Autophagy modulates endothelial junctions to restrain neutrophil diapedesis during inflammation

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Interleukin-6 mediates neutrophil mobilization from bone marrow in pulmonary hypertension

Myeloid cells, such as neutrophils, are produced in the bone marrow in high quantities and are important in the pathogenesis of vascular diseases such as pulmonary hypertension (PH). Although neutrophil recruitment into sites of inflammation has been well studied, the mechanisms of neutrophil egress from the bone marrow are not well understood. Using computational flow cytometry, we observed increased neutrophils in the lungs of patients and mice with PH. Moreover, we found elevated levels of IL-6 in the blood and lungs of patients and mice with PH. We observed that transgenic mice overexpressing Il-6 in the lungs displayed elevated neutrophil egress from the bone marrow and exaggerated neutrophil recruitment to the lungs, resulting in exacerbated pulmonary vascular remodeling, and dysfunctional hemodynamics. Mechanistically, we found that IL-6-induced neutrophil egress from the bone marrow was dependent on interferon regulatory factor 4 (IRF-4)-mediated CX₃CR1 expression in neutrophils. Consequently, Cx₃cr1 genetic deficiency in hematopoietic cells in Il-6-transgenic mice significantly reduced neutrophil egress from bone marrow and decreased neutrophil counts in the lungs, thus ameliorating pulmonary remodeling and hemodynamics. In summary, these findings define a novel mechanism of IL-6-induced neutrophil egress from the bone marrow and reveal a new therapeutic target to curtail neutrophil-mediated inflammation in pulmonary vascular disease.     

Autophagy mediates survival of pancreatic tumour-initiating cells in a hypoxic microenvironment

Involvement of dysregulated autophagy in cancer growth and progression has been shown in different tumour entities, including pancreatic ductal adenocarcinoma (PDA). PDA is an extremely aggressive tumour characterized by a small population of highly therapy-resistant cancer stem cells (CSCs) capable of self-renewal and migration. We examined whether autophagy might be involved in the survival of CSCs despite nutrition and oxygen deprivation typical for the hypoxic tumour microenvironment of PDA. Immunohistochemistry revealed that markers for hypoxia, CSCs and autophagy are co-expressed in patient-derived tissue of PDA. Hypoxia starvation (H/S) enhanced clonogenic survival and migration of established pancreatic cancer cells with stem-like properties (CSC(high)), while pancreatic tumour cells with fewer stem cell markers (CSC(low)) did not survive these conditions. Electron microscopy revealed more advanced autophagic vesicles in CSC(high) cells, which exhibited higher expression of autophagy-related genes under normoxic conditions and relative to CSC(low) cells, as found by RT-PCR and western blot analysis. LC3 was already fully converted to the active LC3-II form in both cell lines, as evaluated by western blot and detection of accumulated GFP-LC3 protein by fluorescence microscopy. H/S increased formation of autophagic and acid vesicles, as well as expression of autophagy-related genes, to a higher extent in CSC(high) cells. Modulation of autophagy by inhibitors and activators resensitized CSC(high) to apoptosis and diminished clonogenicity, spheroid formation, expression of CSC-related genes, migratory activity and tumourigenicity in mice. Our data suggest that enhanced autophagy levels may enable survival of CSC(high) cells under H/S. Interference with autophagy-activating or -inhibiting drugs disturbs the fine-tuned physiological balance of enhanced autophagy in CSC and switches survival signalling to suicide.     

Pathways regulating lipopolysaccharide‐induced neutrophil survival revealed by lentiviral transduction of primary human neutrophils

Human neutrophils express Toll‐like receptor 4 (TLR4) at low levels, and the role of this receptor in neutrophil responses to microbial stimuli has been questioned. Genetic manipulation of these cells to enable the study of the role of proteins such as TLR4 in their function is challenging. Here, we show that primary human neutrophils rapidly express novel proteins such as enhanced green fluorescent protein (eGFP) after transduction with lentivirus. Stimulation of transduced neutrophils with lipopolysaccharide (LPS) resulted in increased cell survival, which was inhibited when neutrophils were transduced with a lentivirus encoding a dominant negative (dn) TLR4 protein. LPS‐induced survival was also inhibited by lentiviruses encoding dnMyD88 or a truncated TRIF (Toll/interleukin‐1R homologous domain‐containing adapter protein inducing interferon‐β) molecule, whilst, in contrast, neutrophil survival was enhanced by overexpression of kinase‐mutated interleukin‐1 receptor‐associated kinase 1 (kmIRAK‐1), which activated nuclear factor (NF)‐κB. These studies provide proof of the role of TLR4 in human neutrophil biology, have begun to elucidate TLR‐dependent pathways regulating neutrophil survival, and demonstrate that neutrophils can be genetically manipulated to enhance or inhibit survival.     

Regulation of human neutrophil chemokine receptor expression and function by activation of Toll-like receptors 2 and 4

Neutrophil chemokine receptor expression can be altered by exposure to Toll-like receptor (TLR) agonists, a process that is thought to have the potential to localize neutrophils to sites of infection. In order to investigate this process in more detail, we examined the regulation of highly pure neutrophil CXCR1 and CXCR2 expression and function by selective agonists of TLR2 (Pam(3)CSK(4)) and TLR4 (lipopolysaccharide, LPS). CXCR1 and CXCR2 were down-regulated by TLR engagement. CXCR2 loss was more rapid and showed a dependence upon soluble helper molecules (LPS binding protein and CD14) that was not evident for CXCR1, suggesting differential coupling of LPS signalling to CXCR1 and CXCR2 loss. However, TLR engagement in highly pure neutrophils did not result in complete loss of chemokine receptors, and LPS-treated neutrophils remained able to mount a respiratory burst to CXCL8 and CXCL1, and were able to migrate towards CXCL8 in assays of under-agarose chemotaxis. Thus, although treatment of purified human neutrophils with TLR2 and TLR4 agonists modifies chemokine receptor expression, remaining receptors remain functionally competent.     

Tanshinone IIA ameliorates chronic arthritis in mice by modulating neutrophil activities

Rheumatoid arthritis (RA) is a chronic immune inflammatory disease mediated by the influx of immune cells into the synovial joint space. As Tanshinone IIA (TIIA) has potent anti‐oxidant and anti‐inflammatory activities, we used the adjuvant‐induced arthritis (AA) murine model of RA to investigate the impact of TIIA on RA and immune cell activation. The anti‐arthritic activity of TIIA was investigated in an adjuvant‐induced arthritis model of RA in mice. Myeloperoxidase and neutrophil elastase expression levels were assessed in ankle joints by immunohistochemistry analysis. Immune cell infiltration was evaluated in air pouch experiments. Proinflammatory cytokines expression levels were determined by quantitative real‐time polymerase chain reaction (PCR) and enzyme‐linked immunosorbent assays. Neutrophil extracellular traps (NETs) were assessed by immunostaining and confocal microscopy. Treatment with TIIA alleviated cartilage erosion and neutrophil infiltration in the ankle joints of AA mice and reduced proinflammatory cytokine expression levels in sera. TIIA suppressed interleukin‐6 and tumour necrosis factor‐α expression and release in neutrophils and promoted neutrophil apoptosis. TIIA also inhibited the NET formation of neutrophils. Our findings demonstrated that TIIA can ameliorate RA effectively by targeting neutrophils, indicating that TIIA may act as a potential therapeutic for RA.     

Decreased bacterial clearance from the lungs of mice following primary respiratory syncytial virus infection

Virus respiratory infections often precede bacterial pneumonia in healthy individuals. In order to determine the potential role of respiratory syncytial virus (RSV) in bacterial secondary infections, a mouse sequential pulmonary infection model was developed. Mice were exposed to RSV then challenged with Streptococcus pneumoniae (StPn). Exposure of BALB/c mice to 10(6)-10(7) plaque forming units (pfu) of virus of RSV significantly decreased StPn clearance 1-7 days following RSV exposure. This finding was not restricted to StPn alone: exposure to RSV followed by Staphylococcus aureus (SA) or Pseudomonas aeruginosa(PA) resulted in similar decreases in bacterial clearance. Both bronchoalveolar lavage (BAL) cell counts and pulmonary histopathology demonstrated that RSV-StPn exposed mice had increased lung cellular inflammation compared to mice receiving StPn or RSV alone. The effect of RSV infection on bacterial clearance was dependent on the mouse genetic background: C57BL/6J mice (relatively resistant to RSV infection) demonstrated a modest change in StPn clearance following RSV exposure, whereas FVBN/J mice (similar to the BALB/cJ mice in RSV susceptibility) demonstrated a similar degree of RSV-associated decrease in StPn clearance 7 days following RSV exposure. Neutrophils from the RSV-StPn sequentially exposed BALB/cJ mice were functionally altered-produced greater levels of peroxide production but less myeloperoxidase (MPO) compared to mice receiving StPn alone. These data demonstrate that RSV infection decreases bacterial clearance, potentially predisposing to secondary bacterial pneumonia despite increased lung cellular inflammation, and suggest that functional changes occur in the recruited neutrophils that may contribute to the decreased bacterial clearance.     

Aging‐related Atg5 defect impairs neutrophil extracellular traps formation

Formation of neutrophil extracellular traps (NETs) is an important function of the innate immune system against infections. It has been proven that aging dysregulates immunity and impairs neutrophil function. However, the influence of aging on the ability to produce NETs has yet to be fully addressed. In this study, we tested the hypothesis that a lower level of autophagy in neutrophils from aged mice was responsible for the decrease in NET formation. We demonstrated that a broad range of Toll‐like receptor 2 (TLR2) ligands could efficiently induce reactive oxygen species (ROS) ‐dependent NET release in young mice, but not in aged ones. We further explored that the difference between young and aged mice in TLR2 ligand‐induced NETosis is the result of an Atg5 defect and subsequent impaired autophagy. Furthermore, we found that lower autophagy capacity led to not only reduced NET formation, but also increased apoptosis. Our results suggest an important role of Atg5 and autophagy in maintaining the function of NETs formation in response to infection and in regulating neutrophil death. Targeting autophagy‐promoted NETs may present a therapeutic strategy to improve infection defence in an aged population.     

Nitric oxide mediates the inhibition of neutrophil migration induced by systemic administration of LPS

To investigate the role of NO in the inhibition of neutrophil migration by circulating endotoxin, mice were pretreated with NO synthase inhibitors or with a free radical scavenger (D-penicillamine), before intravenous LPS injection. LPS dose-dependently inhibited the thioglycollate-induced neutrophil migration into the peritoneal cavities. Aminoguanidine, a selective inducible NO synthase inhibitor, abolished the inhibition of neutrophil migration and the increase in serum nitrate levels induced by a nonlethal dose of LPS. During lethal endotoxemia aminoguanidine partially abolished the neutrophil migration inhibition. Additionally, D-penicillamine prevented the inhibition of neutrophil migration caused by LPS. However, Nitro-L-Arginine, a selective constitutive NO synthase inhibitor, did not prevent neutrophil migration inhibition. Aminoguanidine treatment did not affect the systemic increased levels of TNF-, IL-1, and IL-10, suggesting that NO is the final mediator involved in the inhibition of neutrophil migration. Our results suggest that NO released by the inducible NO synthase mediates the inhibition of neutrophil migration mediated by circulating LPS.     

Underlying chronic inflammation alters the profile and mechanisms of acute neutrophil recruitment

Chronically inflamed tissues show altered characteristics that include persistent populations of inflammatory leukocytes and remodelling of the vascular network. As the majority of studies on leukocyte recruitment have been carried out in normal healthy tissues, the impact of underlying chronic inflammation on ongoing leukocyte recruitment is largely unknown. Here, we investigate the profile and mechanisms of acute inflammatory responses in chronically inflamed and angiogenic tissues, and consider the implications for chronic inflammatory disorders. We have developed a novel model of chronic ischaemia of the mouse cremaster muscle that is characterized by a persistent population of monocyte‐derived cells (MDCs), and capillary angiogenesis. These tissues also show elevated acute neutrophil recruitment in response to locally administered inflammatory stimuli. We determined that Gr1^low MDCs, which are widely considered to have anti‐inflammatory and reparative functions, amplified acute inflammatory reactions via the generation of additional proinflammatory signals, changing both the profile and magnitude of the tissue response. Similar vascular and inflammatory responses, including activation of MDCs by transient ischaemia–reperfusion, were observed in mouse hindlimbs subjected to chronic ischaemia. This response demonstrates the relevance of the findings to peripheral arterial disease, in which patients experience transient exercise‐induced ischaemia known as claudication.These findings demonstrate that chronically inflamed tissues show an altered profile and altered mechanisms of acute inflammatory responses, and identify tissue‐resident MDCs as potential therapeutic targets. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

T cell responses to bacterial infection.

Many exciting advances in our understanding of T cell mediated immunity to bacterial infection have occurred in the past several years. T cell responses have been more fully characterized, due in part to the development of MHC class I tetramers. The importance of cytokines and various effector molecules in defense against infection has come to light. Finally, intracellular bacteria are being exploited to deliver antigens and DNA in an effort to induce immunity to pathogens.     

Delayed inflammatory responses to endotoxin in fibrinogen-deficient mice

Severe inflammation leads to haemostatic abnormalities, such as the development of microvascular thrombi. As a result, ischaemia-related downstream organ damage can occur. The present study demonstrates that mice with a total deficiency of fibrinogen (Fg(-/-)) present with altered responses to challenge with Gram-negative lipopolysaccharide (LPS). Early survival in response to continuous LPS challenge was increased in Fg(-/-) mice and histological findings indicated that this improvement correlated with a lack of fibrin deposition in organs. Neutrophils appeared early in the lungs of challenged wild-type (WT) mice, but occurred in Fg(-/-) mice at later times. This delayed response in Fg(-/-) mice was confirmed by studies that showed a strong dependence on Fg of binding of neutrophils to endothelial cells in the presence of LPS. While cytokines were also elevated in both WT and Fg(-/-) mice, their levels were generally lower at early times in this latter group. The time course of MIP-2 expression correlated with the occurrence of pulmonary leakage after LPS challenge, which was delayed in Fg(-/-) mice. These results suggest that fibrin(ogen) plays a role as an early mediator in the cross-talk between coagulation and inflammation.     

Tolerance to lipopolysaccharide promotes an enhanced neutrophil extracellular traps formation leading to a more efficient bacterial clearance in mice

Tolerance to lipopolysaccharide (LPS) constitutes a stress adaptation, in which a primary contact with LPS results in a minimal response when a second exposure with the same stimulus occurs. However, active important defence mechanisms are mounted during the tolerant state. Our aim was to assess the contribution of polymorphonuclear neutrophils (PMN) in the clearance of bacterial infection in a mouse model of tolerance to LPS. After tolerance was developed, we investigated in vivo different mechanisms of bacterial clearance. The elimination of a locally induced polymicrobial challenge was more efficient in tolerant mice both in the presence or absence of local macrophages. This was related to a higher number of PMN migrating to the infectious site as a result of an increased number of PMN from the marginal pool with higher chemotactic capacity, not because of differences in their phagocytic activity or reactive species production. In vivo, neutrophils extracellular trap (NET) destruction by nuclease treatment abolished the observed increased clearance in tolerant but not in control mice. In line with this finding, in vitro NETs formation was higher in PMN from tolerant animals. These results indicate that the higher chemotactic response from an increased PMN marginal pool and the NETs enhanced forming capacity are the main mechanisms mediating bacterial clearance in tolerant mice. To sum up, far from being a lack of response, tolerance to LPS causes PMN priming effects which favour distant and local anti-infectious responses.     

ANCA-associated vasculitis: is there a role for neutrophil apoptosis in autoimmunity?

The primary small vessel systemic vasculitides are disorders that target small blood vessels, inducing vessel wall inflammation, and are associated with the development of antineutrophil cytoplasmic antibodies. Multiple organs are attacked including the lungs and kidneys. Increasing knowledge of pathogenesis suggests that the antibodies activate neutrophils inappropriately, leading to endothelial and vascular damage. Cytokines, such as tumor necrosis factor, can facilitate damage by priming neutrophils and activating endothelial cells. Apoptosis of infiltrating neutrophils is also disrupted by antineutrophil cytoplasmic antibody activation. Removal of these effete cells occurs in a proinflammatory manner, promoting persistent inflammation. The autoimmune response may be promoted by aberrant phagocytosis of apoptotic neutrophils by dendritic cells. Understanding pathogenesis can help to rationalize existing therapies and indicate new approaches to therapy.     

Autophagy is involved in regulating the immune response of dendritic cells to influenza A (H1N1) pdm09 infection

Autophagy can mediate antiviral immunity. However, it remains unknown whether autophagy regulates the immune response of dendritic cells (DCs) to influenza A (H1N1) pdm09 infection. In this study, we found that infection with the H1N1 virus induced DC autophagy in an endocytosis‐dependent manner. Compared with autophagy‐deficient Beclin‐1^+/? mice, we found that bone‐marrow‐derived DCs from wild‐type mice (WT BMDCs) presented a more mature phenotype on H1N1 infection. Wild‐type BMDCs secreted higher levels of interleukin‐6 (IL‐6), tumour necrosis factor‐ α (TNF‐α), interferon‐β (IFN‐β), IL‐12p70 and IFN‐γ than did Beclin‐1^+/? BMDCs. In contrast to Beclin‐1^+/? BMDCs, H1N1‐infected WT BMDCs exhibited increased activation of extracellular signal‐regulated kinase, Jun N‐terminal kinase, p38, and nuclear factor‐κB as well as IFN regulatory factor 7 nuclear translocation. Blockade of autophagosomal and lysosomal fusion by bafilomycin A1 decreased the co‐localization of H1N1 viruses, autophagosomes and lysosomes as well as the secretion of IL‐6, TNF‐α and IFN‐β in H1N1‐infected BMDCs. In contrast to Beclin‐1^+/? BMDCs, H1N1‐infected WT BMDCs were more efficient in inducing allogeneic CD4⁺ T‐cell proliferation and driving T helper type 1, 2 and 17 cell differentiation while inhibiting CD4⁺ Foxp3⁺ regulatory T‐cell differentiation. Moreover, WT BMDCs were more efficient at cross‐presenting the ovalbumin antigen to CD8⁺ T cells. We consistently found that Beclin‐1^+/? BMDCs were inferior in their inhibition of H1N1 virus replication and their induction of H1N1‐specific CD4⁺ and CD8⁺ T‐cell responses, which produced lower levels of IL‐6, TNF‐α and IFN‐β in vivo. Our data indicate that autophagy is important in the regulation of the DC immune response to H1N1 infection, thereby extending our understanding of host immune responses to the virus.     

Kinetics of pulmonary neutrophil recruitment and clearance in a natural and spontaneously resolving model of airway inflammation

BACKGROUND: Neutrophil apoptosis and phagocytic clearance have been proposed as key determinants affecting the resolution of airway inflammation. Objective To determine the kinetics of neutrophil priming, recruitment, activation and subsequent clearance in a naturally occurring equine disease model of neutrophilic pulmonary inflammation. METHODS AND RESULTS: A 5 h mouldy hay/straw challenge in hypersensitive horses induced transient pulmonary dysfunction lasting 4 days. At 24 h circulating neutrophils were primed and displayed delayed rates of spontaneous apoptosis in vitro. Neutrophil numbers in the airspaces peaked at 5 h and then fell abruptly, returning to pre-challenge levels by 4 days. Airspace neutrophils demonstrated increased respiratory burst activity compared with circulating cells and equine neutrophil elastase 2A concentrations increased in parallel with neutrophil numbers indicating in vivo priming and degranulation. The number of apoptotic neutrophils and proportion of alveolar macrophages containing phagocytosed apoptotic neutrophils increased significantly at 24 h and 4 days post-challenge corresponding to the period of most rapid neutrophil clearance. CONCLUSION: This is the first demonstration of spontaneous neutrophil apoptosis and phagocytic removal in a natural disease model of airway inflammation and provides critical kinetic data to support the hypothesis that this clearance pathway plays a central role in the resolution of neutrophilic inflammation.