Investigation of microvesicle uptake by mouse lung-marginated monocytes in vitro.

Extracellular microvesicles (MVs) are released into the circulation in large numbers during acute systemic inflammation, yet little is known of their intravascular cell/tissue-specific interactions under these conditions. We recently described a dramatic increase in the uptake of intravenously injected MVs by monocytes marginated within the pulmonary vasculature, in a mouse model of low-dose lipopolysaccharide-induced systemic inflammation. To investigate the mechanisms of enhanced MV uptake by monocytes, we developed an in vitro model using in vivo derived monocytes. Although mouse blood is a convenient source, monocyte numbers are too low for in vitro experimentation. In contrast, differentiated bone marrow monocytes are abundant, but they are rapidly mobilized during systemic inflammation, and thus no longer available. Instead, we developed a protocol using marginated monocytes from the pulmonary vasculature as an anatomically relevant and abundant source. Mice are sacrificed by terminal anesthesia, the lungs inflated and perfused via the pulmonary artery. Perfusate cell populations are evaluated by flow cytometry, combined with in vitro generated fluorescently labelled MVs, and incubated in suspension for up to one hour. Washed cells are analyzed by flow cytometry to quantify MV uptake and confocal microscopy to localize MVs within cells (O'Dea et al., 2020). Using this perfusion-based method, substantial numbers of marginated pulmonary vascular monocytes are recovered, allowing multiple in vitro tests to be performed from a single mouse donor. As MV uptake profiles were comparable to those observed in vivo, this method is suitable for physiologically relevant high throughput mechanistic studies on mouse monocytes under in vitro conditions. Graphic abstract: Figure 1. Schematic of lung perfusate cell harvest and co-incubation with in vitro generated MVs. Created with BioRender.com

In vivo compartmental analysis of leukocytes in mouse lungs

The lung has a unique structure consisting of three functionally different compartments (alveolar, interstitial, and vascular) situated in an extreme proximity. Current methods to localize lung leukocytes using bronchoalveolar lavage and/or lung perfusion have significant limitations for determination of location and phenotype of leukocytes. Here we present a novel method using in vivo antibody labeling to enable accurate compartmental localization/quantification and phenotyping of mouse lung leukocytes. Anesthetized C57BL/6 mice received combined in vivo intravenous and intratracheal labeling with fluorophore-conjugated anti-CD45 antibodies, and lung single-cell suspensions were analyzed by flow cytometry. The combined in vivo intravenous and intratracheal CD45 labeling enabled robust separation of the alveolar, interstitial, and vascular compartments of the lung. In naive mice, the alveolar compartment consisted predominantly of resident alveolar macrophages. The interstitial compartment, gated by events negative for both intratracheal and intravenous CD45 staining, showed two conventional dendritic cell populations, as well as a Ly6C(lo) monocyte population. Expression levels of MHCII on these interstitial monocytes were much higher than on the vascular Ly6C(lo) monocyte populations. In mice exposed to acid aspiration-induced lung injury, this protocol also clearly distinguished the three lung compartments showing the dynamic trafficking of neutrophils and exudative monocytes across the lung compartments during inflammation and resolution. This simple in vivo dual-labeling technique substantially increases the accuracy and depth of lung flow cytometric analysis, facilitates a more comprehensive examination of lung leukocyte pools, and enables the investigation of previously poorly defined “interstitial” leukocyte populations during models of inflammatory lung diseases

Alveolar macrophage-derived microvesicles mediate acute lung injury

Background Microvesicles (MVs) are important mediators of intercellular communication, packaging a variety of molecular cargo. They have been implicated in the pathophysiology of various inflammatory diseases; yet, their role in acute lung injury (ALI) remains unknown. Objectives We aimed to identify the biological activity and functional role of intra-alveolar MVs in ALI. Methods Lipopolysaccharide (LPS) was instilled intratracheally into C57BL/6 mice, and MV populations in bronchoalveolar lavage fluid (BALF) were evaluated. BALF MVs were isolated 1 hour post LPS, assessed for cytokine content and incubated with murine lung epithelial (MLE-12) cells. In separate experiments, primary alveolar macrophage-derived MVs were incubated with MLE-12 cells or instilled intratracheally into mice. Results Alveolar macrophages and epithelial cells rapidly released MVs into the alveoli following LPS. At 1 hour, the dominant population was alveolar macrophage-derived, and these MVs carried substantive amounts of tumour necrosis factor (TNF) but minimal amounts of IL-1β/IL-6. Incubation of these mixed MVs with MLE-12 cells induced epithelial intercellular adhesion molecule-1 (ICAM-1) expression and keratinocyte-derived cytokine release compared with MVs from untreated mice (p<0.001). MVs released in vitro from LPS-primed alveolar macrophages caused similar increases in MLE-12 ICAM-1 expression, which was mediated by TNF. When instilled intratracheally into mice, these MVs induced increases in BALF neutrophils, protein and epithelial cell ICAM-1 expression (p<0.05). Conclusions We demonstrate, for the first time, the sequential production of MVs from different intra-alveolar precursor cells during the early phase of ALI. Our findings suggest that alveolar macrophage-derived MVs, which carry biologically active TNF, may play an important role in initiating ALI

The local and systemic immune response to intrauterine LPS in the prepartum mouse

Inflammation plays a key role in human term and preterm labor (PTL). Intrauterine LPS has been widely used to model inflammation-induced complications of pregnancy, including PTL. It has been shown to induce an intense myometrial inflammatory cell infiltration, but the role of LPS-induced inflammatory cell activation in labor onset and fetal demise is unclear. We investigated this using a mouse model of PTL, where an intrauterine injection of 10 μg of LPS (serotype 0111:B4) was given at E16 of CD1 mouse pregnancy. This dose induced PTL at an average of 12.7 h postinjection in association with 85% fetal demise. Flow cytometry showed that LPS induced a dramatic systemic inflammatory response provoking a rapid and marked leucocyte infiltration into the maternal lung and liver in association with increased cytokine levels. Although there was acute placental inflammatory gene expression, there was no corresponding increase in fetal brain inflammatory gene expression until after fetal demise. There was marked myometrial activation of NFκB and MAPK/AP-1 systems in association with increased chemokine and cytokine levels, both of which peaked with the onset of parturition. Myometrial macrophage and neutrophil numbers were greater in the LPS-injected mice with labor onset only; prior to labor, myometrial neutrophils and monocytes numbers were greater in PBS-injected mice, but this was not associated with an earlier onset of labor. These data suggest that intrauterine LPS induces parturition directly, independent of myometrial inflammatory cell infiltration, and that fetal demise occurs without fetal inflammation. Intrauterine LPS provokes a marked local and systemic inflammatory response but with limited inflammatory cell infiltration into the myometrium or placenta

Inhibition of TNF receptor p55 by a domain antibody attenuates the initial phase of acid-induced lung injury in mice

Background: Tumor necrosis factor-α (TNF) is strongly implicated in the development of acute respiratory distress syndrome (ARDS), but its potential as a therapeutic target has been hampered by its complex biology. TNF signals through two receptors, p55 and p75, which play differential roles in pulmonary edema formation during ARDS. We have recently shown that inhibition of p55 by a novel domain antibody (dAb™) attenuated ventilator36 induced lung injury. In the current study we explored the efficacy of this antibody in mouse models of acid-induced lung injury, to investigate the longer consequences of treatment. Methods: We employed two acid-induced injury models, an acute ventilated model and a resolving spontaneously breathing model. C57BL/6 mice were pretreated intratracheally or intranasally with p55-targeting dAb or non-targeting ‘dummy’ dAb, 1 or 4 hours before acid instillation. Results: Acid instillation in the dummy dAb group caused hypoxemia, increased respiratory system elastance, pulmonary inflammation and edema in both the ventilated and resolving models. Pretreatment with p55-targeting dAb significantly attenuated physiological markers of ARDS in both models. p55-targeting dAb also attenuated pulmonary inflammation in the ventilated model, with signs that altered cytokine production and leukocyte recruitment persisted beyond the very acute phase. Conclusions: These results demonstrate that the p55-targeting dAb attenuates lung injury and edema formation in models of ARDS induced by acid aspiration, with protection from a single dose lasting up to 24 hours. Together with our previous data, the current study lends support towards the clinical targeting of p55 for patients with, or at risk of ARDS

ATP redirects cytokine trafficking and promotes novel membrane TNF signaling via microvesicles

Cellular stress or injury induces release of endogenous danger signals such as ATP, which plays a central role in activating immune cells. ATP is essential for the release of nonclassically secreted cytokines such as IL-1β but, paradoxically, has been reported to inhibit the release of classically secreted cytokines such as TNF. Here, we reveal that ATP does switch off soluble TNF (17 kDa) release from LPS-treated macrophages, but rather than inhibiting the entire TNF secretion, ATP packages membrane TNF (26 kDa) within microvesicles (MVs). Secretion of membrane TNF within MVs bypasses the conventional endoplasmic reticulum– and Golgi transport–dependent pathway and is mediated by acid sphingomyelinase. These membrane TNF–carrying MVs are biologically more potent than soluble TNF in vivo, producing significant lung inflammation in mice. Thus, ATP critically alters TNF trafficking and secretion from macrophages, inducing novel unconventional membrane TNF signaling via MVs without direct cell-to-cell contact. These data have crucial implications for this key cytokine, particularly when therapeutically targeting TNF in acute inflammatory diseases.—Soni, S., O'Dea, K. P., Tan, Y. Y., Cho, K., Abe, E., Romano, R., Cui, J., Ma, D., Sarathchandra, P., Wilson, M. R., Takata, M. ATP redirects cytokine trafficking and promotes novel membrane TNF signaling via microvesicles. FASEB J. 33, 6442–6455 (2019). www.fasebj.org

Secreted extracellular cyclophilin a is a novel mediator of ventilator induced lung injury.

RATIONALE: Mechanical ventilation is a mainstay of intensive care but contributes to the mortality of patients through ventilator induced lung injury. Extracellular Cyclophilin A is an emerging inflammatory mediator and metalloproteinase inducer, and the gene responsible for its expression has recently been linked to COVID-19 infection. OBJECTIVES: Here we explore the involvement of extracellular Cyclophilin A in the pathophysiology of ventilator-induced lung injury. METHODS: Mice were ventilated with low or high tidal volume for up to 3 hours, with or without blockade of extracellular Cyclophilin A signalling, and lung injury and inflammation were evaluated. Human primary alveolar epithelial cells were exposed to in vitro stretch to explore the cellular source of extracellular Cyclophilin A, and Cyclophilin A levels were measured in bronchoalveolar lavage fluid from acute respiratory distress syndrome patients, to evaluate clinical relevance. MEASUREMENTS AND MAIN RESULTS: High tidal volume ventilation in mice provoked a rapid increase in soluble Cyclophilin A levels in the alveolar space, but not plasma. In vivo ventilation and in vitro stretch experiments indicated alveolar epithelium as the likely major source. In vivo blockade of extracellular Cyclophilin A signalling substantially attenuated physiological dysfunction, macrophage activation and matrix metalloproteinases. Finally, we found that patients with acute respiratory distress syndrome showed markedly elevated levels of extracellular Cyclophilin A within bronchoalveolar lavage. CONCLUSIONS: Cyclophilin A is upregulated within the lungs of injuriously ventilated mice (and critically ill patients), where it plays a significant role in lung injury. Extracellular Cyclophilin A represents an exciting novel target for pharmacological intervention

Progesterone, the maternal immune system and the onset of parturition in the mouse

The role of progesterone (P4) in the regulation of the local (uterine) and systemic innate immune system, myometrial expression of connexin 43 (Cx-43) and cyclooxygenase 2 (COX-2) and the onset of parturition was examined in: 1) naïve mice delivering at term; 2) E16 mice treated with RU486 (P4-antagonist) to induce preterm parturition; and 3) in mice treated with P4 to prevent term parturition.In naïve mice, myometrial neutrophil and monocyte numbers peaked at E18 and declined with the onset of parturition. In contrast, circulating monocytes did not change and although neutrophils were increased with pregnancy, they did not change across gestation. The myometrial mRNA and protein levels of most chemokines/cytokines, Cx-43 and COX-2 increased with, but not before, parturition.With RU486-induced parturition, myometrial and systemic neutrophil numbers increased before and myometrial monocyte numbers increased with parturition only. Myometrial chemokine/cytokine mRNA abundance increased with parturition, but protein levels peaked earlier at between 4.5 and 9h post RU486. Cx-43, but not COX-2, mRNA expression and protein levels increased prior to the onset of parturition.In mice treated with P4, the gestation-linked increase in myometrial monocyte, but not neutrophil, numbers was prevented and expression of Cx-43 and COX-2 was reduced. On E20 of P4 supplementation, myometrial chemokine/cytokine and leukocyte numbers, but not Cx-43 and COX-2 expression, increased.These data show that during pregnancy P4 controls myometrial monocyte infiltration, cytokine and prolabour factor synthesis via mRNA dependent and independent mechanisms and, with prolonged P4 supplementation, P4 action is repressed resulting in increased myometrial inflammation

Bv8 mediates myeloid cell migration and enhances malignancy of colorectal cancer

Colorectal cancer (CRC) is the third most predominant malignancy in the world. Although the importance of immune system in cancer development has been well established, the underlying mechanisms remain to be investigated further. Here we studied a novel protein prokineticin 2 (Prok2, also known as Bv8) as a key pro-tumoral factor in CRC progression in in vitro and ex vivo settings. Human colorectal tumor tissues, myeloid cell lines (U937 cells and HL60 cells) and colorectal cancer cell line (Caco-2 cells) were used for various studies. Myeloid cell infiltration (especially neutrophils) and Bv8 accumulation were detected in human colorectal tumor tissue with immunostaining. The chemotactic effects of Bv8 on myeloid cells were presented in the transwell assay and chemotaxis assy. Cultured CRC cells treated with myeloid cells or Bv8 produced reactive oxygen species (ROS) and vascular endothelial growth factor (VEGF). Furthermore, ROS and VEGF acted as pro-angiogenesis buffer in myeloid cell-infiltrated CRC microenvironment. Moreover, myeloid cells or Bv8 enhanced energy consumption of glycolysis ATP and mitochondria ATP of CRC cells. Interestingly, myeloid cells increased CRC cell viability, but CRC cells decreased the viability of myeloid cells. ERK signalling pathway in CRC cells was activated in the presence of Bv8 or co-cultured myeloid cells. In conclusion, our data indicated the vital roles of Bv8 in myeloid cell infiltration and CRC development, suggesting that Bv8 may be a potential therapeutic target for colorectal cancer-related immunotherapy

Efficacy and Safety of Inhaled Carbon Monoxide during Pulmonary Inflammation in Mice

Background: Pulmonary inflammation is a major contributor to morbidity in a variety of respiratory disorders, but treatment options are limited. Here we investigate the efficacy, safety and mechanism of action of low dose inhaled carbon monoxide (CO) using a mouse model of lipopolysaccharide (LPS)-induced pulmonary inflammation. Methodology: Mice were exposed to 0–500 ppm inhaled CO for periods of up to 24 hours prior to and following intratracheal instillation of 10 ng LPS. Animals were sacrificed and assessed for intraalveolar neutrophil influx and cytokine levels, flow cytometric determination of neutrophil number and activation in blood, lung and lavage fluid samples, or neutrophil mobilisation from bone marrow. Principal Findings: When administered for 24 hours both before and after LPS, inhaled CO of 100 ppm or more reduced intraalveolar neutrophil infiltration by 40–50%, although doses above 100 ppm were associated with either high carboxyhemoglobin, weight loss or reduced physical activity. This anti-inflammatory effect of CO did not require pre-exposure before induction of injury. 100 ppm CO exposure attenuated neutrophil sequestration within the pulmonary vasculature as well as LPS-induced neutrophilia at 6 hours after LPS, likely due to abrogation of neutrophil mobilisation from bone marrow. In contrast to such apparently beneficial effects, 100 ppm inhaled CO induced an increase in pulmonary barrier permeability as determined by lavage fluid protein content and translocation of labelled albumin from blood to the alveolar space