3D printed hybrid scaffolds do not induce adverse inflammation in mice and direct human BM-MSC chondrogenesis in vitro.

Biomaterials that can improve the healing of articular cartilage lesions are needed. To address this unmet need, we developed novel 3D printed silica/poly(tetrahydrofuran)/poly(ε-caprolactone) (SiO2/PTHF/PCL-diCOOH) hybrid scaffolds. Our aim was to carry out essential studies to advance this medical device towards functional validation in pre-clinical trials. First, we show that the chemical composition, microarchitecture and mechanical properties of these scaffolds were not affected by sterilisation with gamma irradiation. To evaluate the systemic and local immunogenic reactivity of the sterilised 3D printed hybrid scaffolds, they were implanted subcutaneously into Balb/c mice. The scaffolds did not trigger a systemic inflammatory response over one week of implantation. The interaction between the host immune system and the implanted scaffold elicited a local physiological reaction with infiltration of mononuclear cells without any signs of a chronic inflammatory response. Then, we investigated how these 3D printed hybrid scaffolds direct chondrogenesis in vitro. Human bone marrow-derived mesenchymal stem/stromal cells (hBM-MSCs) seeded within the 3D printed hybrid scaffolds were cultured under normoxic or hypoxic conditions, with or without chondrogenic supplements. Chondrogenic differentiation assessed by both gene expression and protein production analyses showed that 3D printed hybrid scaffolds support hBM-MSC chondrogenesis. Articular cartilage-specific extracellular matrix deposition within these scaffolds was enhanced under hypoxic conditions (1.7 or 3.7 fold increase in the median of aggrecan production in basal or chondrogenic differentiation media). Our findings show that 3D printed SiO2/PTHF/PCL-diCOOH hybrid scaffolds have the potential to support the regeneration of cartilage tissue.

Marginalising dyslexic researchers is bad for science.

Communication in the sciences is often based on text, which places researchers with dyslexia at a disadvantage. However, this means that science is missing out on the original insights and specific strengths in exploration that dyslexic researchers bring to their disciplines. Here we discuss how the scientific community can address the challenges that dyslexic researchers face, and how science stands to benefit as a result. We discuss this in the context of a new theoretical framework proposing the existence of complementary learning strategies that could play a key role in scientific progress, particularly with regard to accelerating innovation.

3D printed hybrid scaffolds for bone regeneration using calcium methoxyethoxide as a calcium source.

Introduction: Hybrids consist of inorganic and organic co-networks that are indistinguishable above the nanoscale, which can lead to unprecedented combinations of properties, such as high toughness and controlled degradation. Methods: We present 3D printed bioactive hybrid scaffolds for bone regeneration, produced by incorporating calcium into our "Bouncy Bioglass", using calcium methoxyethoxide (CME) as the calcium precursor. SiO2-CaOCME/PTHF/PCL-diCOOH hybrid "inks" for additive manufacturing (Direct Ink Writing) were optimised for synergy of mechanical properties and open interconnected pore channels. Results and Discussion: Adding calcium improved printability. Changing calcium content (5, 10, 20, 30, and 40 mol.%) of the SiO2-CaOCME/PTHF/PCL-diCOOH hybrids affected printability and mechanical properties of the lattice-like scaffolds. Hybrids containing 30 mol.% calcium in the inorganic network (70S30CCME-CL) printed with 500 µm channels and 100 µm strut size achieved the highest strength (0.90 ± 0.23 MPa) and modulus of toughness (0.22 ± 0.04 MPa). These values were higher than Ca-free SiO2/PTHF/PCL-diCOOH hybrids (0.36 ± 0.14 MPa strength and 0.06 ± 0.01 MPa toughness modulus). Over a period of 90 days of immersion in simulated body fluid (SBF), the 70S30CCME-CL hybrids also kept a stable strain to failure (~30 %) and formed hydroxycarbonate apatite within three days. The extracts released by the 70S30CCME-CL hybrids in growth medium did not cause cytotoxic effects on human bone marrow stromal cells over 24 h of culture.

Drug-based mobilisation of mesenchymal stem/stromal cells improves cardiac function post myocardial infarction.

There is an unmet need for treatments that prevent the progressive cardiac dysfunction following myocardial infarction. Mesenchymal stem/stromal cells (MSCs) are under investigation for cardiac repair; however, culture expansion prior to transplantation is hindering their homing and reparative abilities. Pharmacological mobilisation could be an alternative to MSC transplantation. Here, we report that endogenous MSCs mobilise into the circulation at day 5 post myocardial infarction in male Lewis rats. This mobilisation can be significantly increased by using a combination of the FDA-approved drugs mirabegron (ß3-adrenoceptor agonist) and AMD3100 (CXCR4 antagonist). Blinded cardiac magnetic resonance imaging analysis showed the treated group to have increased left ventricular ejection fraction and decreased end systolic volume at 5 weeks post myocardial infarction. The mobilised group had a significant decrease in plasma IL-6 and TNF-α levels, a decrease in interstitial fibrosis, and an increase in the border zone blood vessel density. Conditioned medium from blood-derived MSCs supported angiogenesis in vitro, as shown by tube formation and wound healing assays. Our data suggest a novel pharmacological strategy that enhances myocardial infarction-induced MSC mobilisation and improves cardiac function after myocardial infarction.

Interaction of monodispersed strontium containing bioactive glass nanoparticles with macrophages.

The cellular response of murine primary macrophages to monodisperse strontium containing bioactive glass nanoparticles (SrBGNPs), with diameters of 90 ± 10 nm and a composition (mol%) of 88.8 SiO2-1.8CaO-9.4SrO (9.4% Sr-BGNPs) was investigated for the first time. Macrophage response is critical as applications of bioactive nanoparticles will involve the nanoparticles circulating in the blood stream and macrophages will be the first cells to encounter the particles, as part of inflammatory response mechanisms. Macrophage viability and total DNA measurements were not decreased by particle concentrations of up to 250 µg/mL. The Sr-BGNPs were actively internalised by the macrophages via formation of endosome/lysosome-like vesicles bordered by a membrane inside the cells. The Sr-BGNPs degraded inside the cells, with the Ca and Sr maintained inside the silica network. When RAW264.7 cells were incubated with Sr-BGNPs, the cells were polarised towards the pro-regenerative M2 population rather than the pro-inflammatory M1 population. Sr-BGNPs are potential biocompatible vehicles for therapeutic cation delivery for applications in bone regeneration.

Lung Marginated and Splenic Murine Resident Neutrophils Constitute Pioneers in Tissue-Defense During Systemic E. coli Challenge.

The rapid response of neutrophils throughout the body to a systemic challenge is a critical first step in resolution of bacterial infection such as Escherichia coli (E. coli). Here we delineated the dynamics of this response, revealing novel insights into the molecular mechanisms using lung and spleen intravital microscopy and 3D ex vivo culture of living precision cut splenic slices in combination with fluorescent labelling of endogenous leukocytes. Within seconds after challenge, intravascular marginated neutrophils and lung endothelial cells (ECs) work cooperatively to capture pathogens. Neutrophils retained on lung ECs slow their velocity and aggregate in clusters that enlarge as circulating neutrophils carrying E. coli stop within the microvasculature. The absolute number of splenic neutrophils does not change following challenge; however, neutrophils increase their velocity, migrate to the marginal zone (MZ) and form clusters. Irrespective of their location all neutrophils capturing heat-inactivated E. coli take on an activated phenotype showing increasing surface CD11b. At a molecular level we show that neutralization of ICAM-1 results in splenic neutrophil redistribution to the MZ under homeostasis. Following challenge, splenic levels of CXCL12 and ICAM-1 are reduced allowing neutrophils to migrate to the MZ in a CD29-integrin dependent manner, where the enlargement of splenic neutrophil clusters is CXCR2-CXCL2 dependent. We show directly molecular mechanisms that allow tissue resident neutrophils to provide the first lines of antimicrobial defense by capturing circulating E. coli and forming clusters both in the microvessels of the lung and in the parenchyma of the spleen.

Biased action of the CXCR4-targeting drug plerixafor is essential for its superior hematopoietic stem cell mobilization.

Following the FDA-approval of the hematopoietic stem cell (HSC) mobilizer plerixafor, orally available and potent CXCR4 antagonists were pursued. One such proposition was AMD11070, which was orally active and had superior antagonism in vitro; however, it did not appear as effective for HSC mobilization in vivo. Here we show that while AMD11070 acts as a full antagonist, plerixafor acts biased by stimulating ß-arrestin recruitment while fully antagonizing G protein. Consequently, while AMD11070 prevents the constitutive receptor internalization, plerixafor allows it and thereby decreases receptor expression. These findings are confirmed by the successful transfer of both ligands' binding sites and action to the related CXCR3 receptor. In vivo, plerixafor exhibits superior HSC mobilization associated with a dramatic reversal of the CXCL12 gradient across the bone marrow endothelium, which is not seen for AMD11070. We propose that the biased action of plerixafor is central for its superior therapeutic effect in HSC mobilization.

Bioglass/carbonate apatite/collagen composite scaffold dissolution products promote human osteoblast differentiation.

OssiMend® Bioactive (Collagen Matrix Inc., NJ) is a three-component porous composite bone graft device of 45S5 Bioglass/carbonate apatite/collagen. Our in vitro studies showed that conditioned media of the dissolution products of OssiMend Bioactive stimulated primary human osteoblasts to form mineralized bone-like nodules in vitro in one week, in basal culture media (no osteogenic supplements). Osteoblast differentiation was followed by gene expression analysis and a mineralization assay. In contrast, the dissolution products from commercial OssiMend (Bioglass-free carbonate apatite/collagen scaffolds), or from 45S5 Bioglass particulate alone, did not induce the mineralization of the extracellular matrix, but did induce osteoblast differentiation to mature osteoblasts, evidenced by the strong upregulation of BGLAP and IBSP mRNA levels. The calcium ions and soluble silicon species released from 45S5 Bioglass particles and additional phosphorus release from OssiMend mediated the osteostimulatory effects. Medium conditioned with OssiMend Bioactive dissolution had a much higher concentration of phosphorus and silicon than media conditioned with OssiMend and 45S5 Bioglass alone. While OssiMend and OssiMend Bioactive led to calcium precipitation in cell culture media, OssiMend Bioactive produced a higher concentration of soluble silicon than 45S5 Bioglass and higher dissolution of phosphorus than OssiMend. These in vitro results suggest that adding 45S5 Bioglass to OssiMend produces a synergistic osteostimulation effect on primary human osteoblasts. In summary, dissolution products of a Bioglass/carbonate apatite/collagen composite scaffold (OssiMend® Bioactive) stimulate human osteoblast differentiation and mineralization of extracellular matrix in vitro without any osteogenic supplements. The mineralization was faster than for dissolution products of ordinary Bioglass.

The Secretive Life of Neutrophils Revealed by Intravital Microscopy.

Neutrophils are the most abundant circulating leukocyte within the blood stream and for many years the dogma has been that these cells migrate rapidly into tissues in response to injury or infection, forming the first line of host defense. While it has previously been documented that neutrophils marginate within the vascular beds of the lung and liver and are present in large numbers within the parenchyma of tissues, such as spleen, lymph nodes, and bone marrow (BM), the function of these tissue resident neutrophils under homeostasis, in response to pathogen invasion or injury has only recently been explored, revealing the unexpected role of these cells as immunoregulators or immune helpers and also unraveling their heterogeneity and plasticity. Neutrophils are highly motile cells and the use of intravital microscopy (IVM) to image cells within their environment with little manipulation has dramatically increased our understanding of the function, migratory behavior, and interaction of these short-lived cells with other innate and adaptive immune cells. Contrary to previous dogma, these studies have shown that marginated and tissue resident neutrophils are the first responders to pathogens and injury, critical in limiting the spread of infection and contributing to the orchestration of the subsequent immune response. The interplay of neutrophils, with other neutrophils, leukocytes, and stroma cells can also modulate and tune their early and late response in order to eradicate pathogens, minimize tissue damage, and, in certain circumstances, contribute to tissue repair. In this review, we will follow the extraordinary journey of neutrophils from their origin in the BM to their death, exploring their role as tissue resident cells in the lung, spleen, lymph nodes, and skin and outlining the importance of neutrophil subsets, their functions under homeostasis, and in response to infection. Finally, we will comment on how understanding these processes in greater detail at a molecular level can lead to development of new therapeutics.

Effect of the CXCR4 antagonist plerixafor on endogenous neutrophil dynamics in the bone marrow, lung and spleen.

Treatment with the CXCR4 antagonist, plerixafor (AMD3100), has been proposed for clinical use in patients with WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome and in pulmonary fibrosis. However, there is controversy with respect to the impact of plerixafor on neutrophil dynamics in the lung, which may affect its safety profile. In this study, we investigated the kinetics of endogenous neutrophils by direct imaging, using confocal intravital microscopy in mouse bone marrow, spleen, and lungs. Neutrophils are observed increasing their velocity and exiting the bone marrow following plerixafor administration, with a concomitant increase in neutrophil numbers in the blood and spleen, while the marginated pool of neutrophils in the lung microvasculature remained unchanged in terms of numbers and cell velocity. Use of autologous radiolabeled neutrophils and SPECT/CT imaging in healthy volunteers showed that plerixafor did not affect GM-CSF-primed neutrophil entrapment or release in the lungs. Taken together, these data suggest that plerixafor causes neutrophil mobilization from the bone marrow but does not impact on lung marginated neutrophil dynamics and thus is unlikely to compromise respiratory host defense both in humans and mice.

Pharmacological tools to mobilise mesenchymal stromal cells into the blood promote bone formation after surgery.

Therapeutic approaches requiring the intravenous injection of autologous or allogeneic mesenchymal stromal cells (MSCs) are currently being evaluated for treatment of a range of diseases, including orthopaedic injuries. An alternative approach would be to mobilise endogenous MSCs into the blood, thereby reducing costs and obviating regulatory and technical hurdles associated with development of cell therapies. However, pharmacological tools for MSC mobilisation are currently lacking. Here we show that ß3 adrenergic agonists (ß3AR) in combination with a CXCR4 antagonist, AMD3100/Plerixafor, can mobilise MSCs into the blood in mice and rats. Mechanistically we show that reversal of the CXCL12 gradient across the bone marrow endothelium and local generation of endocannabinoids may both play a role in this process. Using a spine fusion model we provide evidence that this pharmacological strategy for MSC mobilisation enhances bone formation.

Replicating landmine blast loading in cellular in vitro models.

Trauma arising from landmines and improvised explosive devices promotes heterotopic ossification, the formation of extra-skeletal bone in non-osseous tissue. To date, experimental platforms that can replicate the loading parameter space relevant to improvised explosive device and landmine blast wave exposure have not been available to study the effects of such non-physiological mechanical loading on cells. Here, we present the design and calibration of three distinct in vitro experimental loading platforms that allow us to replicate the spectrum of loading conditions recorded in near-field blast wave exposure. We subjected cells in suspension or in a three-dimensional hydrogel to strain rates up to 6000 s-1 and pressure levels up to 45 MPa. Our results highlight that cellular activation is regulated in a non-linear fashion-not by a single mechanical parameter, it is the combined action of the applied mechanical pressure, rate of loading and loading impulse, along with the extracellular environment used to convey the pressure waves. Finally, our research indicates that PO MSCs are finely tuned to respond to mechanical stimuli that fall within defined ranges of loading.

CXCR4, the master regulator of neutrophil trafficking in homeostasis and disease.

BACKGROUND: Chemokines play a critical role in orchestrating the distribution and trafficking of neutrophils in homeostasis and disease. RESULTS: The CXCR4/CXCL12 chemokine axis has been identified as a central regulator of these processes. CONCLUSION: In this review, we focus on the role of CXCR4/CXCL12 chemokine axis in regulating neutrophil release from the bone marrow and the trafficking of senescent neutrophils back to the bone marrow for clearance under homeostasis and disease. We also discuss the role of CXCR4 in fine-tuning neutrophil responses in the context of inflammation.

Two distinct CXCR4 antagonists mobilize progenitor cells in mice by different mechanisms.

Pharmacological mobilization of hematopoietic progenitor cells (HPCs) is used clinically to harvest HPCs for bone marrow transplants. It is now widely accepted that the CXCR4:CXCL12 chemokine axis plays a critical role in the retention of HPCs in the bone marrow, and CXCR4 antagonists have been developed for their mobilization. The first of this class of drugs to be US Food and Drug Administration-approved was the bicyclam AMD3100. In addition to mobilizing HPCs and leukocytes in naïve mice, AMD3100 has been shown to mobilize mesenchymal progenitor cells (MPCs) in vascular endothelial growth factor (VEGF-A) pretreated mice. AMD3100 binds to the transmembrane region of CXCR4 and is thought to mobilize HPCs by reversing the gradient of CXCL12 across the bone marrow endothelium. Consistent with this hypothesis, our data show that selective neutralization of CXCL12, with chalcone 4-phosphate (C4P), inhibited AMD3100-stimulated mobilization of HPCs and leukocytes in naïve mice and MPCs in VEGF-A pretreated mice. In contrast it is shown here that the CXCR4 antagonist KRH3955 that binds to the extracellular loop of CXCR4 does not reverse the CXCL12 chemokine gradient. However, this drug efficiently mobilizes HPCs, a response that is not inhibited by C4P. In contrast, KRH3955 does not mobilize MPCs in VEGF-A pretreated mice. These data suggest that CXCR4 antagonists that bind to distinct regions of the receptor mobilize progenitor cells by distinct molecular mechanisms.

CD43Lo classical monocytes participate in the cellular immune response to isolated primary blast lung injury.

BACKGROUND: Understanding of the cellular immune response to primary blast lung injury (PBLI) is limited, with only the neutrophil response well documented. Moreover, its impact on the immune response in distal organs remains poorly understood. In this study, a rodent model of isolated primary blast injury was used to investigate the acute cellular immune response to isolated PBLI in the circulation and lung, including the monocyte response, and investigate distal subacute immune effects in the spleen and liver 6 hours after injury. METHODS: Rats were subjected to a shock wave (~135 kPa overpressure, 2 ms duration) inducing PBLI or sham procedure. Rat physiology was monitored, and at 1, 3, and 6 hours thereafter, blood, lung, and bronchoalveolar lavage fluid (BALF) were collected and analyzed by flow cytometry, enzyme-linked immunosorbent assay, and histologic examination. In addition, at 6 hours, spleen and liver were collected and analyzed by flow cytometry. RESULTS: Lung histology confirmed pulmonary barotrauma and inflammation. This was associated with rises in CXCL-1, interleukin 6 (IL-6), tumor necrosis factor α and albumin protein in the BALF. Significant acute increases in blood and lung neutrophils and CD43Lo/His48Hi (classical) monocytes/macrophages were detected. No significant changes were seen in blood or lung "nonclassical" monocyte and in natural killler, B, or T cells. In the BALF, significant increases were seen in neutrophils, CD43Lo monocyte-macrophages and monocyte chemoattractant protein-1. Significant increases in CD43Lo and Hi monocyte-macrophages were detected in the spleen at 6 hours. CONCLUSION: This study reveals a robust and selective response of CD43Lo/His48Hi (classical) monocytes, in addition to neutrophils, in blood and lung tissue following PBLI. An increase in monocyte-macrophages was also observed in the spleen at 6 hours. This profile of immune cells in the blood and BALF could present a new research tool for translational studies seeking to monitor, assess, or attenuate the immune response in blast-injured patients.

Prolonged but not short-duration blast waves elicit acute inflammation in a rodent model of primary blast limb trauma.

BACKGROUND: Blast injuries from conventional and improvised explosive devices account for 75% of injuries from current conflicts; over 70% of injuries involve the limbs. Variable duration and magnitude of blast wave loading occurs in real-life explosions and is hypothesised to cause different injuries. While a number of in vivo models report the inflammatory response to blast injuries, the extent of this response has not been investigated with respect to the duration of the primary blast wave. The relevance is that explosions in open air are of short duration compared to those in confined spaces. METHODS: Hindlimbs of adult Sprauge-Dawley rats were subjected to focal isolated primary blast waves of varying overpressure (1.8-3.65kPa) and duration (3.0-11.5ms), utilising a shock tube and purpose-built experimental rig. Rats were monitored during and after the blast. At 6 and 24h after exposure, blood, lungs, liver and muscle tissues were collected and prepared for histology and flow cytometry. RESULTS: At 6h, increases in circulating neutrophils and CD43Lo/His48Hi monocytes were observed in rats subjected to longer-duration blast waves. This was accompanied by increases in circulating pro-inflammatory chemo/cytokines KC and IL-6. No changes were observed with shorter-duration blast waves irrespective of overpressure. In all cases, no histological damage was observed in muscle, lung or liver. By 24h post-blast, all inflammatory parameters had normalised. CONCLUSIONS: We report the development of a rodent model of primary blast limb trauma that is the first to highlight an important role played by blast wave duration and magnitude in initiating acute inflammatory response following limb injury in the absence of limb fracture or penetrating trauma. The combined biological and mechanical method developed can be used to further understand the complex effects of blast waves in a range of different tissues and organs in vivo.

A Single 9-Colour Flow Cytometric Method to Characterise Major Leukocyte Populations in the Rat: Validation in a Model of LPS-Induced Pulmonary Inflammation.

The rat is a commonly used model for immunological investigation. Yet basic research and characterisation of leukocyte populations and sub-sets lags far behind murine research, with inconsistency on reported leukocyte markers and their overlap. These shortcomings limit the opportunity for more complex and advanced rat immunology research. In this study, we developed a robust 9-colour flow-cytometric protocol to elucidate the major blood and tissue rat leukocyte populations, and validated it in a model of LPS-induced pulmonary inflammation. Blood and tissues (lung, BALF, spleen, liver, bone marrow) from naïve Sprague-Dawley rats were collected and analysed by flow cytometry (FCM). Rats were exposed to aerosolised saline or LPS (1 mg/mL), at 3 and 24 hrs thereafter blood, lung and BALF were collected and analysed using FCM and ELISA. Neutrophils, two monocyte subsets, NK Cells, B Cells, CD4+, CD8+ T Cells and alveolar macrophages can be identified simultaneously across different tissues using a 9-colour panel. Neutrophils and monocytes can be distinguished based upon differential expression of CD43 and His48. Neutrophils and CD43Lo/His48Hi monocyte-macrophages are elevated in the lung at 3 and 24 hrs during LPS-induced pulmonary inflammation. This validated method for leukocyte enumeration will offer a platform for greater consistency in future rat immunology and inflammation research.

Low-dose TNF augments fracture healing in normal and osteoporotic bone by up-regulating the innate immune response.

The mechanism by which trauma initiates healing remains unclear. Precise understanding of these events may define interventions for accelerating healing that could be translated to the clinical arena. We previously reported that addition of low-dose recombinant human TNF (rhTNF) at the fracture site augmented fracture repair in a murine tibial fracture model. Here, we show that local rhTNF treatment is only effective when administered within 24 h of injury, when neutrophils are the major inflammatory cell infiltrate. Systemic administration of anti-TNF impaired fracture healing. Addition of rhTNF enhanced neutrophil recruitment and promoted recruitment of monocytes through CCL2 production. Conversely, depletion of neutrophils or inhibition of the chemokine receptor CCR2 resulted in significantly impaired fracture healing. Fragility, or osteoporotic, fractures represent a major medical problem as they are associated with permanent disability and premature death. Using a murine model of fragility fractures, we found that local rhTNF treatment improved fracture healing during the early phase of repair. If translated clinically, this promotion of fracture healing would reduce the morbidity and mortality associated with delayed patient mobilization.

Pericytes contribute to airway remodeling in a mouse model of chronic allergic asthma.

Myofibroblast accumulation, subepithelial fibrosis, and vascular remodeling are complicating features of chronic asthma, but the mechanisms are not clear. Platelet-derived growth factors (PDGFs) regulate the fate and function of various mesenchymal cells and have been implicated as mediators of lung fibrosis. However, it is not known whether PDGF-BB signaling via PDGFRß, which is critical for the recruitment of pericytes to blood vessels, plays a role in airway remodeling in chronic asthma. In the present study, we used a selective PDGFRß inhibitor (CP-673451) to investigate the role of PDGFRß signaling in the development of airway remodeling and lung dysfunction in an established mouse model of house dust mite-induced chronic allergic asthma. Unexpectedly, we found that pharmacological inhibition of PDGFRß signaling in the context of chronic aeroallergen exposure led to exacerbated lung dysfunction and airway smooth muscle thickening. Further studies revealed that the inflammatory response to aeroallergen challenge in mice was associated with decreased PDGF-BB expression and the loss of pericytes from the airway microvasculature. In parallel, cells positive for pericyte markers accumulated in the subepithelial region of chronically inflamed airways. This process was exacerbated in animals treated with CP-673451. The results indicate that perturbed PDGF-BB/PDGFRß signaling and pericyte accumulation in the airway wall may contribute to airway remodeling in chronic allergic asthma.

A role for Rab27 in neutrophil chemotaxis and lung recruitment.

BACKGROUND: Neutrophils are a critical part of the innate immune system. Their ability to migrate into infected or injured tissues precedes their role in microbial killing and clearance. We have previously shown that Rab27a can promote neutrophil migration by facilitating uropod release through protease secretion from primary granule exocytosis at the cell rear. Rab27b has been implicated in primary granule exocytosis but its role in neutrophil migration has not been investigated. RESULTS: Here we found Rab27b to be expressed in bone marrow derived neutrophils and Rab27b knockout (Rab27b KO) along with Rab27a/b double knockout (Rab27DKO) neutrophils exhibited impaired transwell migration in vitro in response to chemokines MIP-2 and LTB4. Interestingly, no additional defect in migration was observed in Rab27DKO neutrophils compared with Rab27b KO neutrophils. In vivo, Rab27DKO mice displayed severe impairment in neutrophil recruitment to the lungs in a MIP-2 dependent model but not in an LPS dependent model. CONCLUSIONS: These data taken together implicate Rab27b in the regulation of neutrophil chemotaxis, likely through the regulation of primary granule exocytosis.