is still expanding due to the discovery of new evasion proteins and the addition of new functions to already identified evasion proteins. Interestingly, because the different parts of neutrophil attack are redundant, the evasion molecules display redundant functions as well. Knowing how and with which proteins S. aureus is evading the immune system is important in understanding the pathophysiology of this pathogen. This knowledge is crucial for the development of therapeutic approaches that aim to clear staphylococcal infections.

Bergapten (5-methoxypsoralen, 5-MOP) is a plant-derived furocoumarin with demonstrated anti-inflammatory action. The present study investigated its effects on allergic inflammation in two related pathways of mast cell degranulation. Compound 48/80 and lipopolysaccharide (LPS) were used to activate the IgE-independent pathway while bovine serum albumin (BSA) was used as allergen for the IgE-dependent pathway. The modulatory effect of bergapten on mast cell degranulation, neutrophil extravasation, protein concentration, lung histopathology, and oxidative stress was assessed. Bergapten at 10, 30, and 100 μg/ml for 15 min stabilized mast cells in rat mesenteric tissue from disruptionin vitroand when administeredin vivoat 3, 10, and 30 mg kg−1for 1 h protected mice from fatal anaphylaxis induced by compound 48/80. Similarly, treatment of LPS-challenged mice with bergapten (3, 10, and 30 mg kg−1) for 24 h significantly decreased neutrophil infiltration into bronchoalveolar lavage fluid, mean protein concentration, and inflammatory cell infiltration of pulmonary tissues when compared to the saline-treated LPS-challenged control. In addition, lung histology of the bergapten-treated LPS-challenged mice showed significantly less oedema, congestion, and alveolar septa thickening when compared to the saline-treated LPS-challenged disease control. LPS-induced oxidative stress was significantly reduced through increased tissue activities of catalase and superoxide dismutase and reduced malondialdehyde levels on treatment with bergapten. In the triple antigen-induced active anaphylaxis, daily administration of bergapten at 3, 10, and 30 mg kg−1for 10 days, respectively, protected previously sensitized and challenged mice against anaphylactic shock. Overall, our study demonstrates the ability of bergapten to attenuate allergic airway-induced hypersensitivity in murine models of inflammation, suggesting its possible therapeutic benefit in this condition.

Two-photon intravital imaging (2P-IVM) of the murine trachea is a powerful technique for real-time imaging of immune cell recruitment and trafficking during airborne pathogen infections. Neutrophils are an important component of the innate immune response that are able to rapidly infiltrate the airway mucosa in response to Streptococcus pneumoniae infection. Here we describe a protocol to visualize in vivo neutrophil extravasation and cell dynamics in the tracheal tissue of a S. pneumoniae-infected mouse using 2P-IVM. To perform this protocol, we infected and imaged the trachea of a lysozyme M green fluorescent protein (LysM-GFP) mouse, in which neutrophils express GFP. Additionally, we used a custom-designed platform, which allowed the intubation and fixation of the trachea after surgical exposition, and we injected intravenously a fluorescently labeled dextran solution to visualize the blood vessels.

Periodontitis is a common human inflammatory disease. In this condition, microbiota trigger excessive inflammation in oral mucosal tissues surrounding the dentition, resulting in destruction of tooth-supporting structures (connective tissue and bone). While susceptibility factors for common forms of periodontitis are not clearly understood, studies in patients with single genetic defects reveal a critical role for tissue neutrophils in disease susceptibility. Indeed, various genetic defects in the development, egress from the bone marrow, chemotaxis, and extravasation are clearly linked to aggressive/severe periodontitis at an early age. Here, we provide an overview of genetic defects in neutrophil biology that are linked to periodontitis. In particular, we focus on the mechanisms underlying Leukocyte Adhesion Deficiency-I, the prototypic Mendelian defect of impaired neutrophil extravasation and severe periodontitis.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid which modulates vascular integrity through its receptors, S1P1-S1P5. Notably, S1P2 has been shown to mediate the disruption of cerebrovascular integrity in vitro and in vivo. However, the mechanism underlying this process has not been fully elucidated. We evaluated the role of S1P2 in blood-brain barrier (BBB) disruption induced by lipopolysaccharide (LPS)-mediated systemic inflammation and found that BBB disruption and neutrophil infiltration were significantly attenuated in S1pr2-/- mice relative to S1pr2+/- littermates. This is concomitant with attenuation of LPS-induced transcriptional activation of IL-6 and downregulation of occludin. Furthermore, S1pr2-/- mice had significantly reduced expression of genes essential for neutrophil infiltration: Sele, Cxcl1, and Cxcl2. Conversely, pharmacological agonism of S1P2 induced transcriptional activation of E-selectin in vitro and in vivo. Although S1P2 does not appear to be required for activation of microglia, stimulation of microglial cells with the S1P2 potentiated the response of endothelial cells to LPS. These results demonstrate that S1P2 promotes LPS-induced neutrophil extravasation by inducing expression of endothelial adhesion molecule gene, Sele, and potentiating microglial inflammation of endothelial cells. It is likely that S1P2 is a mediator of cerebrovascular inflammation and represents a potential therapeutic target for neurodegenerative disease such as vascular cognitive impairment.

IMPACT: This work identifies host immune perturbations in sepsis mortality that suggests targets for a precision medicine paradigm in the host response to infection. OBJECTIVES/GOALS: To compare the early whole blood transcriptome during sepsis between 30-day survivors and non-survivors in the Intensive Care Unit (ICU), and to evaluate for pathway enrichment that might explain sepsis lethality. METHODS/STUDY POPULATION: We enrolled 162 sepsis patients in the first 24 hours of ICU admission, particularly targeting individuals requiring vasopressors. Peripheral whole blood was collected in PAXgene vacutainers. Isolated RNA was analyzed with Affymetrix Human Genome ST 2.0 microarray. Differential gene expression was performed with Bioconductor/R/limma using log2 fold-change +/-0.6 as a threshold for differential expression, and a Benjamini-Hochberg adjusted p-value

Background/introduction The neuroinflammatory response plays a major role in the secondary injury cascade after traumatic spinal cord injury (SCI). To date, systemic anti-inflammatory medications such as methylprednisolone sodium succinate (MPSS) have shown promise in SCI. However, systemic immunosuppression can have detrimental side effects. Therefore, immunomodulatory approaches including the use of human immunoglobulin G (hIgG) could represent an attractive alternative. While emerging preclinical data suggests that hIgG is neuroprotective after SCI, the optimal time window of administration and the mechanism of action remain incompletely understood. These knowledge gaps were the focus of this research study. Methods Female adult Wistar rats received a clip compression-contusion SCI at the C7/T1 level of the spinal cord. Injured rats were randomized, in a blinded manner, to receive a single intravenous bolus of hIgG (2 g/kg) or control buffer at 15 minutes (min), 1 hour (h) or 4 h post-SCI. At 24 h and 8 weeks post-SCI, molecular, histological and neurobehavioral analyses were undertaken. Results At all 3 administration time points, hIgG (2 g/kg) resulted in significantly better short-term and long-term outcomes as compared to control buffer. No significant differences were observed when comparing outcomes between the different time points of administration. At 24 h post-injury, hIgG (2 g/kg) administration enhanced the integrity of the blood spinal cord barrier (BSCB) by increasing expression of tight junction proteins and reducing inflammatory enzyme expression. Improvements in BSCB integrity were associated with reduced immune cell infiltration, lower amounts of albumin and Evans Blue in the injured spinal cord and greater expression of anti-inflammatory cytokines. Furthermore, hIgG (2 g/kg) increased expression of neutrophil chemoattractants in the spleen and sera. After hIgG (2 g/kg) treatment, there were more neutrophils in the spleen and fewer neutrophils in the blood. hIgG also co-localized with endothelial cell ligands that mediate neutrophil extravasation into the injured spinal cord. Importantly, short-term effects of delayed hIgG (2 g/kg) administration were associated with enhanced tissue and neuron preservation, as well as neurobehavioral and sensory recovery at 8 weeks post-SCI. Discussion and conclusion hIgG (2 g/kg) shows promise as a therapeutic approach for SCI. The anti-inflammatory effects mediated by hIgG (2 g/kg) in the injured spinal cord might be explained in twofold. First, hIgG might antagonize neutrophil infiltration into the spinal cord by co-localizing with endothelial cell ligands that mediate various steps in neutrophil extravasation. Second, hIgG could traffic neutrophils towards the spleen by increasing expression of neutrophil chemoattractants in the spleen and sera. Overall, we demonstrate that delayed administration of hIgG (2 g/kg) at 1 and 4-h post-injury enhances short-term and long-term benefits after SCI by modulating local and systemic neuroinflammatory cascades.