Murine Alveolar Macrophages Research Articles

Isorhynchophylline exerts anti-inflammatory and anti-oxidative activities in LPS-stimulated murine alveolar macrophages

AimsExcessive inflammatory response and oxidative stress are considered as important pathogenic factors in the development of acute lung injury. Isorhynchophylline (IRN), a tetracyclic oxindole alkaloid isolated from Uncaria rhynchophylla, possesses anti-inflammatory and anti-oxidant activities. Our study aimed to investigate the effects and potential mechanisms of IRN on lipopolysaccharide (LPS)-stimulated murine alveolar macrophage cell lines MH-S and NR8383. Main methodsCCK-8 assay was used to evaluate the cytotoxicity of IRN and LPS. Inflammatory response was assessed by detecting the mRNA expressions and release of tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6, and plasminogen activator inhibitor-1 (PAI-1) using qRT-PCR and ELISA. The expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 were examined by qRT-PCR and western blot. Oxidative stress was evaluated by detecting malondialdehyde (MDA) level and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT). The changes of the toll like receptor (TLR4)/nuclear factor-kappa B (NF-κB)/nod-like receptor protein 3 (NLRP3) inflammasome pathway was detected by western blot. Key findingsTreatment with LPS or IRN for 24 h showed no cytotoxicity on MH-S and NR8383 cells. IRN pretreatment inhibited LPS-induced production of inflammatory cytokines, expressions of iNOS and COX-2, and oxidative stress in murine alveolar macrophages. Additionally, IRN inhibited LPS-induced activation of TLR4/NF-κB/NLRP3 inflammasome pathway in MH-S cells. Mechanistically, inhibition of TLR4/NF-κB/NLRP3 inflammasome pathway by si-TLR4 suppressed LPS-induced inflammation and oxidative stress in murine alveolar macrophages. SignificanceIRN exerted anti-inflammatory and anti-oxidant effects on LPS-stimulated murine alveolar macrophages via inhibition of the TLR4/NF-κB/NLRP3 inflammasome pathway.

3582 Scavenger Receptor Expression is Differentially Affected by DNAzyme-Gold Nanoparticle Conjugates

OBJECTIVES/SPECIFIC AIMS: Scavenger receptor (SR) surface proteins are highly conserved motifs and are implicated in the uptake of nanotherapies. Gold nanoparticles functionalized with DNAzymes (DzNP) represent a promising novel nanotherapy for lung diseases such as asthma, particularly because they can be delivered directly to the lung. Our lab has been studying the therapeutic potential of a DzNP targeting GATA-3, a master transcription factor regulating Th2 inflammation. Although nanoparticle uptake through scavenger receptors has been described in macrophages in other models, the role of SRs in DzNP uptake in the lung is poorly understood. We hypothesize that scavenger receptors mediate DzNP uptake in alveolar macrophages. To begin examining this hypothesis, we examined whether DzNP exposure and uptake regulates gene expression of MARCO and MSR1, two class A scavenger receptors. METHODS/STUDY POPULATION: Using a silver stain, we measured dose dependent DzNP uptake in murine alveolar macrophages (MH-S). Using qRT-PCR, we measured gene expression of scavenger receptors MSR1 and MARCO in murine alveolar macrophages (MH-S) and after 24 hour exposure to 2251 DzNP, a DzNP targeting GATA-3, and dextran sulfate sodium (DSS), a known SR-A blocker. RESULTS/ANTICIPATED RESULTS: 2251 DzNP uptake in alveolar macrophages is dose dependent. MARCO gene expression levels significantly increase in murine alveolar macrophages when cultured with increasing concentrations of 2251 DzNP (10 pM-2 nM) or DSS 25-200 ug/ml) for 24 hours. However, MSR1 gene expression levels have minimal change when exposed to low concentrations of 2251 DzNP and DSS. At higher concentrations of 2251 DzNP and DSS, MSR1 expression levels are decreased. DISCUSSION/SIGNIFICANCE OF IMPACT: Alveolar macrophages exhibit a dose dependent increase in MARCO gene expression levels with increasing concentrations of 2251 DzNP and DSS, but MSR1 gene expression is not affected in a similar fashion. 2251 DzNP-induced increases in MARCO gene expression suggests that 2251 DzNP may facilitate its own uptake through MARCO. 2251 DzNP exposure negatively regulates MSR1 expression at higher doses and suggests that 2251 DzNP may inhibit its own uptake thought MSR1.

Targeting murine alveolar macrophages by the intratracheal administration of locked nucleic acid containing antisense oligonucleotides

The pulmonary delivery of locked nucleic acid containing antisense oligonucleotides (LNA-ASOs) is expected to be a novel therapeutic approach for lung diseases. However, there are two concerns to be considered: immune responses, as the lung has a distinct immune mechanism to protect it from inhaled pathogens; and leakage into blood, since the lung is permeable to small molecules. As phagocytic alveolar macrophages reside in the alveolar space, it is hypothesized that inhaled LNA-ASOs effectively accumulate and exert a knockdown (KD) effect on these cells at low doses. Thus, targeting alveolar macrophages by inhaled LNA-ASOs may reduce these risks. To test this hypothesis, LNA-ASOs targeting Scarb1 or Hprt1 were intratracheally administered to mice. We confirmed the remarkable accumulation of intratracheally administered LNA-ASOs in murine alveolar macrophages and found that they exerted a significant and sequence-dependent KD effect. The dose required for KD in alveolar macrophages was lower than that required to induce KD in the whole lung. Furthermore, when a KD effect was observed in alveolar macrophages, no KD effect was observed in the liver or kidney; however, several inflammatory cytokines were increased in the lung. These results suggest the potential application of LNA-ASOs as an inhaled drug specific to alveolar macrophages. However, further studies on the immuno-stimulatory effects of LNA-ASOs will be necessary for the development of novel inhaled therapeutic agents.

Intracellular Invasion and Killing Assay to Investigate the Effectsof Binge Alcohol Toxicity in Murine Alveolar Macrophages.

Alcohol consumption has diverse and well-documented effects on the human immune system and its ability to defend against infective agents. While pulmonary related infections can occur in healthy humans, binge alcohol use is recognized as a major health risk factor (Nelson et al., 1991). Although binge alcohol consumption has been considered as a risk factor for the development of pulmonary infections, no experimental studies have investigated the outcomes of a single binge alcohol exposure during infection. A key assay to assess the effects of a single binge alcohol exposure on the interactions between bacteria and alveolar macrophage is a binge alcohol intracellular invasion and killing assay. MH-S alveolar macrophages (AMs) are exposed to a single binge alcohol dose prior to infection for 3 h. The macrophage monolayer is then infected to allow for engulfment, followed by removal of extracellular bacteria to assess the intracellular killing capacity of infected macrophages over time. We have utilized this assay to demonstrate that low alcohol exposure significantly suppressed the uptake and killing of less virulent Burkholderia thailandensis (B. thailandensis) by AMs. More recently we found that activated AMs with interferon (IFN)-γ incubated in alcohol (0.08%) for 3 h prior to infection showed significantly lower bacterial uptake at 2 and 8 h post infection, which lead to B. thailandensis survival and a ~2.5-fold replication increase compared to controls (Jimenez et al., 2017). These results provide insights into binge alcohol consumption, a culturally prevalent risk factor, as a predisposing factor for pulmonary bacterial infections. This assay can be adapted to other bacterial species and host cell types to assess tissue specific effects of alcohol during infection.

Isolation and Long-term Cultivation of Mouse Alveolar Macrophages.

Alveolar macrophages (AM) are tissue-resident macrophages that colonize the lung around birth and can self-maintain long-term in an adult organism without contribution of monocytes. AM are located in the pulmonary alveoli and can be harvested by washing the lungs using the method of bronchoalveolar lavage (BAL). Here, we compared different conditions of BAL to obtain high yields of murine AM for in vitro culture and expansion of AM. In addition, we describe specific culture conditions, under which AM proliferate long-term in liquid culture in the presence of granulocyte-macrophage colony-stimulating factor. This method can be used to obtain large numbers of AM for in vivo transplantation or for in vitro experiments with primary mouse macrophages.

MiR-146b overexpression ameliorates lipopolysaccharide-induced acute lung injury in vivo and in vitro.

Acute respiratory distress syndrome (ARDS) is a type of acute lung injury (ALI), which causes high morbidity and mortality. So far, effective clinical treatment of ARDS is still limited. Recently, miR-146b has been reported to play a key role in inflammation. In the present study, we evaluated the functional role of miR-146b in ARDS using the murine model of lipopolysaccharide (LPS)-induced ALI. The miR-146b expression could be induced by LPS stimulation, and miR-146b overexpression was required in the maintenance of body weight and survival of ALI mice; after miR-146b overexpression, LPS-induced lung injury, pulmonary inflammation, total cell and neutrophil counts, proinflammatory cytokines, and chemokines in bronchial alveolar lavage (BAL) fluid were significantly reduced. The promotive effect of LPS on lung permeability through increasing total protein, albumin and IgM in BAL fluid could be partially reversed by miR-146b overexpression. Moreover, in murine alveolar macrophages, miR-146b overexpression reduced LPS-induced TNF-α and interleukin (IL)-1β releasing. Taken together, we demonstrated that miR-146b overexpression could effectively improve the LPS-induced ALI; miR-146b is a promising target in ARDS treatment.

29 - Nrf2 independent heme oxygenase-1 induction by auranofin is in lung macrophages

Inhibition of thioredoxin reductase-1 (TXNRD1) activates nuclear factor (erythroid-derived 2)-like 2 (Nrf2) responses, attenuates experimental lung injury, and disproportionally increases heme oxygenase-1 (HO-1) levels. HO-1 has been investigated as a potential therapeutic target to prevent lung injury; however, the mechanisms by with TXNRD1 inhibition increases of HO-1 levels in macrophages is unclear. We recently demonstrated that the TXNRD1 inhibitor auranofin (AFN) induces HO-1 in lung epithelial cells via Nrf2-dependent mechanisms. TXNRD1 is abundantly expressed in both lung epithelia and macrophages. The present studies used murine alveolar macrophages (MH-S) to test the hypothesis that AFN-mediated HO-1 induction is Nrf2 dependent. Nrf2 expression was decreased in MH-S cells using siRNA. Knockdown was confirmed by qPCR and western blotting. Within 24 h of transfection, Nrf2 mRNA levels were 56% lower than in scramble siRNA transfected controls. In control cells, treatment with 0.5 µM AFN for 1 h decreased TXNRD1 activity by 90% and increased HO-1 mRNA levels by 2-fold when compared to vehicle-treated cells. In Nrf2 siRNA-treated cells, NQO1 mRNA levels were 80% less than in scramble siRNA transfected controls which is consistent with Nrf2 knockdown. Conversely, HO-1 mRNA levels were not different between scramble and Nrf2 siRNA-treated cells. Treatment with 0.5 µM AFN for 2h did not increase Nqo1 mRNA levels in Nrf2 siRNA-treated cells; however, HO-1 mRNA levels were 3-fold greater when compared to vehicle-treated knockdown cells. In conclusion, AFN induces HO-1 expression in MH-S cells which is consistent with our findings in previous studies. In contrast to lung epithelia, AFN-mediated HO-1 induction in lung macrophages is Nrf2 independent. The mechanisms by which TXNRD1 inhibition elicits HO-1 induction in alveolar macrophages are currently being investigated.

Modification of nano-silver bioactivity by adsorption on carbon nanotubes and graphene oxide

Objective: The toxicity of silver nanomaterials in various forms has been extensively evaluated, but the toxicity of silver nanocarbon composites is less well understood. Therefore, silver-carbon nanotube composites (Ag-MWCNT-COOH) and silver-graphene oxide composites (Ag-GO) were synthesized by microwave irradiation and evaluated in two in vitro cell models.Materials/Methods: Toxicity of silver nanosphere (Ag), Ag-MWCNT-COOH and Ag-GO were analyzed by MTS assay and LDH assay in primary C57BL/6 murine alveolar macrophages and human THP-1 cells. Activation of NLRP3 inflammasome by particle variants in these models was done by proxy using LPS co-culture and IL-1β release.Results: The results depended on the model, as the amount of Ag on the modified carbon resulted in slightly increased toxicity for the murine cells, but did not appear to affect toxicity in the human cell model. IL-1β release from carbon particle-exposures was decreased by the presence of Ag in both cell models. Suspensions of Ag-MWCNT-COOH, Ag-GO and Ag in artificial lysosomal fluid were prepared and ICP-MS was used to detect Ag ions concentration in three silver suspension/solutions. The amount of Ag ions released from Ag-MWCNT-COOH and Ag-GO were similar, which were both lower than that of Ag nanospheres.Conclusions: The results suggest the bioactivity of silver composites may be related to the amount of Ag ions released, which can be dependent on the cell model under investigation.

Metformin Targets Mitochondrial Electron Transport to Reduce Air-Pollution-Induced Thrombosis

Urban particulate matter air pollution induces the release of pro-inflammatory cytokines including interleukin-6 (IL-6) from alveolar macrophages, resulting in an increase in thrombosis. Here, we report that metformin provides protection in this murine model. Treatment of mice with metformin or exposure of murine or human alveolar macrophages to metformin prevented the particulate matter-induced generation of complex III mitochondrial reactive oxygen species, which were necessary for the opening of calcium release-activated channels (CRAC) and release of IL-6. Targeted genetic deletion of electron transport or CRAC channels in alveolar macrophages in mice prevented particulate matter-induced acceleration of arterial thrombosis. These findings suggest metformin as a potential therapy to prevent some of the premature deaths attributable to air pollution exposure worldwide.

Antimicrobial and immunomodulatory activity induced by loperamide in mycobacterial infections

Loperamide is an antidiarrheal drug that targets μ-opioid receptors and calcium channels. A previous report demonstrated that loperamide induces autophagy and enhances antimicrobial activity towards M. tuberculosis in murine and human alveolar macrophages. The aim of this study was to evaluate the immunomodulatory effects of loperamide on macrophages with respect to cytokine and antimicrobial peptide production during mycobacterial infection. We infected monocyte-derived macrophages (macrophages) with M. tuberculosis H37Rv at a multiplicity of infection (MOI) of 5 and treated the cells with 3 μM loperamide. Cytokine production in the supernatants of 24-h cultures and gene expression of the cytokines TNFα, IL1β and IL10 and the antimicrobial peptides LL37 and bactericidal/permeability increasing protein (BPI) in the cell lysates was measured. Intracellular bacterial loads were evaluated by enumerating colony-forming units 3 days posttreatment for M. tuberculosis and 24 h posttreatment for M. smegmatis. We observed that loperamide exerted an immunomodulatory effect on TNFα production in human macrophages infected with M. tuberculosis and that these responses were independent of the bacteria, as they also occurred when macrophages were infected with M. smegmatis and to a lesser extent with M. bovis. In addition, antibacterial mechanisms triggered by loperamide induced a significant reduction in bacterial load and an upregulation of BPI and LL37 gene expression. Thus, our results show that loperamide exerts immunomodulatory effects, which supports its use for additional medical conditions other than diarrhea.

Knockdown of LncRNA MALAT1 contributes to the suppression of inflammatory responses by up-regulating miR-146a in LPS-induced acute lung injury

ABSTRACTBackground: Acute lung injury (ALI) is a type of severe pulmonary inflammatory disease with high rates of morbidity and mortality. Now, an increasing number of studies suggest that lncRNAs may act as key regulators of the inflammatory response and play a crucial role in the pathogenesis of many inflammatory diseases. Our study firstly explored the function and underlying mechanism of lncRNA metastasis-associated lung adenocarcinoma transcription 1 (MALAT1) in regulating the inflammatory response of lipopolysaccharide (LPS)-induced ALI in rats. Methods: The ALI rats were constructed by intratracheal instillation with LPS. Hematoxylin and eosin (HE) for histological examination were performed to detect histopathological changes in the lung tissues. Enzyme-linked immunosorbent assay (ELISA) was used to determine the concentrations of cytokines TNF-α, IL-6, and IL-1β in the supernatants of the bronchoalveolar lavage fluid (BALF). Quantitative real-time PCR (qRT-PCR) analysis was employed to assess the expression of MALAT1, miR-146a, TNF-α, IL-6, and IL-1β in lung tissues. Luciferase reporter assay and RNA immunoprecipitation (RIP) assay were used to detect the relationship between MALAT1 and miR-146a. Results: The results revealed that MALAT1 knockdown played a protective role in the LPS-induced ALI rat model. In addition, knockdown of MALAT1 in vitro inhibited LPS-induced inflammatory response in murine alveolar macrophages cell line MH-S and murine alveolar epithelial cell line MLE-12. This study found that MALAT1 acts as a molecular sponge for miR-146a and MALAT1 negatively regulated miR-146a expression. Mechanistically, MALAT1 overexpression alleviated the inhibitory effect of miR-146a on LPS-induced inflammatory response in MH-S. Conclusions: Together, our study provided the first evidence that MALAT1 knockdown could suppress inflammatory response by up-regulating miR-146a in LPS-induced ALI, which provided a potential therapeutic target for the treatment of ALI.

Soluble mucus component CLCA1 modulates expression of leukotactic cytokines and BPIFA1 in murine alveolar macrophages but not in bone marrow-derived macrophages

The secreted airway mucus cell protein chloride channel regulator, calcium-activated 1, CLCA1, plays a role in inflammatory respiratory diseases via as yet unidentified pathways. For example, deficiency of CLCA1 in a mouse model of acute pneumonia resulted in reduced cytokine expression with less leukocyte recruitment and the human CLCA1 was shown to be capable of activating macrophages in vitro. Translation of experimental data between human and mouse models has proven problematic due to several CLCA species-specific differences. We therefore characterized activation of macrophages by CLCA1 in detail in solely murine ex vivo and in vitro models. Only alveolar but not bone marrow-derived macrophages freshly isolated from C57BL6/J mice increased their expression levels of several pro-inflammatory and leukotactic cytokines upon CLCA1 stimulation. Among the most strongly regulated genes, we identified the host-protective and immunomodulatory airway mucus component BPIFA1, previously unknown to be expressed by airway macrophages. Furthermore, evidence from an in vivo Staphylococcus aureus pneumonia mouse model suggests that CLCA1 may also modify BPIFA1 expression in airway epithelial cells. Our data underscore and specify the role of mouse CLCA1 in inflammatory airway disease to activate airway macrophages. In addition to its ability to upregulate cytokine expression which explains previous observations in the Clca1-deficient S. aureus pneumonia mouse model, modulation of BPIFA1 expression expands the role of CLCA1 in airway disease to involvement in more complex downstream pathways, possibly including liquid homeostasis, airway protection, and antimicrobial defense.

A role for heat shock factor 1 in hypercapnia-induced inhibition of inflammatory cytokine expression.

Hypercapnia, elevated levels of CO2 in the blood, is a known marker for poor clinical prognosis and is associated with increased mortality in patients hospitalized with both bacterial and viral pneumonias. Although studies have established a connection between elevated CO2 levels and poor pneumonia outcomes, a mechanistic basis of this association has not yet been established. We previously reported that hypercapnia inhibits expression of key NF-κB-regulated, innate immune cytokines, TNF-α, and IL-6, in LPS-stimulated macrophages in vitro and in mice during Pseudomonas pneumonia. The transcription factor heat shock factor 1 (HSF1) is important in maintaining proteostasis during stress and has been shown to negatively regulate NF-κB activity. In this study, we tested the hypothesis that HSF1 activation in response to hypercapnia results in attenuated NF-κB-regulated gene expression. We found that hypercapnia induced the protein expression and nuclear accumulation of HSF1 in primary murine alveolar macrophages and in an alveolar macrophage cell line (MH-S). In MH-S cells treated with short interfering RNA targeting Hsf1, LPS-induced IL-6 and TNF-α release were elevated during exposure to hypercapnia. Pseudomonas-infected Hsf1+/+ (wild-type) mice, maintained in a hypercapnic environment, showed lower levels of IL-6 and TNF-α in bronchoalveolar lavage fluid and IL-1β in lung tissue than did infected mice maintained in room air. In contrast, infected Hsf1+/- mice exposed to either hypercapnia or room air had similarly elevated levels of those cytokines. These results suggest that hypercapnia-mediated inhibition of NF-κB cytokine production is dependent on HSF1 expression and/or activation.-Lu, Z., Casalino-Matsuda, S. M., Nair, A., Buchbinder, A., Budinger, G. R. S., Sporn, P. H. S., Gates, K. L. A role for heat shock factor 1 in hypercapnia-induced inhibition of inflammatory cytokine expression.

Inhibition of Macrophage Complement Receptor CRIg by TRIM72 Polarizes Innate Immunity of the Lung.

The complement system plays a critical role in immune responses against pathogens. However, its identity and regulation in the lung are not fully understood. This study aimed to explore the role of tripartite motif protein (TRIM) 72 in regulating complement receptor (CR) of the Ig superfamily (CRIg) in alveolar macrophage (AM) and innate immunity of the lung. Imaging, absorbance quantification, and flow cytometry were used to evaluate in vitro and in vivo AM phagocytosis with normal, or altered, TRIM72 expression. Pulldown, coimmunoprecipitation, and gradient binding assays were applied to examine TRIM72 and CRIg interaction. A pneumonia model was established by intratracheal injection of Pseudomonas aeruginosa. Mortality, lung bacterial burden, and cytokine levels in BAL fluid and lung tissues were examined. Our data show that TRIM72 inhibited CR-mediated phagocytosis, and release of TRIM72 inhibition led to increased AM phagocytosis. Biochemical assays identified CRIg as a binding partner of TRIM72, and TRIM72 inhibited formation of the CRIg-phagosome. Genetic ablation of TRIM72 led to improved pathogen clearance, reduced cytokine storm, and improved survival in murine models of severe pneumonia, specificity of which was confirmed by adoptive transfer of wild-type or TRIM72KO AMs to AM-depleted TRIM72KO mice. TRIM72 overexpression promoted bacteria-induced NF-κB activation in murine alveolar macrophage cells. Our data revealed a quiescent, noninflammatory bacterial clearance mechanism in the lung via AM CRIg, which is suppressed by TRIM72. In vivo data suggest that targeted suppression of TRIM72 in AM may be an effective measure to treat fatal pulmonary bacterial infections.

Mesenchymal Stromal Cells Modulate Macrophages in Clinically Relevant Lung Injury Models by Extracellular Vesicle Mitochondrial Transfer.

Acute respiratory distress syndrome (ARDS) remains a major cause of respiratory failure in critically ill patients. Mesenchymal stromal cells (MSCs) are a promising candidate for a cell-based therapy. However, the mechanisms of MSCs' effects in ARDS are not well understood. In this study, we focused on the paracrine effect of MSCs on macrophage polarization and the role of extracellular vesicle (EV)-mediated mitochondrial transfer. To determine the effects of human MSCs on macrophage function in the ARDS environment and to elucidate the mechanisms of these effects. Human monocyte-derived macrophages (MDMs) were studied in noncontact coculture with human MSCs when stimulated with LPS or bronchoalveolar lavage fluid (BALF) from patients with ARDS. Murine alveolar macrophages (AMs) were cultured ex vivo with/without human MSC-derived EVs before adoptive transfer to LPS-injured mice. MSCs suppressed cytokine production, increased M2 macrophage marker expression, and augmented phagocytic capacity of human MDMs stimulated with LPS or ARDS BALF. These effects were partially mediated by CD44-expressing EVs. Adoptive transfer of AMs pretreated with MSC-derived EVs reduced inflammation and lung injury in LPS-injured mice. Inhibition of oxidative phosphorylation in MDMs prevented the modulatory effects of MSCs. Generating dysfunctional mitochondria in MSCs using rhodamine 6G pretreatment also abrogated these effects. In the ARDS environment, MSCs promote an antiinflammatory and highly phagocytic macrophage phenotype through EV-mediated mitochondrial transfer. MSC-induced changes in macrophage phenotype critically depend on enhancement of macrophage oxidative phosphorylation. AMs treated with MSC-derived EVs ameliorate lung injury in vivo.

Suppression of inflammatory and infection responses in lung macrophages by eucalyptus oil and its constituent 1,8-cineole: Role of pattern recognition receptors TREM-1 and NLRP3, the MAP kinase regulator MKP-1, and NFκB.

Eucalyptus oil (EO) used in traditional medicine continues to prove useful for aroma therapy in respiratory ailments; however, there is a paucity of information on its mechanism of action and active components. In this direction, we investigated EO and its dominant constituent 1,8–cineole (eucalyptol) using the murine lung alveolar macrophage (AM) cell line MH-S. In an LPS-induced AM inflammation model, pre-treatment with EO significantly reduced (P ≤0.01or 0.05) the pro-inflammatory mediators TNF-α, IL-1 (α and β), and NO, albeit at a variable rate and extent; 1,8-cineole diminished IL-1 and IL-6. In a mycobacterial-infection AM model, EO pre-treatment or post-treatment significantly enhanced (P ≤0.01) the phagocytic activity and pathogen clearance. 1,8-cineole also significantly enhanced the pathogen clearance though the phagocytic activity was not significantly altered. EO or 1,8-cineole pre-treatment attenuated LPS-induced inflammatory signaling pathways at various levels accompanied by diminished inflammatory response. Among the pattern recognition receptors (PRRs) involved in LPS signaling, the TREM pathway surface receptor (TREM-1) was significantly downregulated. Importantly, the pre-treatments significantly downregulated (P ≤0.01) the intracellular PRR receptor NLRP3 of the inflammasome, which is consistent with the decrease in IL-1β secretion. Of the shared downstream signaling cascade for these PRR pathways, there was significant attenuation of phosphorylation of the transcription factor NF-κB and p38 (but increased phosphorylation of the other two MAP kinases, ERK1/2 and JNK1/2). 1,8-cineole showed a similar general trend except for an opposite effect on NF-κB and JNK1/2. In this context, either pre-treatment caused a significant downregulation of MKP-1 phosphatase, a negative regulator of MAPKs. Collectively, our results demonstrate that the anti-inflammatory activity of EO and 1,8-cineole is modulated via selective downregulation of the PRR pathways, including PRR receptors (TREM-1 and NLRP3) and common downstream signaling cascade partners (NF-κB, MAPKs, MKP-1). To our knowledge, this is the first report on the modulatory role of TREM-1 and NLRP3 inflammasome pathways and the MAPK negative regulator MKP-1 in context of the anti-inflammatory potential of EO and its constituent 1,8-cineole.

Differential exposure and acute health impacts of inhaled solid-fuel emissions from rudimentary and advanced cookstoves in female CD-1 mice

BackgroundThere is an urgent need to provide access to cleaner end user energy technologies for the nearly 40% of the world's population who currently depend on rudimentary cooking and heating systems. Advanced cookstoves (CS) are designed to cut emissions and solid-fuel consumption, thus reducing adverse human health and environmental impacts. Study premiseWe hypothesized that, compared to a traditional (Tier 0) three-stone (3-S) fire, acute inhalation of solid-fuel emissions from advanced natural-draft (ND; Tier 2) or forced-draft (FD; Tier 3) stoves would reduce exposure biomarkers and lessen pulmonary and innate immune system health effects in exposed mice. ResultsAcross two simulated cooking cycles (duration ~ 3h), emitted particulate mass concentrations were reduced 80% and 62% by FD and ND stoves, respectively, compared to the 3-S fire; with corresponding decreases in particles visible within murine alveolar macrophages. Emitted carbon monoxide was reduced ~ 90% and ~ 60%, respectively. Only 3-S-fire-exposed mice had increased carboxyhemoglobin levels. Emitted volatile organic compounds were FD ≪ 3-S-fire ≤ ND stove; increased expression of genes involved in xenobiotic metabolism (COX-2, NQO1, CYP1a1) was detected only in ND- and 3-S-fire-exposed mice. Diminished macrophage phagocytosis was observed in the ND group. Lung glutathione was significantly depleted across all CS groups, however the FD group had the most severe, ongoing oxidative stress. ConclusionsThese results are consistent with reports associating exposure to solid fuel stove emissions with modulation of the innate immune system and increased susceptibility to infection. Lower respiratory infections continue to be a leading cause of death in low-income economies. Notably, 3-S-fire-exposed mice were the only group to develop acute lung injury, possibly because they inhaled the highest concentrations of hazardous air toxicants (e.g., 1,3-butadiene, toluene, benzene, acrolein) in association with the greatest number of particles, and particles with the highest % organic carbon. However, no Tier 0–3 ranked CS group was without some untoward health effect indicating that access to still cleaner, ideally renewable, energy technologies for cooking and heating is warranted.

Mycobacterium tuberculosis ESAT6 induces IFN-β gene expression in Macrophages via TLRs-mediated signaling

Mycobacterium tuberculosis is a highly virulent bacterium that causes tuberculosis. It infects about one third of the world’s population. Type I interferons (IFNs) play a detrimental role in host defense against M. tuberculosis infection. Proteins secreted by M. tuberculosis through ESX-1 secretion system contribute to type I IFNs production. However, the precise mechanism by which 6-kDa early secretory antigen target (ESAT6), one of ESX-1-mediated secretory proteins, induces type I IFNs production in host cells is currently unclear. Therefore, the objective of the present study was to determine the underlying molecular mechanism regulating ESAT6-mediated gene expression of IFN-β in macrophages. Recombinant ESAT6 produced from E. coli expression system induced IFN-β gene expression in various types of macrophages such as mouse bone marrow-derived macrophages (BMDMs), peritoneal macrophages, and MH-S cells (murine alveolar macrophage cell line). Deficiency of TLR4 and TRIF absolutely abrogated ESAT6-induced IFN-β gene expression. TLR2 and MyD88 were partially involved in IFN-β gene expression in response to low dose of ESAT6. Another recombinant ESAT6 produced from baculovirus system also upregulated IFN-β gene expression via TLR4-dependent pathway. Polymyxin B (PMB) treatment impaired LPS-induced IFN-β expression. However, IFN-β expression induced by ESAT6 was not influenced by PMB. This suggests that ESAT6-mediated IFN-β expression is not due to LPS contamination. Treatment with ESAT6 resulted in activation of TBK1 and IRF3 in macrophages. Such activation was abolished in TLR4- and TRIF-deficient cells. Moreover, inhibition of IRF3 and TBK1 suppressed IFN-β gene expression in response to ESAT6. Our results suggest that ESAT6 might contribute to virulence of M. tuberculosis by regulating type I IFNs production through TLR4-TRIF signaling pathway.

Modulation of alveolar macrophage innate response in proinflammatory-, pro-oxidant-, and infection- models by mint extract and chemical constituents: Role of MAPKs

There is a continuing need for discovering novel primary or adjunct therapeutic agents to treat inflammatory conditions and infections. Natural products have inspired the discovery of several modern therapeutics; however, there is a paucity of mechanistic information on their mode of action. This study investigated the therapeutic potential and mode of action of corn mint’s (Mentha arvensis) leaf extract (ME) in alveolar macrophages (AMs) challenged with model pro-inflammatory (LPS), pro- oxidant (LPS or H2O2), and infection (Mycobacterium) agents and contribution of its dominant constituents rosmarinic acid, l-menthol, and l-menthone. LPS-induced inflammatory response in the murine AM cell line MH-S was significantly reduced in terms of pro-inflammatory cytokines (TNF-α, IL-1α) and nitric oxide (NO) when pre- or post-treated with ME. The ME pretreatment of macrophages led to a significant increase (P≤0.05) in phagocytic activity toward Mycobacterium smegmatis and a greater pathogen clearance in 24h in both ME pre-treated (P≤0.05) and post-treated cells. Significant attenuation (P≤0.01) of reactive oxygen species (ROS) production in LPS- or H2O2-treated macrophages by pretreatment with whole mint extract (ME) was accounted for in part by the mint constituents rosmarinic acid and l-menthone. Attenuation of pro-inflammatory response by ME pretreatment coincided with the significant reduction in total and phosphorylated JNK1/2, decrease in total p38, and increase in phospho-ERK1/2 thereby implying a role of differential modulation of MAPKs. Taken together, the results demonstrate that corn mint leaf components cause potent anti-inflammatory, anti-oxidant, and anti–infection effects in AMs via suppression of the production of cytokines/soluble mediators and ROS and increased pathogen clearance, respectively. To our knowledge, this is the first report on the mode of action of corn mint targeting the alveolar macrophages and on the potential role of MAPKs in immunomodulation by this product.

Rhesus θ-Defensin-1 Attenuates Endotoxin-induced Acute Lung Injury by Inhibiting Proinflammatory Cytokines and Neutrophil Recruitment.

Acute lung injury (ALI) is a clinical syndrome characterized by acute respiratory failure and is associated with substantial morbidity and mortality. Rhesus θ-defensin (RTD)-1 is an antimicrobial peptide with immunomodulatory activity. As airway inflammation and neutrophil recruitment and activation are hallmarks of ALI, we evaluated the therapeutic efficacy of RTD-1 in preclinical models of the disease. We investigated the effect of RTD-1 on neutrophil chemotaxis and macrophage-driven pulmonary inflammation with human peripheral neutrophils and LPS-stimulated murine alveolar macrophage (denoted MH-S) cells. Treatment and prophylactic single escalating doses were administered subcutaneously in a well-established murine model of direct endotoxin-induced ALI. We assessed lung injury by histopathology, pulmonary edema, inflammatory cell recruitment, and inflammatory cytokines/chemokines in the BAL fluid. In vitro studies demonstrated that RTD-1 suppressed CXCL8-induced neutrophil chemotaxis, TNF-mediated neutrophil-endothelial cell adhesion, and proinflammatory cytokine release in activated murine alveolar immortalized macrophages (MH-S) cells. Treatment with RTD-1 significantly inhibited in vivo LPS-induced ALI by reducing pulmonary edema and histopathological changes. Treatment was associated with dose- and time-dependent inhibition of proinflammatory cytokines (TNF, IL-1β, and IL-6), peroxidase activity, and neutrophil recruitment into the airways. Antiinflammatory effects were demonstrated in animals receiving RTD-1 up to 12 hours after LPS challenge. Notably, subcutaneously administered RTD-1 demonstrates good peptide stability as demonstrated by the long in vivo half-life. Taken together, these studies demonstrate that RTD-1 is efficacious in an experimental model of ALI through inhibition of neutrophil chemotaxis and adhesion, and the attenuation of proinflammatory cytokines and gene expression from alveolar macrophages.