Proinflammatory Cytokines In Lungs Research Articles

Elevated Circulating Cell-Free Hemoglobin Drives Endothelial Glycocalyx Destruction and Inflammation in Sepsis

Rationale: Endothelial injury is a hallmark of sepsis-induced acute respiratory distress syndrome (ARDS). Endothelial barrier integrity is regulated by the endothelial glycocalyx, a matrix of proteoglycans and glycosaminoglycans (GAGs) that lines the vascular lumen. In sepsis, this glycocalyx is degraded by heparanase, which cleaves heparan sulfate, the primary glycocalyx GAG. Glycocalyx breakdown is linked to worse sepsis outcomes, but the mechanisms inducing heparanase activity in sepsis are unknown. We previously found that plasma levels of hemoglobin released from red blood cells (cell-free hemoglobin, CFH) are elevated in clinical sepsis and correlate with higher rates of ARDS and death. CFH also worsens endothelial injury and microvascular permeability in murine sepsis. We therefore hypothesized that CFH upregulates heparanase production to drive glycocalyx shedding and inflammation in sepsis. Methods: To test the association between CFH levels, heparanase expression, and glycocalyx degradation, circulating levels of heparanase, heparan sulfate, and total GAGs were measured in 77 sepsis patients and compared across CFH quartiles. In mice with intraabdominal polymicrobial sepsis with elevated CFH (CS [cecal slurry] + intravenous [IV] CFH), plasma heparanase, total GAGs, and syndecan-1 were quantified. Whole lung and plasma pro-inflammatory cytokines were also determined to investigate the impact of CFH on inflammation in sepsis. Results: In sepsis patients, higher circulating CFH levels were associated with increased plasma heparanase (p=0.0304), total GAGs (p=0.0178), and heparan sulfate (p=0.0112). CFH also exacerbated glycocalyx degradation in murine sepsis; circulating syndecan-1 (p=0.0152) and total GAGs (p=0.0094) were elevated in animals that received CS+IV CFH compared to CS alone. This effect may be mediated by upregulation of active heparanase, which was higher in the plasma of CS+IV CFH treated animals (p=0.1333). CFH also worsened illness severity, as CS+IV CFH animals had worse sepsis severity scores (p=0.0074). Whole lung tumor necrosis factor-alpha (TNF-a; p=0.0129) and interferon-gamma (IFN-g; p=0.0264) transcript levels were increased in the CS+IV CFH group, indicating greater lung inflammation. Systemic inflammation was also enhanced in the CS+IV CFH animals, demonstrated by higher plasma TNF-a (p<0.001) and IFN-g (p=0.0221) concentrations. Conclusions: In sepsis, CFH is associated with upregulation of heparanase and endothelial glycocalyx degradation, which may disrupt the endothelial barrier, causing downstream organ injury. Future studies to define the molecular pathways that underlie these findings and their effect on endothelial permeability will be an important next step towards identifying novel treatment targets to improve outcomes for patients with sepsis-associated ARDS. Funding F30HL170483, R01HL164937, R01HL158906, R01HL150783, R01HL125371. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dynasore Alleviates LPS-Induced Acute Lung Injury by Inhibiting NLRP3 Inflammasome-Mediated Pyroptosis.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are clinically severe respiratory disorders without available pharmacological therapies. Dynasore is a cell-permeable molecule that inhibits GTPase activity and exerts protective effects in several disease models. However, whether dynasore can alleviate lipopolysaccharide (LPS)-induced ALI is unknown. This study investigated the effect of dynasore on macrophage activation and explored its potential mechanisms in LPS-induced ALI in vitro and in vivo. Bone marrow-derived macrophages (BMDMs) were activated classically with LPS or subjected to NLRP3 inflammasome activation with LPS+ATP. A mouse ALI model was established by the intratracheal instillation (i.t.) of LPS. The expression of PYD domains-containing protein 3 (NLRP3), caspase-1, and gasdermin D (GSDMD) protein was detected by Western blots. Inflammatory mediators were analyzed in the cell supernatant, in serum and bronchoalveolar lavage fluid (BALF) by enzyme-linked immunosorbent assays. Morphological changes in lung tissues were evaluated by hematoxylin and eosin staining. F4/80, Caspase-1 and GSDMD distribution in lung tissue was detected by immunofluorescence. Dynasore downregulated nuclear factor (NF)-κB signaling and reduced proinflammatory cytokine production in vitro and inhibited the production and release of interleukin (IL)-1β, NLRP3 inflammasome activation, and macrophage pyroptosis through the Drp1/ROS/NLRP3 axis. Dynasore significantly reduced lung injury scores and proinflammatory cytokine levels in both BALF and serum in vivo, including IL-1β and IL-6. Dynasore also downregulated the co-expression of F4/80, caspase-1 and GSDMD in lung tissue. Collectively, these findings demonstrated that dynasore could alleviate LPS-induced ALI by regulating macrophage pyroptosis, which might provide a new therapeutic strategy for ALI/ARDS.

Effect of Enteromorpha polysaccharides on gut-lung axis in mice infected with H5N1 influenza virus

Enteromorpha polysaccharides (EPPs) have been reported to have antiviral and anti-inflammatory properties. To explore the effect of EPPs on H5N1-infected mice, mice were pretreated with EPPs before being infected with the H5N1 influenza virus intranasally. H5N1 infection resulted in body-weight loss, pulmonary and intestinal damage, and an imbalance of gut microbiota in mice. As a result of the inclusion of EPPs, the body weight of mice recovered and pathological damage to the lung and intestine was reduced. EPPs also diminished inflammation by drastically lowering the expression of proinflammatory cytokines in lungs and intestines. H5N1 infection reduced bacterial diversity, and the abundance of pathogenic bacteria such as Desulfovibrio increased. However, the beneficial bacteria Alistipes rebounded in the groups which received EPPs before the infection. The modulation of the gut-lung axis may be related to the mechanism of EPPs in antiviral and anti-inflammatory responses. EPPs have shown potential in protecting the host from the influenza A virus infection.

Lung proinflammatory microRNA and cytokine expression in a mouse model of allergic inflammation: role of sex chromosome complement and gonadal hormones.

Epigenetic alterations such as dysregulation of miRNAs have been reported to play important roles in interactions between genetic and environmental factors. In this study, we tested the hypothesis that induction of lung inflammation by inhaled allergens triggers a sex-specific miRNA regulation that is dependent on chromosome complement and hormonal milieu. We challenged the four core genotypes (FCGs) model through intranasal sensitization with a house dust mite (HDM) solution (or PBS as a control) for 5 wk. The FCG model allows four combinations of gonads and sex chromosomes: 1) XX mice with ovaries (XXF), 2) XY mice with testes (XYM), 3) XX mice with testes (XXM), and 4) XY mice with ovaries (XYF). Following the challenge (n = 5-7/group), we assessed the expression of 84 inflammatory miRNAs in lung tissue using a PCR array and cytokine levels in bronchoalveolar lavage fluid (BAL) by a multiplex protein assay (n = 4-7 animals/group). Our results showed higher levels of the chemokine KC (an Il-8 homolog) and IL-7 in BAL from XYF mice challenged with HDM. In addition, IL-17A was significantly higher in BAL from both XXF and XYF mice. A three-way interaction among treatment, gonads, and sex chromosome revealed 60 of 64 miRNAs that differed in expression depending on genotype; XXF, XXM, XYF, and XYM mice had 45, 32, 4, and 52 differentially expressed miRNAs, respectively. Regulatory networks of miRNAs identified in this study were implicated in pathways associated with asthma. Female gonadal hormonal effects may alter miRNA expression and contribute to the higher susceptibility of females to asthma.NEW & NOTEWORTHY miRNAs play important roles in regulating gene and environmental interactions. However, their role in mediating sex differences in allergic responses and lung diseases has not been elucidated. Our study used a targeted omics approach to characterize the contributions of gonadal hormones and chromosomal components to lung responses to an allergen challenge. Our results point to the influence of sex hormones in miRNA expression and proinflammatory markers in allergic airway inflammation.

Protective effect of olopatadine hydrochloride against LPS-induced acute lung injury: via targeting NF-κB signaling pathway.

Morbidity and mortality rates associated with acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are high (30-40%). Nuclear factor-kappa B (NF-κB) is a transcription factor, associated with transcription of numerous cytokines leading to cytokine storm, and thereby, plays a major role in ALI/ARDS and in advanced COVID-19 syndrome. Considering the role of NF-κB in ALI, cost-effective in silico approaches were utilized in the study to identify potential NF-κB inhibitor based on the docking and pharmacokinetic results. The identified compound was then pharmacologically validated in lipopolysaccharide (LPS) rodent model of acute lung injury. LPS induces ALI by altering alveolar membrane permeability, recruiting activated neutrophils and macrophages to the lungs, and compromising the alveolar membrane integrity and ultimately impairs the gaseous exchange. Furthermore, LPS exposure is associated with exaggerated production of various proinflammatory cytokines in lungs. Based on in silico studies Olopatadine Hydrochloride (Olo), an FDA-approved drug was found as a potential NF-κB inhibitor which has been reported for the first time, and consideredfurther for the pharmacological validation. Intraperitoneal LPS administration resulted in ALI/ARDS by fulfilling 3 out of the 4 criteria described by ATS committee (2011) published workshop report. However, treatment with Olo attenuated LPS-induced elevation of proinflammatory markers (IL-6 and NF-κB), oxidative stress, neutrophil infiltration, edema, and damage in lungs. Histopathological studies also revealed that Olo treatment significantly ameliorated LPS-induced lung injury, thus conferring improvement in survival. Especially, the effects produced by Olo medium dose (1mg/kg) were comparable to dexamethasone standard. In nutshell, inhibition of NF-κB pathway by Olo resulted in protection and reduced mortality in LPS- induced ALI and thus has potential to be used clinically to arrest disease progression in ALI/ARDS, since the drug is already in the market. However, the findings warrant further extensive studies, and also future studies can be planned to elucidate its role in COVID-19-associated ARDS or cytokine storm.

STING mediates SU5416/hypoxia-induced pulmonary arterial hypertension in rats by regulating macrophage NLRP3 inflammasome activation

BackgroundThe NLRP3 inflammasome in macrophages is known to promote infection-related vascular growth, and NLRP3 inflammasome activation interacts with PAH. STING is a crucial inflammatory reaction link that can increase the overexpression of NLRP3. However, the expression and effect of STING in PAH have not been elucidated. We examined the expression and articulation of STING in PAH and researched its hidden mechanism. MethodsA SU5416 plus hypoxia (Su/Hy)-induced rat model of PAH was constructed to examine STING activation. Su/Hy induced PAH rats were given a prophylactic injection of STING the inhibitor C-176. After modeling, hemodynamic changes, right ventricular hypertrophy index, lung morphological features, inflammasome activation, and proinflammatory cytokine secretion levels were assessed. In addition, the STING agonist DMXAA or inhibitor C-176 was used to interfere with LPS-induced BMDMs, NLRP3 inflammasome activation and cytokine secretion were examined, and the effect on PASMCs was evaluated in a coculture system. ResultsSTING expression increased significantly in the lung tissue of Su/Hy-treated PAH rats compared with normoxia-treated rats. Moreover, STING inhibitors can alleviate the Su/Hy-induced increase in pulmonary artery pressure and restrain the activation of the NLRP3 inflammasome and proinflammatory cytokines. In vitro experiments confirmed that STING affected the expression of the NLRP3 inflammasome and the secretion of inflammatory cytokines in BMDMs and promoted the proliferation of PASMCs in the coculture system. ConclusionOur study shows that STING is activated in Su/Hy-induced PAH and boosts the actuation of the macrophage NLRP3 inflammasome to advance the inflammatory response and vascular proliferation in rats with Su/Hy-induced pulmonary hypertension.

High Concentration Hydrogen Mitigates Sepsis-Induced Acute Lung Injury in Mice by Alleviating Mitochondrial Fission and Dysfunction.

Background: Multiple organ failure (MOF) is the main cause of early death in septic shock. Lungs are among the organs that are affected in MOF, resulting in acute lung injury. A large number of inflammatory factors and stress injury in sepsis can lead to alterations in mitochondrial dynamics. Numerous studies have confirmed that hydrogen can alleviate sepsis in the animal model. The purpose of this experiment was to explore the therapeutic effect of high concentration (67%) hydrogen on acute lung injury in septic mice and its mechanism. Methods: The moderate and severe septic models were prepared by cecal ligation and puncture. Hydrogen with different concentrations was inhaled for one hour at 1 h and 6 h after the corresponding surgery. The arterial blood gas of mice during hydrogen inhalation was monitored in real time, and the 7-day survival rate of mice with sepsis was recorded. The pathological changes of lung tissues and functions of livers and kidneys were measured. The changes of oxidation products, antioxidant enzymes and pro-inflammatory cytokines in lungs and serums were detected. Mitochondrial function was measured. Results: The inhalation of 2% or 67% hydrogen improves the 7-day survival rate and reduces acute lung injury as well as liver and kidney injury in sepsis. The therapeutic effect of 67% hydrogen inhalation on sepsis was related to increasing antioxidant enzyme activity, reducing oxidation products and pro-inflammatory cytokines in lungs and serums. Compared with the Sham group, mitochondrial dysfunction was alleviated in hydrogen groups. Conclusions: Hydrogen inhalation by high or low concentration can both significantly improve sepsis; however, a high concentration demonstrates a better protective effect. High concentration hydrogen inhalation can significantly improve the mitochondrial dynamic balance and reduce the lung injury in septic mice.

Lung inflammation induced by silica particles triggers hippocampal inflammation, synapse damage and memory impairment in mice

BackgroundConsiderable evidence indicates that a signaling crosstalk between the brain and periphery plays important roles in neurological disorders, and that both acute and chronic peripheral inflammation can produce brain changes leading to cognitive impairments. Recent clinical and epidemiological studies have revealed an increased risk of cognitive impairment and dementia in individuals with impaired pulmonary function. However, the mechanistic underpinnings of this association remain unknown. Exposure to SiO2 (silica) particles triggers lung inflammation, including infiltration by peripheral immune cells and upregulation of pro-inflammatory cytokines. We here utilized a mouse model of lung silicosis to investigate the crosstalk between lung inflammation and memory.MethodsSilicosis was induced by intratracheal administration of a single dose of 2.5 mg SiO2/kg in mice. Molecular and behavioral measurements were conducted 24 h and 15 days after silica administration. Lung and hippocampal inflammation were investigated by histological analysis and by determination of pro-inflammatory cytokines. Hippocampal synapse damage, amyloid-β (Aβ) peptide content and phosphorylation of Akt, a proxy of hippocampal insulin signaling, were investigated by Western blotting and ELISA. Memory was assessed using the open field and novel object recognition tests.ResultsAdministration of silica induced alveolar collapse, lung infiltration by polymorphonuclear (PMN) cells, and increased lung pro-inflammatory cytokines. Lung inflammation was followed by upregulation of hippocampal pro-inflammatory cytokines, synapse damage, accumulation of the Aβ peptide, and memory impairment in mice.ConclusionThe current study identified a crosstalk between lung and brain inflammatory responses leading to hippocampal synapse damage and memory impairment after exposure to a single low dose of silica in mice.

CIRCKLHL2 KNOCKDOWN ALLEVIATES SEPSIS-INDUCED ACUTE LUNG INJURY BY REGULATING MIR-29B-3P MEDIATED ROCK1 EXPRESSION DOWN-REGULATION.

Background: Acute lung injury (ALI) induced by sepsis is distinguished by an inflammatory progression. Herein, we investigated the action of circular RNA kelch like family member 2 (circKlhl2) in sepsis-induced ALI. Methods: The animal or cell model of sepsis ALI was established by LPS stimulation. The contents of circKlhl2, microRNA-29b-3p (miR-29b-3p), rho-associated coiled-coil containing protein kinase 1 (ROCK1), CyclinD1, B-cell lymphoma-2 (Bcl-2), and cleaved-caspase 3 (C-caspase-3) were detected by quantitative real-time polymerase chain reaction and western blot, respectively. Cell viability was assessed by cell counting kit 8 assay. Cell cycle and apoptosis were evaluated by flow cytometry. The abundances of proinflammatory cytokines were detected by enzyme-linked immunosorbent assay. Besides, the targeted relationship between miR-29b-3p and circKlhl2 or ROCK1 was verified by dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA pull-down assay. Results: Loss of circKlhl2 mitigated lung injury and proinflammatory cytokine expression in sepsis-ALI mice model and alleviated LPS-induced apoptosis and inflammatory response in microvascular endothelial cell (MPVECs) in vitro . The abundances of circKlhl2 and ROCK1 were boosted, while the miR-29b-3p level was diminished in the animal or cell model of sepsis-ALI. MiR-29b-3p inhibition abrogated circKlhl2 knockdown-mediated effects on MPVECs injury. Moreover, miR-29b-3p overexpression promoted cell proliferation and inhibited apoptosis and inflammation in LPS-treated MPVECs, while ROCK1 enhancement reversed these effects. Conclusion: CircKlhl2 expedited the sepsis-induced ALI by adjusting miR-29b-3p/ROCK1 axis.

Angiotensin-(1-7) alleviates acute lung injury by activating the Mas receptor in neutrophil.

Acute lung injury (ALI) is a major cause of mortality and morbidity in the clinic. None of the current pharmacological interventions has achieved a detectable benefit. The renin-angiotensin system (RAS) is a complex humoral system essentially involved in the regulation of ALI. In the RAS family, angiotensin (Ang)-(1-7) was found to provide protection by counteracting the effects of Ang II in various cardiopulmonary disease models. The downstream receptor of Ang-(1-7) is the G protein-coupled receptor (GPCR) Mas. We hypothesize that the Ang-(1-7)-Mas pathway would protect patients from ALI. To establish a 2-hit ALI model, the mice underwent intratracheal instillation of hydrochloric acid followed by ventilator-induced lung injury (VILI). ALI was evaluated based on lung edema, histology, myeloperoxidase activity, and proinflammatory cytokine production. The effects of the infusion or inhalation of Ang-(1-7) and Mas receptor blocker A779 were examined. The human neutrophils were isolated, and Mas receptor expression was examined. The neutrophil responses to platelet-activating factor (PAF) stimulation were tested by measuring the formation of reactive oxygen species (ROS), neutrophil adhesion, and chemotaxis. Next, in the mouse model, the neutrophils were depleted using an anti-ly6G antibody. The infusion or inhalation of Ang-(1-7) protected mice from ALI as evidenced by decreases in lung edema, the histological lung injury score, myeloperoxidase activity, and proinflammatory cytokine production. Such effects were largely blocked by the Mas receptor blocker A779. Mas receptor expression in the neutrophils was identified at both the messenger ribonucleic acid and protein levels. Ang-(1-7) prevented neutrophil responses to PAF stimulation, including the formation of ROS, neutrophil adhesion, and chemotaxis, while A779 alleviated these effects. The importance of neutrophils in ALI was further confirmed by neutrophil depletion using the anti-ly6G antibody; however, A779 partially reversed the protective role of neutrophil depletion in ALI, indicating the critical role of Ang-(1-7)-Mas signaling in other pulmonary cells. Ang-(1-7)/Mas receptor attenuates the key features of ALI by regulating neutrophil activation. Our study provides new evidence of their role in the pathogenesis of ALI and may lead to the development of a promising therapeutic strategy.

MicroRNA-31 mediated by interferon regulatory factor 7 signaling facilitates control of Mycobacterium tuberculosis infection

Tuberculosis (TB) induced by Mycobacterium tuberculosis (M. tuberculosis) infection remains a global most deadly infectious disease. While development of more effective TB vaccines and therapeutics relies on identifications of true biomarkers designating an immune protection against M. tuberculosis infection, exact protective immune components against M. tuberculosis infection remain largely unidentified. We previously found that severe TB induced remarkable up-regulation of interferon regulatory factor 7 (IRF7) and IRF7-related gene signatures, implicating that some unknown downstream molecules in IRF7 signaling cascades may determine the M. tuberculosis infection outcomes and serve as a protective immune component against M. tuberculosis infection. Indeed, here, we observe that genetic ablation of IRF7 leads to more severe lung pathology, increased M. tuberculosis burdens, impaired differentiation of effector/memory T subsets, and extensively elevated expression of pro-inflammatory cytokines in lungs. Importantly, IRF7 is vital for sustaining expression of PD-1/PD-L1 and PD-1/PD-L1-modulated miRNA-31. Moreover, interventions of miRNA-31 expressions via administration of miRNA-31 agomir reduces lung pathology and bacilli burdens via inducing up-regulation of gene sets involved in biological processes of defense response or cellular and chemical homeostasis in lungs. Thus, this study uncovers previously unrecognized importance and mechanisms of IRF7-mediated miRNA-31 as a protective immune component against M. tuberculosis infection.

Intranasal administration of a single dose of MVA-based vaccine candidates against COVID-19 induced local and systemic immune responses and protects mice from a lethal SARS-CoV-2 infection

Current coronavirus disease-19 (COVID-19) vaccines are administered by the intramuscular route, but this vaccine administration failed to prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infection in the upper respiratory tract, mainly due to the absence of virus-specific mucosal immune responses. It is hypothesized that intranasal (IN) vaccination could induce both mucosal and systemic immune responses that blocked SARS-CoV-2 transmission and COVID-19 progression. Here, we evaluated in mice IN administration of three modified vaccinia virus Ankara (MVA)-based vaccine candidates expressing the SARS-CoV-2 spike (S) protein, either the full-length native S or a prefusion-stabilized [S(3P)] protein; SARS-CoV-2-specific immune responses and efficacy were determined after a single IN vaccine application. Results showed that in C57BL/6 mice, MVA-based vaccine candidates elicited S-specific IgG and IgA antibodies in serum and bronchoalveolar lavages, respectively, and neutralizing antibodies against parental and SARS-CoV-2 variants of concern (VoC), with MVA-S(3P) being the most immunogenic vaccine candidate. IN vaccine administration also induced polyfunctional S-specific Th1-skewed CD4+ and cytotoxic CD8+ T-cell immune responses locally (in lungs and bronchoalveolar lymph nodes) or systemically (in spleen). Remarkably, a single IN vaccine dose protected susceptible K18-hACE2 transgenic mice from morbidity and mortality caused by SARS-CoV-2 infection, with MVA-S(3P) being the most effective candidate. Infectious SARS-CoV-2 viruses were undetectable in lungs and nasal washes, correlating with high titers of S-specific IgGs and neutralizing antibodies against parental SARS-CoV-2 and several VoC. Moreover, low histopathological lung lesions and low levels of pro-inflammatory cytokines in lungs and nasal washes were detected in vaccinated animals. These results demonstrated that a single IN inoculation of our MVA-based vaccine candidates induced potent immune responses, either locally or systemically, and protected animal models from COVID-19. These results also identified an effective vaccine administration route to induce mucosal immunity that should prevent SARS-CoV-2 host-to-host transmission.

Higher susceptibility of males to bleomycin-induced pulmonary inflammation is associated with sex-specific transcriptomic differences in myeloid cells

Idiopathic pulmonary fibrosis, a condition with likely genetic and environmental etiology, is relatively more prevalent with poor prognosis in human males. However, the underlying mechanisms for these gender-associated differences in the severity of fibrosis remain unknown. Here, we tested the hypothesis that the transcriptomic repertoire of myeloid cells determines the higher susceptibility of male mice to bleomycin (BLM)-induced lung fibrosis. Adult mice were oropharyngeally challenged with saline or BLM. Lung injury, inflammation, and fibrosis outcomes were assessed, and airspace myeloid-cells were subjected to RNA-sequencing. As compared with the female mice, the male mice manifested significantly increased lung injury, inflammation, proinflammatory cytokines (IL-6, IL-1β, IL-7, and IP-10), and fibrosis in response to BLM challenge. Interestingly, several pro-inflammatory and extracellular matrix-associated genes were significantly up-regulated in male myeloid-cells compared to female myeloid-cells in the saline-control group. Similarly, BLM challenge resulted in greater pro-inflammatory and pro-fibrotic transcriptomic changes in male compared to female myeloid cells. On the other hand, anti-inflammatory and regulatory cytokine, Il10 and Ifng respectively, were uniquely upregulated in BLM-challenged female but not in male myeloid cells when compared to their respective saline-control groups. Further, cross-sex bone marrow transplantation experiments revealed that male hematopoietic progenitor cells (HPCs) increased the granulocytic infiltration in female mice while female HPCs decreased the granulocytic infiltration in male mice post-BLM challenge. These findings suggest that there are inherent transcriptomic differences between the male and female lung myeloid cells and that the pro-inflammatory nature of male myeloid cells is sufficient to increase the susceptibility of female mice to BLM-induced inflammation.

Differential Oral Microbial Input Determines Two Microbiota Pneumo-Types Associated with Health Status.

The oral and upper respiratory tracts are closely linked anatomically and physiologically with the lower respiratory tract and lungs, and the influence of oral and upper respiratory microbes on the lung microbiota is increasingly being recognized. However, the ecological process and individual heterogeneity of the oral and upper respiratory tract microbes shaping the lung microbiota remain unclear owing to the lack of controlled analyses with sufficient sample sizes. Here, the microbiomes of saliva, nasal cavity, oropharyngeal area, and bronchoalveolar lavage samples are profiled and the shaping process of multisource microbes on the lung microbiota is measured. It is found that oral and nasal microbial inputs jointly shape the lung microbiota by occupying different ecological niches. It is also observed that the spread of oral microbes to the lungs is heterogeneous, with more oral microbes entering the lungs being associated with decreased lung function and increased lung proinflammatory cytokines. These results depict the external shaping process of lung microbiota and indicate the great value of oral samples, such as saliva, in monitoring and assessing lung microbiota status in clinical settings.

A 2A ADENOSINE RECEPTORS REGULATE MULTIPLE ORGAN FAILURE AFTER HEMORRHAGIC SHOCK IN MICE.

Trauma hemorrhagic shock (T/HS) is a clinical condition that causes multiple organ failure that needs rapid intervention. Restricted oxygen at the cellular level causes inflammation and subsequent cell death. Adenosine triphosphate is the universal intracellular energy currency and an important extracellular inflammatory signaling molecule. Adenosine, an endogenous nucleotide formed as a result of the breakdown of adenosine triphosphate, is also released during T/HS. Adenosine binds to four G protein-coupled receptors (A 1R , A 2a , A 2b , A 3R ) called adenosine receptors or P1 receptors. In the present study, we evaluated the effect of activation, inactivation, and genetic absence of A2aR (A2aR -/- mice) on T/HS-induced multiple organ failure. Wild-type mice were pretreated (30 min before shock induction) with an agonist or antagonist and then subjected to T/HS by withdrawing arterial blood and maintaining the blood pressure between 28 and 32 mm Hg. A2aR -/- mice were subjected to T/HS in the absence of pharmacologic treatment. Neutrophil sequestration was assessed by detecting myeloperoxidase, and Evans blue dye (EBD) method was used to analyze lung permeability. Blood and lung inflammatory cytokine levels were determined by sandwich enzyme-linked immunosorbent assay. The liver enzymes aspartate aminotransferase and alanine aminotransferase were determined spectrophotometrically from plasma. Activation of the apoptotic cascade was evaluated using a mouse apoptosis array. Our results demonstrate that the selective A2aR agonist CGS21680 decreases lung neutrophil sequestration, lung proinflammatory cytokines IL-6 and TNF-α, and bronchoalveolar lavage EBD. Pretreatment with the selective antagonist ZM241385 and genetic blockade in A2aR -/- mice increased neutrophil sequestration, proinflammatory cytokine levels, and bronchoalveolar lavage fluid EBD. The myeloperoxidase level in the lung was also increased in A2aR -/- mice. We observed that antiapoptotic markers decreased significantly with the absence of A2aR in the lung and spleen after T/HS. In conclusion, our data demonstrate that activation of A2aR regulates organ injury and apoptosis in the setting of T/HS.

Protective role of crocin against sepsis-induced injury in the liver, kidney and lungs via inhibition of p38 MAPK/NF-κB and Bax/Bcl-2 signalling pathways

Context Crocin has been reported to have multiple bioactivities. However, the effect of crocin administration on caecal ligation and puncture (CLP)-induced sepsis remains unknown. Objective We investigated the effects of crocin on CLP-induced sepsis in mice and the underlying mechanism of action. Materials and methods Five experimental groups (n = 10) of BALB/c mice were used: control, CLP (normal saline) and CLP + crocin (50, 100 and 250 mg/kg, 30 min prior to CLP). Mice were sacrificed 24 h after CLP. Liver, kidney and lung histopathology, indicator levels, apoptotic status, pro-inflammatory cytokines and relative protein levels were evaluated. Results Compared to the CLP group, crocin treatment significantly increased the survival rate (70%, 80%, 90% vs. 30%). Crocin groups exhibited protection against liver, kidney and lung damage with mild-to-moderate morphological changes and lower indicator levels: liver (2.80 ± 0.45, 2.60 ± 0.55, 1.60 ± 0.55 vs. 5.60 ± 0.55), kidney (3.00 ± 0.71, 2.60 ± 0.55, 1.40 ± 0.55 vs. 6.20 ± 0.84) and lungs (8.00 ± 1.59, 6.80 ± 1.64, 2.80 ± 0.84 vs. 14.80 ± 1.79). The proinflammatory cytokines (IL-1β, TNF-α, IL-6 and IL-10 levels in the crocin groups) were distinctly lower and the apoptotic index showed a significant decrease. Crocin administration significantly suppressed p38 MAPK phosphorylation and inhibited NF-κB/IκBα and Bcl-2/Bax activation. Discussion and conclusions Pre-treatment with crocin confers protective effects against CLP-induced liver, kidney and lung injury, implying it to be a potential therapeutic agent.

Differential Lung Protective Capacity of Exosomes Derived from Human Adipose Tissue, Bone Marrow, and Umbilical Cord Mesenchymal Stem Cells in Sepsis-Induced Acute Lung Injury.

Exosomes derived from human mesenchymal stem cells (hMSCs) have the capacity to regulate various biological events associated with sepsis-induced acute respiratory distress syndrome (ARDS), including cellular immunometabolism, the production of proinflammatory cytokines, allowing them to exert therapeutic effects. However, little is known about which type of hMSC-derived exosomes (hMSC-exo) is more effective and suitable for the treatment of sepsis-induced ARDS. The purpose of this study is to compare the efficacy of hMSC-derived exosomes from human adipose tissue (hADMSC-exo), human bone marrow (hBMMSC-exo), and human umbilical cord (hUCMSC-exo) in the treatment of sepsis-induced ARDS. We cocultured lipopolysaccharide- (LPS-) stimulated RAW264.7 macrophage cells with the three kinds of hMSCs and found that all hMSCs reduced the glycolysis level and the content of lactic acid in macrophages. Accordingly, the expression of proinflammatory cytokines also decreased. Notably, the protective effects of hMSCs from adipose tissue were more obvious than those of bone marrow and umbilical cord hMSCs. However, this protective effect was eliminated when an exosome inhibitor, GW4869, was added. Subsequently, we extracted and cocultured hMSC-derived exosomes with LPS-stimulated RAW264.7 cells and found that all three kinds of exosomes exerted a similar protective effect as their parental cells, with exosomes from adipose hMSCs showing the strongest protective effect. Finally, an experimental sepsis model in mice was established, and we found that all three types of hMSCs have obvious lung-protective effects, in reducing lung injury scores, lactic acid, and proinflammatory cytokine levels in the lung tissues and decreasing the total protein content and inflammatory cell count in the bronchoalveolar lavage fluid (BALF), and also can attenuate the systemic inflammatory response and improve the survival rate of mice. Intravenous injection of three types of hMSC-exo, in particular those derived from adipose hADMSCs, also showed lung-protective effects in mice. These findings revealed that exosomes derived from different sources of hMSCs can effectively downregulate sepsis-induced glycolysis and inflammation in macrophages, ameliorate the lung pathological damage, and improve the survival rate of mice with sepsis. It is worth noting that the protective effect of hADMSC-exo is better than that of hBMMSC-exo and hUCMSC-exo.

Cell cycle kinase CHEK2 in macrophages alleviates the inflammatory response to Staphylococcus aureus-induced pneumonia

Background Excessive macrophage-mediated inflammation participates in the development of Staphylococcus aureus (S. aureus)-induced pneumonia. Checkpoint kinase 2 (Chek2) was screened out as macrophage-related infantile pneumonia gene after the differentially expressed analysis of RNAseq data derived from pam3CSK4 stimulated bone marrow-derived macrophages (BMDMs). Methods RAW264.7 macrophage cells were transfected with Chek2-specific gRNA, which were further overexpressed with wide-type Chek2 or Chek2 kinase activity mutant (Chek2 KD, D368N). At the same time, the relative protein and mRNA expression of inflammatory cytokines were determined. C57BL/6J WT mice were intranasally infected with S. aureus to induce S. aureus-induced pneumonia, which was treated with BML-277, an inhibitor of Chek2. The symptoms of pneumonia mice and inflammatory cytokines associated with the nuclear factor kappa B (NF-κB) signaling pathways were further examined. Results In vivo, BML-277 significantly promoted pneumonia symptoms, including mortality, lung infiltration of immune cells, and the abundance of lung pro-inflammatory cytokines. Mechanically, BML-277 did not affect BMDMs survival but up-regulated the mRNA expression of tumor necrosis factor (Tnf), nitric oxide synthase 2 (Nos2), interleukin (Il)23a, and the secretion of Tnf-α and Il-23a. At the same time, genetic complementation experiment testified that Chek2 KD did not inhibit NF-κB and relevant inflammatory cytokines expression. Conclusion Chek2 functions through the kinase mechanism to down-regulate the NF-κB pathway in macrophages to alleviate S. aureus-induced pneumonia in mice.

Tissue Specific Transcriptome Changes Upon Influenza A Virus Replication in the Duck.

Ducks are the natural host and reservoir of influenza A virus (IAV), and as such are permissive to viral replication while being unharmed by most strains. It is not known which mechanisms of viral control are globally regulated during infection, and which are specific to tissues during infection. Here we compare transcript expression from tissues from Pekin ducks infected with a recombinant H5N1 strain A/Vietnam 1203/04 (VN1203) or an H5N2 strain A/British Columbia 500/05 using RNA-sequencing analysis and aligning reads to the NCBI assembly ZJU1.0 of the domestic duck (Anas platyrhynchos) genome. Highly pathogenic VN1203 replicated in lungs and showed systemic dissemination, while BC500, like most low pathogenic strains, replicated in the intestines. VN1203 infection induced robust differential expression of genes all three days post infection, while BC500 induced the greatest number of differentially expressed genes on day 2 post infection. While there were many genes globally upregulated in response to either VN1203 or BC500, tissue specific gene expression differences were observed. Lungs of ducks infected with VN1203 and intestines of birds infected with BC500, tissues important in influenza replication, showed highest upregulation of pattern recognition receptors and interferon stimulated genes early in the response. These tissues also appear to have specific downregulation of inflammatory components, with downregulation of distinct sets of proinflammatory cytokines in lung, and downregulation of key components of leukocyte recruitment and complement pathways in intestine. Our results suggest that global and tissue specific regulation patterns help the duck control viral replication as well as limit some inflammatory responses in tissues involved in replication to avoid damage.

Capsaicin Protects Against Lipopolysaccharide-Induced Acute Lung Injury Through the HMGB1/NF-κB and PI3K/AKT/mTOR Pathways.

PurposeCapsaicin (8-methyl-N-geranyl-6-nonamide; CAP) is an alkaloid isolated from chili peppers, which has complex pharmacological properties, including beneficial effects against various diseases. The aim of this study was to investigate the role of CAP in lipopolysaccharide (LPS)-induced acute lung injury (ALI), and the possible underlying mechanisms.Materials and MethodsALI was induced by intranasal administration of LPS (0.5 mg/kg), and CAP (1 mg/kg) injected intraperitoneally 3 days before exposure to LPS. Then, the histopathological changes were evaluated by hematoxylin and eosin staining. Enzyme-linked immunosorbent assay and qPCR were used to detect pro-inflammatory cytokines in serum and lung tissue. The expressions of HMGB1/NF-κB, PI3K/AKT/mTOR signaling pathways and apoptosis-associated molecules were determined by Western blot and/or qPCR. In addition, the lung cell apoptosis was analyzed by TUNEL staining, and the expression and location of cleaved caspase-3 were detected by immunofluorescence analysis.ResultsCAP pretreatment significantly protected mice from LPS-induced ALI, with reduced lung wet/dry weight ratio, lung histological damage, myeloperoxidase (MPO) activity, malondialdehyde (MDA) content and pro-inflammatory cytokine levels, and significant increased superoxide dismutase (SOD) activity. In addition, CAP pretreatment significantly inhibited the high-mobility group protein B1 (HMGB1) expression, nuclear factor-kappa B (NF-κB) activation, and the PI3K/AKT/mTOR signaling pathway. Furthermore, mice pre-treated with CAP exhibited reduced apoptosis of lung tissues, with associated down-regulation of caspase-3, cleaved caspase-3, and BAX expression, and up-regulation of BCL-2.ConclusionOur data demonstrate that CAP can protect against LPS-induced ALI by inhibiting oxidative stress, inflammatory responses and apoptosis through down-regulation of the HMGB1/NF-κB and PI3K/AKT/mTOR pathways.