Model Of Pulmonary Inflammation Research Articles

Lung Volumes in a Mouse Model of Pulmonary Allergic Inflammation.

Air trapping, often attested in humans by elevated residual volume (RV) and ratio of RV on total lung capacity (RV/TLC), is frequently observed in asthma. Confirming these alterations in experimental asthma would be important for translational purposes. Herein, lung volumes were investigated in a mouse model of pulmonary allergic inflammation. Eight- to 10-week-old male C57BL/6 and BALB/c mice were exposed once daily to intranasal house dust mite (HDM) for 10 consecutive days. All readouts were measured 24h after the last exposure. Lung volumes were assessed with the flexiVent using a new automated method consisting of degassing the lungs followed by a full-range pressure-volume maneuver. The weight and the volume of the lungs were also measured ex vivo and a lobe was further processed for histological analyses. HDM exposure led to tissue infiltration with inflammatory cells, goblet cell hyperplasia, thickening of the airway epithelium, and elevated ex vivo lung weight and volume. It also decreased TLC and vital capacity but without affecting RV and RV/TLC. These observations were similar between the two mouse strains. Alterations of lung volumes in a murine model of pulmonary allergic inflammation are inconsistent with observations made in human asthma. These discrepancies reflect the different means whereby lung volumes are measured between species. The invasive method used herein enables RV to be measured more precisely and without the confounding effect of air trapping, suggesting that changes in RV and RV/TLC using this method in mice should be interpreted differently than in humans.

Helminth infection induces a distinct subset of CD101hi lung tissue-infiltrating eosinophils that are differentially regulated by type 2 cytokines.

Eosinophils play divergent roles in health and disease, contributing to both immunoregulatory and proinflammatory responses. Helminth infection is strongly associated with eosinophilia and the induction of the type 2 cytokines interleukin (IL)-5, IL-4 and IL-13. This study aimed to elucidate the heterogeneity of pulmonary eosinophils in response to helminth infection and the roles of IL-5, IL-4 and IL-13 in driving pulmonary eosinophil responses. Using the murine helminth model Nippostrongylus brasiliensis (Nb), we characterize a subtype of eosinophils, defined by high expression of CD101, that is induced in the lungs of Nb-infected mice and are phenotypically distinct from lung eosinophils that express low levels of CD101. Strikingly, we show that the two eosinophil subtypes have distinct anatomical localization within the lung: CD101low eosinophils are predominantly localized in the lung vasculature, whereas Nb-induced CD101hi eosinophils are predominantly localized in the extravascular lung niche. We show that CD101hi eosinophils are also induced across other models of pulmonary infection and inflammation, including a nonlung-migrating helminth infection, house dust mite-induced allergic inflammation and influenza infection. Furthermore, we demonstrate that the induction of CD101hi tissue eosinophils is independent of IL-5 and IL-4 signaling, but is dependent on intact IL-13 signaling. These results suggest that IL-13 produced during helminth infection and other disease states promotes a pulmonary tissue-infiltrating program in eosinophils defined by high expression of CD101.

Absence of sympathetic innervation hampers the generation of tertiary lymphoid structures upon acute lung inflammation

Tertiary lymphoid structures (TLS) are lymphoid organs present in inflammatory non-lymphoid tissues. Studies have linked TLS to favorable outcomes for patients with cancers or infectious diseases, but the mechanisms underlying their formation are not fully understood. In particular, secondary lymphoid organs innervation raises the question of sympathetic nerve fibers involvement in TLS organogenesis. We established a model of pulmonary inflammation based on 5 daily intranasal instillations of lipopolysaccharide (LPS) in immunocompetent mice. In this setting, lung lymphoid aggregates formed transiently, evolving toward mature TLS and disappearing when inflammation resolved. Sympathetic nerve fibers were then depleted using 6-hydroxydopamine. TLS quantification by immunohistochemistry showed a decrease in LPS-induced TLS number and surface in denervated mouse lungs. Although a reduction in alveolar space was observed, it did not impair overall pulmonary content of transcripts encoding TNF-α, IL-1β and IFN-γ inflammation molecules whose expression was induced by LPS instillations. Immunofluorescence analysis of immune infiltrates in lungs of LPS-treated mice showed a drop in the proportion of CD23+ naive cells among CD19+ B220+ B cells in denervated mice whereas the proportion of other cell subsets remained unchanged. These data support the existence of neuroimmune crosstalk impacting lung TLS neogenesis and local naive B cell pool.

4-hydroxysesamin protects rat with right ventricular failure due to pulmonary hypertension by inhibiting JNK/p38 MAPK signaling.

The specific mechanism of 4-hydroxysesamin (4-HS), a modification of Sesamin, on right ventricular failure due to pulmonary hypertension (PH) is ominous. By creating a rat model of PH in vivo and a model of pulmonary artery smooth muscle cell (PASMC) hypoxia and inflammation in vitro, the current work aimed to investigate in depth the molecular mechanism of the protective effect of 4-HS. In an in vitro model of hypoxia PASMC, changes in cell proliferation and inflammatory factors were detected after treatment with 4-HS, followed by changes in the JNK/p38 MAPK signaling pathway as detected by Western blot signaling pathway. The findings demonstrated that 4-HS was able to minimize PASMC cell death, block the JNK/p38 MAPK signaling pathway, and resist the promoting effect of hypoxia on PASMC cell proliferation. Following that, we found that 4-HS could both mitigate the right ventricular damage brought on by MCT and had a protective impact on rats Monocrotaline (MCT)-induced PH in in vivo investigations. The key finding of this study is that 4-HS may protect against PH by inhibiting the JNK/p38 MAPK signaling pathway.

MiR-616-3p alleviates inflammatory response by targeting C-X-C motif chemokine ligand 5

C/EBP homologous protein (CHOP) is a key regulator in ER stress-mediated signaling pathway via PERK-dependent unfolded protein response. It has been known that microRNA-616 (miR-616) is produced from the intron of the human DDIT3 gene encoding CHOP and increased by ER stress. However, the role of miR-616 and its targets are not fully addressed yet. Here we try to identify a novel target of miR-616 in human lung epithelial cells. Microarray analysis showed that CXCL5 is the most downregulated gene by miR-616 overexpression in A549 cells. We also found that CXCL5 mRNA and protein levels were significantly reduced by miR-616 mimic in the presence or absence of TNFα, while anti-miR-616 enhanced CXCL5 expression. In addition, miR-616-3p targeting sequence in 3′UTR of CXCL5 was confirmed by luciferase reporter assay suggesting that miR-616-3p directly binds to 3′UTR of CXCL5 and inhibits CXCL5 expression. Finally, we confirmed that conditioned medium from A549 cells treated with TNFα or Streptococcus pneumoniae lysates increased intra-alveolar neutrophil infiltration in a mouse model of pulmonary inflammation, while this induction was significantly reduced in a conditioned medium from cells transfected with miR-616-3p. These results suggest that miR-616-3p can alleviate CXCL5-induced pulmonary inflammatory response via targeting 3′UTR of CXCL5 gene.

Increased m6A-RNA methylation and demethylase FTO suppression is associated with silica-induced pulmonary inflammation and fibrosis

Silicosis is a severe worldwide occupational hazard, characterized with lung tissue inflammation and irreversible fibrosis caused by crystalline silicon dioxide. As the most common and abundant internal modification of messenger RNAs or noncoding RNAs, N6-methyladenosine (m6A) methylation is dysregulated in the chromic period of silicosis. However, whether m6A modification is involved in the early phase of silica-induced pulmonary inflammation and fibrosis and its specific effector cells remains unknown. In this study, we established a pulmonary inflammation and fibrosis mouse model by silica particles on day 7 and day 28. Then, we examined the global m6A modification level by m6A dot blot and m6A RNA methylation quantification kits. The key m6A regulatory factors were analyzed by RTqPCR, Western blot, and immunohistochemistry (IHC) in normal and silicosis mice. The results showed that the global m6A modification level was upregulated in silicosis lung tissues with the demethylase FTO suppression after silica exposure for 7 days and 28 days. METTL3, METTL14, ALKBH5, and other m6A readers had no obvious differences between the control and silicosis groups. Then, single-cell sequencing analysis revealed that thirteen kinds of cells were recognized in silicosis lung tissues, and the mRNA expression of FTO was downregulated in epithelial cells, endothelial cells, fibroblasts, and monocytes. These results were further confirmed in mouse lung epithelial cells (MLE-12) exposed to silica and in the peripheral blood mononuclear cells of silicosis patients. In conclusion, the high level of global m6A modification in the early stage of silicosis is induced by the downregulation of the demethylase FTO, which may provide a novel target for the diagnosis and treatment of silicosis.

In Vivo Efficacy and Toxicity of an Antimicrobial Peptide in a Model of Endotoxin-Induced Pulmonary Inflammation.

SET-M33 is a synthetic peptide that is being developed as a new antibiotic against major Gram-negative bacteria. Here we report two in vivo studies to assess the toxicity and efficacy of the peptide in a murine model of pulmonary inflammation. First, we present the toxicity study in which SET-M33 was administered to CD-1 mice by snout inhalation exposure for 1 h/day for 7 days at doses of 5 and 20 mg/kg/day. The results showed adverse clinical signs and effects on body weight at the higher dose, as well as some treatment-related histopathology findings (lungs and bronchi, nose/turbinates, larynx and tracheal bifurcation). On this basis, the no observable adverse effect level (NOAEL) was considered to be 5 mg/kg/day. We then report an efficacy study of the peptide in an endotoxin (LPS)-induced pulmonary inflammation model. Intratracheal administration of SET-M33 at 0.5, 2 and 5 mg/kg significantly inhibited BAL neutrophil cell counts after an LPS challenge. A significant reduction in pro-inflammatory cytokines, KC, MIP-1α, IP-10, MCP-1 and TNF-α was also recorded after SET-M33 administration.

C5aR1 activation in mice controls inflammatory eosinophil recruitment and functions in allergic asthma.

Pulmonary eosinophils comprise at least two distinct populations of resident eosinophils (rEOS) and inflammatory eosinophils (iEOS), the latter recruited in response to pulmonary inflammation. Here, we determined the impact of complement activation on rEOS and iEOS trafficking and function in two models of pulmonary inflammation. BALB/c wild-type and C5ar1-/- mice were exposed to different allergens or IL-33. Eosinophil populations in the airways, lung, or mediastinal lymph nodes (mLN) were characterized by FACS or immunohistochemistry. rEOS and iEOS functions were determined in vivo and in vitro. HDM and IL-33 exposure induced a strong accumulation of iEOS but not rEOS in the airways, lungs, and mLNs. rEOS and iEOS expressed C3/C5 and C5aR1, which were significantly higher in iEOS. Initial pulmonary trafficking of iEOS was markedly reduced in C5ar1-/- mice and associated with less IL-5 production from ILC2 cells. Functionally, adoptively transferred pulmonary iEOS from WT but not from C5ar1-/- mice-induced airway hyperresponsiveness (AHR), which was associated with significantly reduced C5ar1-/- iEOS degranulation. Pulmonary iEOS but not rEOS were frequently associated with T cells in lung tissue. After HDM or IL-33 exposure, iEOS but not rEOS were found in mLNs, which were significantly reduced in C5ar1-/- mice. C5ar1-/- iEOS expressed less costimulatory molecules, associated with a decreased potency to drive antigen-specific T cell proliferation and differentiation into memory T cells. We uncovered novel roles for C5aR1 in iEOS trafficking and activation, which affects key aspects of allergic inflammation such as AHR, ILC2, and T cell activation.

Uticaj mikrobiote creva na imunske reakcije relevantne za patologiju pluća

Bacterial microbiota of the gastrointestinal tract is known to prevent the invasion of pathogenic microorganisms and regulate intestinal permeability, digestion, metabolism, and immune response. It affects function, homeostasis, and disease outcomes in the gastrointestinal tract and extra-intestinal sites such as the lungs. This review summarizes the currently available knowledge regarding the gut-lung axis. The association of bacterial composition and/or dysbiosis in the gut with asthma, chronic obstructive lung disease, cystic fibrosis, recurrent respiratory tract infections, and lung cancer in humans is highlighted, as well as data obtained from animal models of pulmonary inflammation, which indicated that modulation of immune system activity lies at the base of this interaction. Additionally, the potential use of prebiotics, probiotics, and postbiotics in the treatment of lung inflammation is presented.

Role of S100A8/A9 in Platelet–Neutrophil Complex Formation during Acute Inflammation

Acute respiratory distress syndrome (ARDS) due to pulmonary infections is associated with high morbidity and mortality. Upon inflammation, the alarmin S100A8/A9 is released and stimulates neutrophil recruitment mainly via binding to Toll-like receptor 4 (TLR4). TLR4 is also expressed on platelets, which modulate the immune response through direct interaction with leukocytes. In a murine model of Klebsiella pneumoniae-induced pulmonary inflammation, global S100A9 deficiency resulted in diminished neutrophil recruitment into the lung alveoli and neutrophil accumulation in the intravascular space, indicating an impaired neutrophil migration. A lack of TLR4 on platelets resulted in reduced neutrophil counts in the whole lung, emphasising the impact of TLR4-mediated platelet activity on neutrophil behaviour. Flow cytometry-based analysis indicated elevated numbers of platelet-neutrophil complexes in the blood of S100A9-/- mice. Intravital microscopy of the murine cremaster muscle confirmed these findings and further indicated a significant increase in neutrophil-platelet complex formation in S100A9-/- mice, which was reversed by administration of the S100A8/A9 tetramer. An in vitro bilayer assay simulated the murine alveolar capillary barrier during inflammation and validated significant differences in transmigration behaviour between wild-type and S100A9-/- neutrophils. This study demonstrates the role of S100A8/A9 during platelet-neutrophil interactions and neutrophil recruitment during pulmonary inflammation.

Cellular stress modulates severity of the inflammatory response in lungs via cell surface BiP.

Inflammation is a central pathogenic feature of the acute respiratory distress syndrome (ARDS) in COVID-19. Previous pathologies such as diabetes, autoimmune or cardiovascular diseases become risk factors for the severe hyperinflammatory syndrome. A common feature among these risk factors is the subclinical presence of cellular stress, a finding that has gained attention after the discovery that BiP (GRP78), a master regulator of stress, participates in the SARS-CoV-2 recognition. Here, we show that BiP serum levels are higher in COVID-19 patients who present certain risk factors. Moreover, early during the infection, BiP levels predict severe pneumonia, supporting the use of BiP as a prognosis biomarker. Using a mouse model of pulmonary inflammation, we observed increased levels of cell surface BiP (cs-BiP) in leukocytes during inflammation. This corresponds with a higher number of neutrophiles, which show naturally high levels of cs-BiP, whereas alveolar macrophages show a higher than usual exposure of BiP in their cell surface. The modulation of cellular stress with the use of a clinically approved drug, 4-PBA, resulted in the amelioration of the lung hyperinflammatory response, supporting the anti-stress therapy as a valid therapeutic strategy for patients developing ARDS. Finally, we identified stress-modulated proteins that shed light into the mechanism underlying the cellular stress-inflammation network in lungs.

Integrating network pharmacology and experimental verification to decipher the immunomodulatory effect of Bu-Zhong-Yi-Qi-Tang against poly (I:C)-induced pulmonary inflammation.

Pulmonary inflammation caused by respiratory tract viral infections is usually associated with acute exacerbation of respiratory diseases, such as asthma and chronic obstructive pulmonary disease (COPD). Therefore, maintaining the pulmonary immune homeostasis is particular important for prevention of the acute exacerbation. Bu-Zhong-Yi-Qi-Tang (BZYQT), a traditional Chinese medicine formula, has been broadly used to improve respiratory and gastrointestinal disorders in China for over 700years. Previously, we have found the regulatory activity of BZYQT on the lower respiratory immune system, while its potential effects during pulmonary inflammation remain unknown. Thus, the current study focused on deciphering its immunomodulatory effect and potential mechanism against pulmonary inflammation by using a viral RNA analogue, poly (I:C), induced murine pulmonary inflammation model and BEAS-2B cell model coupled with network pharmacology. Inflammatory cells in the bronchoalveolar lavage fluid were counted through microscope examination according to the cell's morphology and staining characteristics; protein and gene levels of inflammatory mediators were determined with Elisa and quantitative PCR, respectively; network pharmacology was conducted based on 46 BZYQT-related potential bioactive components, pulmonary inflammation and immune-related targets. Our results indicated that the recruitment of neutrophils and the expression of Adgre1 (encoding the F4/80, which is a macrophage marker) in the lung induced by poly (I:C) were significantly reduced after BZYQT treatment, and these effects were further demonstrated to be related to the interference of leukocyte transendothelial migration from the decreased levels of CXCL10, IL-6, TNF-α, CXCL2, ICAM-1, VCAM-1, and E/P-selectins. Furthermore, BZYQT inhibited the CXCL10, TNF-α, and IFN-β expression of poly (I:C)-challenged BEAS-2B cells in a dose-dependent manner. Through integrating results from network pharmacology, experiments, and the published literature, isoliquiritigenin, Z-ligustilide, atractylenolide I, atractylenolide III, formononetin, ferulic acid, hesperidin, and cimigenoside were presumed as the bioactive components of BZYQT against pulmonary inflammation. Overall, our findings demonstrated that BZYQT possesses a pronounced immunomodulatory effect on poly (I:C)-induced pulmonary inflammation, which provides a pharmacological basis for BZYQT in the treatment of respiratory disorders.

Effects of Purinergic Receptor Deletion or Pharmacologic Modulation on Pulmonary Inflammation in Mice.

COVID-19 disease is associated with progressive accumulation of SARS-CoV-2-specific mRNA, which is recognized by innate immune receptors, such as TLR3. This in turn leads to dysregulated production of multiple cytokines, including IL-6, IFN-γ, CXCL1, and TNF-α. Excessive production of these cytokines leads to acute lung injury (ALI), which consequently compromises alveolar exchange of O2 and CO2. It is therefore of considerable interest to develop novel therapies that reduce pulmonary inflammation and stem production of pro-inflammatory cytokines, potentially for COVID-19 patients that are at high risk of developing severe disease. Purinergic signaling has a central role in fine-tuning the innate immune system, with P2 (nucleotide) receptor antagonists and adenosine receptor agonists having anti-inflammatory effects. Accordingly, we focused here on the potential role of purinergic receptors in driving neutrophilic inflammation and cytokine production in a mouse model of pulmonary inflammation. To mimic the effects of SARS-CoV-2-specific RNA accumulation in mice, we administered progressively increasing daily doses of a viral mimetic, polyinosinic:polycytidylic acid [poly(I:C)] into the airways of mice over the course of 1 week. Some mice also received increasing daily doses of ovalbumin to mimic virus-encoded protein accumulation. Animals receiving both poly(I:C) and ovalbumin displayed particularly high cytokine levels and neutrophilia, suggestive of both innate and antigen-specific, adaptive immune responses. The extent of these responses was diminished by genetic deletion (P2Y14R, P2X7R) or pharmacologic modulation (P2Y14R antagonists, A3AR agonists) of purinergic receptors. These results suggest that pharmacologic modulation of select purinergic receptors might be therapeutically useful in treating COVID-19 and other pulmonary infections.

Cigarette smoke extract combined with LPS reduces ABCA3 expression in chronic pulmonary inflammation may be related to PPARγ/ P38 MAPK signaling pathway.

ABCA3 (ATP-binding cassette class A3) is a transmembrane transporter that plays a positive role in chronic pulmonary inflammation by regulating lipid metabolism. However, it is not completely clear whether ABCA3 and its signaling factors are involved in chronic pulmonary inflammation induced by the combination of CSE (cigarette smoke extract) and LPS (lipopolysaccharide). In this study, we used the method of combining CSE and LPS which was widely used to study lung inflammation-related diseases and has been proven effective in our group's studies to create in vivo and in vitro pulmonary inflammation models. The result showed that, after CSE in combination with LPS treatment, ABCA3 expression was downregulated in rat lung in vivo and in a human alveolar cell line in vitro. ABCA3 expression was upregulated, and related inflammatory factors were downregulated in the state of overexpression of PPARγ or inhibition of the p38 MAPK pathway, while PPARγ deletion or MAPK14 overexpression showed the opposite results. The level of PPARγ remained unchanged, and the expression of ABCA3 was upregulated in the state of the p38 MAPK pathway was inhibited under overexpression of PPARγ. These results indicate that CSE combined with LPS can result in downregulation of ABCA3 under conditions of inflammation, and that the p38 MAPK signaling pathway mediated by PPARγ can regulate the expression changes of ABCA3, thus providing new targets for treating chronic pulmonary inflammation.

Circulating Myeloid Cell-derived Extracellular Vesicles as Mediators of Indirect Acute Lung Injury.

Blood-borne myeloid cells, neutrophils and monocytes, play a central role in the development of indirect acute lung injury (ALI) during sepsis and noninfectious systemic inflammatory response syndrome. By contrast, the contribution of circulating myeloid cell-derived extracellular vesicles (EVs) to ALI is unknown, despite acute increases in their numbers during sepsis and systemic inflammatory response syndrome. Here, we investigated the direct role of circulating myeloid-EVs in ALI using a mouse isolated perfused lung system and a human cell coculture model of pulmonary vascular inflammation consisting of lung microvascular endothelial cells and peripheral blood mononuclear cells. Total and immunoaffinity-isolated myeloid (CD11b+) and platelet (CD41+) EVs were prepared from the plasma of intravenous LPS-injected endotoxemic donor mice and transferred directly into recipient lungs. Two-hour perfusion of lungs with unfractionated EVs from a single donor induced pulmonary edema formation and increased perfusate concentrations of RAGE (receptor for advanced glycation end products), consistent with lung injury. These responses were abolished in the lungs of monocyte-depleted mice. The isolated myeloid- but not platelet-EVs produced a similar injury response and the acute intravascular release of proinflammatory cytokines and endothelial injury markers. In the invitro human coculture model, human myeloid- (CD11b+) but not platelet- (CD61+) EVs isolated from LPS-stimulated whole blood induced acute proinflammatory cytokine production and endothelial activation. These findings implicate circulating myeloid-EVs as acute mediators of pulmonary vascular inflammation and edema, suggesting an alternative therapeutic target for attenuation of indirect ALI.

Neutrophil‐Derived Microvesicles Enhance Pulmonary Vascular Inflammation via a Toll‐like Receptor 4 Signaling‐Dependent Mechanism

BackgroundCirculating neutrophil‐derived microvesicles (NMVs) are markedly elevated during sepsis and therefore could have a role in the development of indirect acute lung injury (ALI). We recently found that NMV‐enriched CD11b+ MVs, immunoaffinity isolated from lipopolysaccharide (LPS)‐stimulated healthy volunteer blood, have potent pro‐inflammatory activity in a human peripheral blood mononuclear cell (PBMC) and lung microvascular endothelial cell (HLMEC) coculture model of pulmonary vascular inflammation (1). By contrast, immunoaffinity isolated platelet‐MVs (CD61+) produced negligible responses in these assays, suggesting specificity of the NMV‐enriched CD11b+ MV, activity. Here, we investigated the signaling mechanisms responsible for NMV‐mediated activation of monocytes and HLMECs in this model.MethodsHeparinized blood from healthy donors was treated with LPS (100 ng/ml, 3 h) and NMVs (CD66b+) were isolated by positive immunoaffinity selection. NMVs were then incubated in PBMC‐HLMEC cocultures for 4 h. NMV‐induced responses were determined by flow cytometric quantification of cell surface activation markers (ICAM‐1 for HLMECs, ICAM‐1 and tissue factor for monocytes) and pro‐inflammatory cytokine release by ELISA (TNFα, IL‐8, IL‐6, MCP‐1).ResultsNMV‐induced HLMEC activation required the presence of monocytes and was completely attenuated by anti‐TNFα neutralizing antibody (ICAM‐1: isotype control 302±103 vs anti‐TNFα 66±29; p<0.01). Pharmacological inhibition of mitogen‐activated protein kinases p38, MEK1‐MEK2 and phosphoinositide‐3 kinase ablated monocyte and HLMEC activation. Monocyte activation was prevented in the presence of the Toll‐like receptor 4 (TLR4) inhibitor TAK‐242 (e.g., TNFα release: vehicle 1108±257 vs TAK‐242 16±12; p<0.001) and partially inhibited by an anti‐TLR4 neutralizing antibody. However, the LPS inhibitor polymyxin‐B had no effect on these responses, indicating that LPS contamination was unlikely. Furthermore, NMV activity was found to be sensitive to proteinase K and heat‐treatment (55 °C, 15 min) (e.g., TNFα release: untreated 474±267 vs proteinase K 12±15; p<0.0001 vs heat‐treatment 200±84; p<0.01).ConclusionsWe have demonstrated that NMVs produced within LPS‐stimulated blood are potent mediators of lung microvascular endothelial cells via monocyte‐derived TNFα signaling. NMV‐induced monocyte activation involves TLR4 signaling and is protease sensitive, suggesting that MVs serve as vehicles of damage‐associated molecular pattern (DAMP) activity, mediated by surface proteins. Together these findings support a major role for NMVs as novel long‐range mediators for systemic propagation of inflammation, contributing to the pathogenesis of sepsis‐induced indirect acute lung injury.(1) Tsiridou et al. European Respiratory Journal. 2020;56:4468

Stimulation of Neutrophils in Whole Blood Enhances the Pro‐inflammatory Activity of Neutrophil‐Derived Microvesicles

BackgroundAcute increases in circulating levels of neutrophil‐derived microvesicles (N‐MVs) during sepsis combined with evidence of enhanced uptake of MVs within the pulmonary microvasculature during endotoxemia in mice, suggests a role for N‐MVs in development of indirect acute lung injury (ALI). However, evidence for the pro‐inflammatory activity of N‐MVs is unclear, with various in vitro studies demonstrating anti‐inflammatory effects. Most in vitro investigations of N‐MV activity involve stimulation of purified neutrophils with single agonists such as formyl‐methionyl‐leucyl‐phenylalanine (fMLP). This reductionist approach excludes the complex in vivo interplay between different stimuli and other vascular cells contributing to neutrophil responses during sepsis. Here we investigated the proinflammatory activities of N‐MVs under more clinically relevant in vitro conditions, treating healthy volunteer whole blood with lipopolysaccharide (LPS) as a sepsis‐relevant inflammatory stimulus and fMLP as an acute N‐MV inducing stimulus. The activities of N‐MVs derived from whole blood as well as isolated neutrophils were evaluated in a monocyte‐neutrophil‐endothelial cell ‘tri‐culture’ model of pulmonary vascular inflammation.MethodsN‐MVs were generated from whole blood or neutrophils isolated by two‐step density gradient centrifugation, by stimulation with fMLP (1 µM, 30 min) alone or combined with LPS pre‐treatment (100 ng/ml, 1 h). N‐MVs were isolated from blood by anti‐CD66b immunomagnetic‐bead selection. For the tri‐culture assay, isolated neutrophils and immunomagnetic‐bead sorted monocytes were cultured with human lung microvascular endothelial cells. N‐MVs were incubated in the tri‐cultures for 3 h, and MV‐induced responses were determined by ELISA and flow cytometric quantification of cell activation markers on monocytes and neutrophils.ResultsN‐MVs derived from LPS‐fMLP‐stimulated (but not fMLP‐stimulated) whole blood induced significant responses in the tri‐culture assay, i.e. substantive tumour necrosis factor (TNF) release (375.2±251.4 vs untreated 2.2±1.4 pg/ml, p<0.01), increased tissue factor expression on monocytes (MFI: 231.1±90.0 vs untreated 9.2±2.5, p<0.001), and increased expression of CD45 (MFI: 478.3±131.2 vs untreated 281.4±37.5, p<0.05), CD11b (MFI: 208.0±33.0 vs untreated 112.7±20.1, p<0.05) and CD63 (MFI: 77.3±36.5 vs untreated 16.23±5.3, p<0.05) on neutrophils. In contrast, N‐MVs derived from isolated neutrophils showed no activity, with either the LPS‐fMLP or the fMLP stimulation protocols.ConclusionsWe demonstrated that LPS inflammatory ‘priming’ of neutrophils within a whole blood environment was necessary for generation of leukocyte‐activating N‐MVs in our in vitro model of pulmonary vascular inflammation. These findings support a role for circulating N‐MVs in the pathogenesis of indirect ALI, and also highlight the crucial importance of using physiologically relevant in vitro models to evaluate the function of circulating intravascular cell‐derived MVs.

LTi-like type 3 innate lymphoid cells are associated with the transient induction of tertiary lymphoid structures in inflamed pulmonary tissue

Abstract Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates observed in inflamed tissues. Their presence in pulmonary tissue has been associated with good prognosis in patients with lung cancer. Although TLS formation shares common features with secondary lymphoid organogenesis, the mechanisms underlying this process are yet to be fully elucidated. Presence of ILC3 in the vicinity of tumor-associated TLS correlates with the density of lymphoid structures. We hypothesized that RORγt+ ILC3 play a major role in TLS induction in inflamed lungs, as do RORγt+ fetal lymphoid tissue inducer cells (LTi) during lymph node formation. We developed a murine model of pulmonary inflammation induced by LPS intranasal instillation that triggers TLS formation. ILC3 presence in lungs was observed with a specific flow cytometry antibody panel. Three RORgt+ILC3 subsets (NCR− CCR6−, NCR−CCR6+, NCR+CCR6−) were detected. In particular, NCR−CCR6+ LTi-like cells represented about 25% of CD117+CD127+ILC (ILC2/ILC3) and less than 0.04% among CD45+ cells. Analyses of ILC subsets at different time points following LPS instillation showed an increase in the number and % (up to 0.59%) of ILC3 among CD45+ cells, as well as an increase in the % of LTi-like ILC3 among CD45+ cells, that peaked around day 10 (from 0.02% up to 0.15%). Quantification of TLS by histochemistry showed that they are induced in lung tissue in an LPS dose-dependent manner. TLS density increases between days 3 and 10 and decreases thereafter. They were no longer detected at day 25, while LTi-like ILC3 % returned to baseline. Gene expression analysis and functional assays of sorted LTi-like ILC3 are essential to determine if these cells are key players in TLS induction. Supported by grants from La Ligue Nationale contre le cancer (TAHG21042) and Fondation ARC (R19232DD).

Experimental Model of Pulmonary Inflammation Induced by SARS-CoV-2 Spike Protein and Endotoxin.

COVID-19 is characterized by a dysregulated and excessive inflammatory response and, in severe cases, acute respiratory distress syndrome. We have recently demonstrated a previously unknown high-affinity interaction between the SARS-CoV-2 spike (S) protein and bacterial lipopolysaccharide (LPS), leading to the boosting of inflammation. Here we present a mouse inflammation model employing the coadministration of aerosolized S protein together with LPS to the lungs. Using NF-κB-RE-Luc reporter and C57BL/6 mice followed by combinations of bioimaging, cytokine, chemokine, fluorescence-activated cell sorting, and histochemistry analyses, we show that the model yields severe pulmonary inflammation and a cytokine profile similar to that observed in COVID-19. Therefore, the model offers utility for analyses of the pathophysiological features of COVID-19 and the development of new treatments.

Thrombin cleaves IL‐33 and modulates IL‐33‐activated allergic lung inflammation

Organisms have orchestrated coagulation and immune systems. Although a link between inflammation and haemostasis has been reported in asthma, the interaction mechanism has not been completely elucidated. Here, we investigated the direct link between the mammalian immune and coagulation systems. Mice were administered protease or antigens intranasally to induce airway inflammation with or without thrombin inhibitors treatment. The effects of thrombin and its inhibitors on interleukin (IL)-33 were investigated both in vivo and in vitro. Peripheral blood mononuclear cells (PBMCs) and plasma from asthma patients are collected to verify the correlation between thrombin and group 2 innate lymphocytes (ILC2s). Low-molecular-weight heparin (LMWH, an indirect inhibitor of thrombin) restrained both papain- and fungus-induced type 2 immune responses in mice by inhibiting IL-33cleavage. Upon examining the potential thrombin protease consensus sites, we found that IL-33 was directly cleaved by thrombin at specific amino acids (R48 and R106) to generate a mature form of IL-33 with potent biological activity. In addition, we found that bivalirudin TFA (a direct inhibitor of thrombin) inhibited a variety of type 2 inflammatory responses, such as those in house dust mite (HDM)- and ovalbumin (OVA)-mediated pulmonary inflammation models. We found that plasma thrombin-antithrombin complex (TATc) levels in asthma patients were positively associated with the number and function of IL-33-responder group 2 innate lymphocytes (ILC2s) among peripheral blood mononuclear cells (PBMCs) from asthma patients. The data suggested that thrombin inhibitors administration could be effective in treating lung inflammation by regulating ILC2s via IL-33maturation, indicating that targeting thrombin is a potential way to treat allergic diseases.