Lipopolysaccharide Pretreatment Research Articles

Lipopolysaccharide pretreatment increases the sensitivity of the TRPV1 channel and promotes an anti-inflammatory phenotype of capsaicin-activated macrophages

BackgroundThe transient receptor potential vanilloid 1 (TRPV1) is well-established in neuronal function, yet its role in immune reactions remains enigmatic. The conflicting data on its inflammatory role, suggesting both pro-inflammatory and anti-inflammatory effects upon TRPV1 stimulation in immune cells, adds complexity. To unravel TRPV1 immunomodulatory mechanisms, we investigated how the TRPV1 agonist capsaicin influences lipopolysaccharide (LPS)-induced pro-inflammatory macrophage phenotypes.ResultsChanges in the surface molecules, cytokine production, and signaling cascades linked to the phenotype of M1 or M2 macrophages of the J774 macrophage cell line and bone marrow-derived macrophages, treated with capsaicin before or after the LPS-induced inflammatory reaction were determined. The functional capacity of macrophages was also assessed by infecting the stimulated macrophages with the intracellular parasite Leishmania mexicana.ConclusionOur findings reveal that TRPV1 activation yields distinct macrophage responses influenced by the inflammatory context. LPS pre-treatment followed by capsaicin activation prompted increased calcium influx, accompanied by a shift toward an anti-inflammatory M2b-like polarization state.

Gastrodin regulates the expression of renin-angiotensin system-SIRT3 and proinflammatory mediators in reactive astrocytes via activated microglia.

Gastrodin, an anti-inflammatory herbal agent, is known to suppress microglia activation. Here, we investigated whether it would exert a similar effect in reactive astrocytes and whether it might act through the renin-angiotensin system (RAS) and sirtuin 3 (SIRT3). Angiotensinogen (ATO), angiotensin-converting enzyme (ACE), angiotensin II type 1 (AT1) and type 2 (AT2) receptor and SIRT3 expression was detected in TNC-1 astrocytes treated with BV-2 microglia conditioned medium (CM) with or without gastrodin and lipopolysaccharide (LPS) pre-treatment by RT-PCR, immunofluorescence and western blotting analysis. Expression of C3 (A1 astrocyte marker), S100A10 (A2 astrocyte marker), proinflammatory cytokines and neurotrophic factors was then evaluated. The results showed a significant increase of ATO, ACE, AT1, SIRT3, C3, proinflammatory cytokines and neurotrophic factors expression in TNC-1 astrocytes incubated in CM + LPS when compared with cells incubated in the CM, but AT2 and S100A10 expression was reduced. TNC-1 astrocytes responded vigorously to BV-2 CM treated with gastrodin + LPS as compared with the control. This was evident by the decreased expression of the abovementioned protein markers, except for AT2 and S100A10. Interestingly, SIRT3, IGF-1 and BDNF expression was enhanced, suggesting that gastrodin inhibited the expression of RAS and proinflammatory mediators but promoted the expression of neurotrophic factors. And gastrodin regulated the phenotypic changes of astrocytes through AT1. Additionally, azilsartan (a specific inhibitor of AT1) inhibited the expression of C3 and S100A10, which remained unaffected in gastrodin and azilsartan combination treatment. These findings provide evidence that gastrodin may have a therapeutic effect via regulating RAS-SIRT3.

Lipopolysaccharide-Educated Cancer-Associated Fibroblasts Facilitate Malignant Progression of Ovarian Cancer Cells via the NF-kB/IL-6/JAK2 Signal Transduction.

Gram-negative bacteria increase in ovarian cancer (OC) tissues, but its association with OC progression remains largely unknown. The present study aimed to investigate whether and how cancer-associated fibroblasts (CAFs) pretreated by the main components of bacterial outer membrane lipopolysaccharide (LPS) influence the malignancy of OC cells. Specifically, the culture medium of LPS-preconditioned CAFs (LPS-CM) further accelerated cell proliferation, colony formation and tumorigenesis of OC cells SKOV3 and HEY A8, compared with culture medium of CAFs. Next, we found that LPS pretreatment activated the nuclear factor-kappa B (NF-kB) pathway in CAFs to secret cytokines, including interleukin 1β (IL-1β), interleukin 6 (IL-6), vascular endothelial growth factor (VEGF), etc. Neutralization of IL-6 in LPS-CM abolished the promoting effect of LPS-CM on cell proliferation, survival and epithelial-mesenchymal transition (EMT) in SKOV3 and HEY A8 cells. Mechanistically, LPS-CM activated the Janus kinases 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway, while application with JAK2 inhibitor also reversed the promoting effect of LPS-CM on malignancy of OC cells. In summary, LPS-pretreated CAFs IL-6-dependently accelerate OC progression via activating the JAK2/STAT3 signal pathway, which enriches our understanding of the molecular mechanisms underlying ovaries-colonized gram-negative bacteria in OC progression.

Cryptotanshinone Inhibits Bladder Cancer Cell Malignant Progression in a Lipopolysaccharide-Induced Inflammatory Microenvironment through NLRP3 Inhibition

Background. Bladder cancer (BC) is one of the most common malignancies of the urogenital system. This study assessed the nucleotide-binding oligomerization domain and leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3) in BC as well as the effects of cryptotanshinone on changes in BC malignant behaviors and NLRP3 expression under a lipopolysaccharide (LPS)-induced inflammatory microenvironment. Methods. BC tissue specimens from 62 patients were collected for immunohistochemical detection of NLRP3 protein. BC and normal urothelial cell lines were cultured for the detection of NLRP3 mRNA and protein. Then, BC cells were pretreated with LPS to mimic the inflammatory tumor microenvironment. Next, these cells were incubated with a low or high dose of cryptotanshinone to assess its effects on tumor cell malignant behaviors as well as transfected with NLRP3 cDNA to confirm the role of NLRP3 in BC cells in vitro. Results. High NLRP3 expression was associated with larger tumor diameters (>2 cm), muscle invasion, and metastasis. The levels of NLRP3 mRNA and protein were greater in BC cells than in normal urothelial cells. LPS pretreatment significantly promoted NLRP3 and inflammatory cytokine expression in BC cells, and induced cell viability, migration, and invasion. However, cryptotanshinone was able to reduce the LPS-induced increase of NLRP3 and inflammatory cytokine expression as well as the BC cell malignant progression. NLRP3 overexpression using NLRP3 cDNA further promoted BC cell malignant progression after LPS stimulation and reversed cryptotanshinone-reduced LPS-induced BC cell malignant behaviors. Conclusion. NLRP3 might possess oncogenic activity in BC, and the antitumor activity of cryptotanshinone in BC in vitro might be related to its inhibition of NLRP3 expression.

Abstract 12972: Trained Innate Immunity Through Toll-Like Receptor 4/Interferon Signaling Establishes Cardioprotection in a Stress-Induced Cardiomyopathy Model

Introduction: Although extensively studied, the mechanisms responsible for the development of cardioprotection following preconditioning stimuli are not known. Our group showed that isoproterenol (ISO) mediated cardiac injury induces potent cardioprotection against a second ISO challenge for up to 5 weeks. The lack of an immune response after the second ISO dose and the durability of protection suggested that trained innate immunity might represent a novel mechanism for cardioprotection. Hypothesis: We hypothesized that ISO confers cardioprotection through trained immunity via Toll-like receptor 4 (TLR4) signaling that is activated by damage-associated molecular patterns released by ISO-induced cell necrosis. Methods: Wild-type C57BL/6J mice were intraperitoneally injected with TLR agonists or diluent (saline), and then 300 mg/kg ISO a week later. Mice were assessed before and after receiving ISO via serum cardiac troponin analysis, flow cytometry, and 2-D echocardiography. Results: The TLR4 agonist lipopolysaccharide (LPS), but not TLR1/2 or TLR3 agonists, induced cardioprotection as shown by decreased troponin I release (p<0.01), reduced cardiac neutrophil influx (p<0.05), and decreased left ventricle wall motion abnormalities (p<0.05) following ISO injury compared to saline treated controls . RNA sequencing identified interferon signaling as commonly upregulated (p<0.05) in mouse hearts a week after LPS or ISO pre-treatment. Blocking type I/II interferon receptors partially abolished LPS-induced cardioprotection, while pre-treatment with recombinant interferon β+γ conferred cardioprotection as shown by decreased troponin I release post-ISO (p<0.01). Given that β-glucan reverses the epigenetic tolerizing effects of LPS in immune cells, we treated mice with β-glucan following LPS pre-treatment and observed that the cardioprotection to ISO was abolished. Conclusions: Viewed together this study shows for the first time that TLR4/interferon signaling confers cardioprotection against ISO-induced injury. Our findings further suggest that this cardioprotective response may be secondary to TLR4 mediated epigenetic changes that dampen immune responses following tissue injury, consistent with trained innate immunity.

Dual-regulation by Cx32 in hepatocyte to trigger and worsen liver graft injury

Liver transplantation is the ultimate treatment option for end-stage liver failure. However, liver graft injury remains a challenge. This study aimed to investigate the role of connexin32 (Cx32) in liver graft injury and elucidate its mechanism of action. Through detecting liver graft samples from 6 patients, we observed that changes in the Cx32 level coincided with liver graft injury. Therefore, we established autologous orthotopic liver transplantation (AOLT) models using Cx32-knockout and wild-type mice and hypoxia/reoxygenation (H/R) and lipopolysaccharide (LPS) pretreatment models using alpha mouse liver 12 (AML12) cells, to explore Cx32 mechanisms in liver graft injury. Following in vivo and in vitro Cx32 knockout, oxidative stress and inflammatory response were inhibited through the regulation of PKC-α/NF-κB/NLRP3 and Nrf2/NOX4/ROS signaling pathways, thereby reducing Bak/Bax-related apoptosis and ameliorating liver graft injury. When the Cx32-based gap junction (GJ) was blocked with 2-aminoethoxydiphenyl borate (2-APB), ROS transfer was attenuated between neighboring cells, exacerbated oxidative stress and inflammatory response were prevented, and aggravation of liver graft injury was mitigated. These results highlight the dual regulation mechanism of Cx32 in liver graft injury. Through interaction with PKC-α, Cx32 regulated the NF-κB/NLRP3 and Nrf2/NOX4/ROS signaling pathways, thus directly triggering oxidative stress and inflammatory response. Simultaneously, mass-produced ROS were transferred to neighboring cells through Cx32 channels, for which oxidative stress and the inflammatory response were aggravated indirectly. Finally, Bak/Bax-related apoptosis was activated, thereby worsening liver graft injury. Our findings propose Cx32 as a dual mechanistic factor for oxidative stress and inflammatory signaling pathways in regulating cell apoptosis on liver graft injury, which suggests a promising therapeutic targets for liver graft injury.

Tolerized Microglia Protect Neurons Against Endotoxin-Induced TNF-α Production via an LBP-Dependent Intracellular p38 MAPK Signaling Pathway.

The development of microglial endotoxin tolerance (ET) is a critical event in protecting neurons against excessive immune responses when microglia are administered two consecutive lipopolysaccharide (LPS) challenges. However, the intrinsic mechanisms of microglia shape ET programs and protect neurons remain unclear. This study aimed to determine whether extracellular autocrine cascades or intracellular signaling pathways are involved in ET microglia-mediated tumor necrosis factor-alpha (TNF-α) reduction and neuroprotection. Neuron-glia cultures composed of astroglia, neurons, and microglia were performed in different conditions: with or without serum or LPS-binding proteins (LBP), along with an induction approach of ET. Enzyme-linked immunosorbent assay results revealed that LPS induced TNF-α tolerance of microglia in an LBP-dependent manner. Furthermore, we determined whether the early pro-inflammatory cytokines induced by LPS might contribute to the development of microglial ET. Our data showed that the neutralization of TNF-α using an anti-TNF-α antibody had no change in the TNF-α tolerance of microglia during the ET challenge. Furthermore, pre-incubation of TNF-α, interleukin-1 beta, and prostaglandin E2 failed to induce any TNF-α tolerance in microglia after LPS treatment. Moreover, using three specific chemical inhibitors that respectively blocked the activities of the mitogen-activated protein kinases (MAPKs) namely p38, c-Jun N-terminal kinase and extracellular signal-related kinases revealed that inhibition of p38 MAPK by SB203580 disrupted the tolerated microglia-mediated TNF-α reduction and neuroprotection. In summary, our findings demonstrated that the LPS pre-treatment immediately programmed the microglial ET to prevent endotoxin-induced TNF-α production and neuronal damage through the intracellular p38 MAPK signaling pathway.

Regulatory Effects of Three-Dimensional Cultured Lipopolysaccharide-Pretreated Periodontal Ligament Stem Cell-Derived Secretome on Macrophages.

Phenotypic transformation of macrophages plays important immune response roles in the occurrence, development and regression of periodontitis. Under inflammation or other environmental stimulation, mesenchymal stem cells (MSCs) exert immunomodulatory effects through their secretome. It has been found that secretome derived from lipopolysaccharide (LPS)-pretreated or three-dimensional (3D)-cultured MSCs significantly reduced inflammatory responses in inflammatory diseases, including periodontitis, by inducing M2 macrophage polarization. In this study, periodontal ligament stem cells (PDLSCs) pretreated with LPS were 3D cultured in hydrogel (termed SupraGel) for a certain period of time and the secretome was collected to explore its regulatory effects on macrophages. Expression changes of immune cytokines in the secretome were also examined to speculate on the regulatory mechanisms in macrophages. The results indicated that PDLSCs showed good viability in SupraGel and could be separated from the gel by adding PBS and centrifuging. The secretome derived from LPS-pretreated and/or 3D-cultured PDLSCs all inhibited the polarization of M1 macrophages, while the secretome derived from LPS-pretreated PDLSCs (regardless of 3D culture) had the ability to promote the polarization of M1 to M2 macrophages and the migration of macrophages. Cytokines involved in the production, migration and polarization of macrophages, as well as multiple growth factors, increased in the PDLSC-derived secretome after LPS pretreatment and/or 3D culture, which suggested that the secretome had the potential to regulate macrophages and promote tissue regeneration, and that it could be used in the treatment of inflammation-related diseases such as periodontitis in the future.

E. coli LPS/TLR4/NF-κB Signaling Pathway Regulates Th17/Treg Balance Mediating Inflammatory Responses in Oral Lichen Planus.

Oral lichen planus (OLP) is a chronic inflammatory autoimmune disease mediated by T cells. The imbalance of microflora has potential impacts on the onset and development of OLP, but the mechanism is still unclear. Here, we investigated the effects of Escherichia coli (E. coli) lipopolysaccharide (LPS) simulating the microbial enrichment state of OLP on T cell immune functions in vitro. Effect of E. coli LPS on the viability of T cell using CCK8 assay. After E. coli LPS pretreatment, the expression of the toll-like receptor 4 (TLR4), nuclear factor-kappa B p65 (NF-κB p65), cytokines, retinoic acid-related orphan receptor γt (RORγt), and forkhead box p3 (Foxp3) in the peripheral blood of OLP patients and normal controls (NC) were assessed using quantitative RT-PCR (qRT-PCR), western blot, and enzyme-linked immunosorbent assay (ELISA). Finally, Th17 and Treg cells were detected by flow cytometry. We found that the TLR4/NF-κB pathway was activated and the expression of interleukin (IL)-6 and IL-17 was increased in both groups after E. coli LPS stimulation. CC chemokine ligand (CCL)20 and CC chemokine receptor (CCR)4 expression was increased in OLP after E. coli LPS treatment, while no difference was found in CCR6 and CCL17 expression of both groups. Moreover, E. coli LPS treatment enhanced the proportion of Th17 cells, Th17/Treg ratio, and RORγt/Foxp3 ratio in OLP. In conclusion, E. coli LPS regulated Th17/Treg balance to mediate the inflammatory responses of OLP through the TLR4/NF-κB pathway in vitro, indicating that oral microbiota dysbiosis affected the chronic inflammatory state of OLP.

ATG2B upregulated in LPS-stimulated BMSCs-derived exosomes attenuates septic liver injury by inhibiting macrophage STING signaling.

Pretreated mesenchymal stem cells (MSCs)-derived exosomes have shown great potential in the treatment of various inflammatory diseases. Recent evidence suggests that macrophage stimulator of interferon genes (STING) signal activation plays a critical role in sepsis and septic liver injury. Here, we aimed to investigate the role and effects of lipopolysaccharide (LPS)-pretreated bone marrow mesenchymal stem cells (BMSCs)-derived exosomes (L-Exo) on macrophage STING signaling in septic liver injury. Exosomes were collected from the BMSCs medium via ultracentrifugation. Liver injury, intrahepatic inflammation, and the activation of macrophage STING signaling were analyzed. Mitophagy and the release of mitochondrial DNA (mtDNA) into the cytosol were investigated. Through in vivo and in vitro experiments, L-Exo could markedly attenuate cecal ligation and puncture-induced septic liver injury and inhibit macrophage STING signaling. Mechanistically, L-Exo inhibited macrophage STING signaling by enhancing mitophagy and inhibiting the release of mtDNA into the cytosol. Furthermore, autophagy-related protein 2 homolog B (ATG2B) may be a major factor involved in this effect of L-Exo. These findings reveal that macrophage STING signaling plays an important role in septic liver injury and may be a therapeutic target. In addition, LPS pretreatment is an effective and promising approach for optimizing the therapeutic efficacy of MSCs-derived exosomes in septic liver injury, providing new strategies for treatment.

Neuroprotective effect of lipopolysaccharides in a dual-hit rat pup model of preterm hypoxia–ischemia

The combination of lipopolysaccharide (LPS) and hypoxia–ischemia (HI) has been used to model the brain injury sustained by sick pre-term infants in order to study the pathological conditions of diffuse white matter injury, which is a major cause of preterm morbidity. Prior studies have shown that the timing and dose of LPS administration will determine whether the injury is reduced or exacerbated. Here we show that administering a single injection of LPS (0.1 mg/kg) to postnatal-day-2 rat pups 14 h before inducing HI effectively protects the brain from HI-associated damage. We show that the LPS-treated HI rat pups have significantly less histopathology compared to the saline-treated HI rat pups. Apoptotic deaths were dramatically curtailed in both the neocortex and white matter when evaluated at 2 days of recovery. Microglial activation was reduced when the percentage of CD68+/Iba1+ cells was quantified in the neocortex of the LPS-treated vs the saline-treated HI rat pups. One mechanism through which LPS pre-treatment appears to be preventing injury is through the AKT-endothelial nitric oxide synthase (eNOS) pathway as LPS induced an increase in both the expression and phosphorylation of eNOS. Altogether these data show that the neocortex, as well as the white matter sustain damage after HI at this timepoint in forebrain development and that acutely activating the immune system can protect the brain from brain injury.

LPS preconditioning of MSC-CM improves protection against hypoxia/reoxygenation-induced damage in H9c2 cells partly via HMGB1/Bach1 signalling.

Mesenchymal stem cell-derived conditioned medium (MSC-CM) improves cardiac function after myocardial infarction; however, this cardioprotective effect is moderate and transient. Lipopolysaccharide (LPS) pretreatment partially improves MSC-CM-mediated cardioprotective effects owing to the presence of paracrine factors. However, the mechanism underlying these improved effects remains unknown. To study the effect of LPS-pretreated MSC-CM on hypoxia/reoxygenation (H/R)-induced injury, MSCs were treated with or without LPS (400 ng/mL) for 48 h, and the supernatant was collected (MSC-CM). Subsequently, H9c2 cells were co-cultured with Nor-CM (CM derived from LPS-untreated MSCs) and LPS-CM (CM derived from LPS-pretreated MSCs) for 24 h and subjected to H/R. MSC-CM inhibited the progression of H/R-induced injury in H9c2 cells, and this protective effect was enhanced via LPS pretreatment as evidenced by the improved apoptosis assessment index (i.e. caspase-3 and B-cell lymphoma-2 [Bcl-2] expression) and decreased levels of lactic dehydrogenase (LDH) and cardiac troponin (cTn). In addition, the results of haematoxylin-eosin staining (H&E), transmission electron microscopy (TEM) and TdT-mediated dUTP nick-end labelling (TUNEL) validated that MSC-CM inhibited H/R-induced injury in H9c2 cardiomyocytes. LPS pretreatment downregulated the expression of high mobility group box-1 (HMGB1) and BTB and CNC homology-1 (Bach1) proteins in MSCs but upregulated the expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and insulin-like growth factor (IGF). HMGB1 knockdown (MSC/siHMGB1-CM) significantly decreased the expression of Bach1 and increased the expression of VEGF, HGF and IGF. Bach1 knockdown (MSC/siBach1-CM) did not alter the production of HMGB1 but increased the expression of VEGF and IGF. LPS pretreatment did not alter the expression of the paracrine factors VEGF and HGF in the MSC/siHMGB1 group but increased their expression in the MSC/siBach1 group. The myocyte anti-apoptotic effects of MSCs/siBach1-CM were similar to those of untreated MSCs, which were not enhanced by LPS. LPS-pretreated MSC-CM protects H9c2 cells against H/R-induced injury partly through the HMGB1/Bach1 signalling pathway.

Lipopolysaccharide pre-treatment prevents viral induced death by priming macrophages for a robust anti-viral immune response

Abstract Rationale Respiratory viruses are an increasing public health burden. Using Sendai virus (SeV, murine parainfluenza virus type 1), previous data indicate that pretreatment of C57BL6 wild-type (WT) mice with 0.1μg of lipopolysaccharide (LPS) prevented mortality from a normally lethal SeV infection. However, the mechanism by which LPS pre-treatment confers this survival advantage is unknown. LPS is a potent activator of macrophages. We hypothesized that LPS prevents viral induced death by stimulating the recruitment of macrophages, which could drive a more robust anti-viral immune response and subsequent survival. Therefore, we examined the change in lung macrophage subsets with LPS pretreatment of SeV infected mice. Methods WT mice were inoculated with 30 μL of 0.1 μg LPS or PBS i.n. 24 hours before SeV (2e6 pfu). After LPS sensitization, lungs were harvested at 6 hours, 24 hours, and days 1,3,5, and 7 post SeV infection (PI SeV); single lung cell suspensions isolated and stained for various innate immune cells by flow cytometry to assess the immune cells recruited by LPS. Results Mice pre-treated with LPS had a significant increase of alveolar macrophages (GR1- Siglec F+ CD11c+) at days 1 and 3 PI SeV (p=0.080 and p=0.0026, respectively; n≥6). Additionally, interstitial macrophages (GR1- Siglec F− CD11b+ CD11c+ F4/80+ MHCII+) were significantly increased at days 0 and 3 PI SeV (p=0.025 and p=0.019 respectively; n≥6). The LPS effect was no longer significant by day 5 PI SeV. Conclusion . LPS pretreatment of mice increases macrophages early during the SeV infection. Further studies will be needed to determine if the increase in these macrophages provides the protection from viral induced mortality seen in our model and the cytokines produced. This work was supported by the Abigail Wexner Research Institute at Nationwide Children’s Hospital (MHG).

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

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

Delayed Reperfusion of the Renal Outer Medulla Attenuates Ischemia Reperfusion‐Induced Vascular Congestion

Vascular congestion, or red blood cell (RBC) trapping, of the renal outer medulla (OM) is a common finding in acute tubular necrosis caused by ischemia reperfusion (IR). Work from our laboratory suggests that vascular congestion originates in the renal venous vasculature of the cortex and OM during the ischemic phase. Following reperfusion, RBCs then fill the OM plexus capillaries as the venous drainage vessels of this region remain blocked. We have also previously reported that pretreatment with low dose lipopolysaccharide (LPS) attenuates IR‐induced vascular congestion, however the mechanisms remain unknown. In the current study, we hypothesized that pretreatment with LPS prevents vascular congestion by delayed reperfusion of blood to the OM following ischemia.To test this hypothesis, WKY rats (male, 12 weeks) were pretreated (i.p) with 1mg/kg LPS (n=6) or saline control (n=7) once daily for 3 days. Rats were then anesthetized, and Transonic Laser Doppler probes were inserted in the renal cortex and OM. Doppler flux, as a determinate of regional kidney blood flow was then measured over 10 minutes of baseline, 45 minutes of renal artery clamping and 30 minutes of reperfusion.We found no differences in baseline blood flow of the cortex (pTREATMENT=0.0543) or OM (pTREATMENT=0.5085) between rats pretreated with low dose LPS or saline control. Following removal of the renal artery clamp, the return of blood flow to the cortex was gradual over the first several minutes of reperfusion (pTIME<0.0001), reaching a plateau following 10 minutes of reperfusion in both treatment groups (pINTERACTION=0.9999) (Fig. A). The reperfusion of blood to the OM, however, rapidly returned to baseline levels within 1 minute of reperfusion in control animals (1 min: 0 to 0.43AU) (Fig. B). In contrast, OM blood flow returned slowly in LPS treated rats (1 min: 0 to ‐0.08AU) and did not return to baseline levels during the 30‐minute reperfusion period (pINTERACTION=0.0127) (Fig. B).These data indicate that LPS pretreatment, paradoxically, delayed early reperfusion of the renal OM via a regional vasoconstriction response. We speculate that delayed reperfusion of the OM attenuates medullary plexus congestion by allowing time for the RBCs in the shared venous circulation to clear. These findings support the hypothesis that reperfusion of the renal outer medullary plexus before cortical perfusion is restored, is responsible for the development of prolonged vascular congestion. New therapeutics focused on renal hemodynamics to prevent congestion after ischemia may prevent much of the injury associated with acute kidney injury.

Microvesicle-Mediated Communication Within the Alveolar Space: Mechanisms of Uptake by Epithelial Cells and Alveolar Macrophages.

Intra-alveolar microvesicles (MVs) are important mediators of inter-cellular communication within the alveolar space, and are key components in the pathophysiology of lung inflammation such as acute respiratory distress syndrome (ARDS). Despite the abundance of data detailing the pro-inflammatory effects of MVs, it remains unclear how MVs interact or signal with target cells in the alveolus. Using both in vivo and in vitro alveolar models, we analyzed the dynamics of MV uptake by resident alveolar cells: alveolar macrophages and epithelial cells. Under resting conditions, the overwhelming majority of MVs were taken up by alveolar macrophages. However, following lipopolysaccharide (LPS)-mediated inflammation, epithelial cells internalized significantly more MVs (p<0.01) whilst alveolar macrophage internalization was significantly reduced (p<0.01). We found that alveolar macrophages adopted a pro-inflammatory phenotype after internalizing MVs under resting conditions, but reduction of MV uptake following LPS pre-treatment was associated with loss of inflammatory phenotype. Instead, MVs induced significant epithelial cell inflammation following LPS pre-treatment, when MV internalization was most significant. Using pharmacological inhibitors, we interrogated the mechanisms of MV internalization to identify which endocytic pathways and cell surface receptors are involved. We demonstrated that epithelial cells are exclusively dependent on the clathrin and caveolin dependent endocytotic pathway, whereas alveolar macrophage uptake may involve a significant phagocytic component. Furthermore, alveolar macrophages predominantly engulf MVs via scavenger receptors whilst, epithelial cells internalize MVs via a phosphatidylserine/integrin receptor mediated pathway (specifically alpha V beta III), which can be inhibited with phosphatidylserine-binding protein (i.e. annexin V). In summary, we have undertaken a comprehensive evaluation of MV internalization within the alveolar space. Our results demonstrate that different environmental conditions can modulate MV internalization, with inflammatory stimuli strongly enhancing epithelial cell uptake of MVs and inducing epithelial cell activation. Our data reveal the unique mechanisms by which alveolar macrophages and epithelial cells internalize MVs thereby elucidating how MVs exert their pathophysiological effect during lung inflammation and injury. As MVs are potential novel therapeutic targets in conditions such as ARDS, these data provide crucial insights into the dynamics of MV-target cell interactions and highlight potential avenues for researchers to modulate and inhibit their pro-inflammatory actions within the alveolar space.

Lipopolysaccharide Pretreatment Prevents Medullary Vascular Congestion following Renal Ischemia by Limiting Early Reperfusion of the Medullary Circulation.

Vascular congestion of the renal medulla-trapped red blood cells in the medullary microvasculature-is a hallmark finding at autopsy in patients with ischemic acute tubular necrosis. Despite this, the pathogenesis of vascular congestion is not well defined. In this study, to investigate the pathogenesis of vascular congestion and its role in promoting renal injury, we assessed renal vascular congestion and tubular injury after ischemia reperfusion in rats pretreated with low-dose LPS or saline (control). We used laser Doppler flowmetry to determine whether pretreatment with low-dose LPS prevented vascular congestion by altering renal hemodynamics during reperfusion. We found that vascular congestion originated during the ischemic period in the renal venous circulation. In control animals, the return of blood flow was followed by the development of congestion in the capillary plexus of the outer medulla and severe tubular injury early in reperfusion. Laser Doppler flowmetry indicated that blood flow returned rapidly to the medulla, several minutes before recovery of full cortical perfusion. In contrast, LPS pretreatment prevented both the formation of medullary congestion and its associated tubular injury. Laser Doppler flowmetry in LPS-pretreated rats suggested that limiting early reperfusion of the medulla facilitated this protective effect, because it allowed cortical perfusion to recover and clear congestion from the large cortical veins, which also drain the medulla. Blockage of the renal venous vessels and a mismatch in the timing of cortical and medullary reperfusion results in congestion of the outer medulla's capillary plexus and promotes early tubular injury after renal ischemia. These findings indicate that hemodynamics during reperfusion contribute to the renal medulla's susceptibility to ischemic injury.

'Candidatus Liberibacter asiaticus'-Encoded BCP Peroxiredoxin Suppresses Lipopolysaccharide-Mediated Defense Signaling and Nitrosative Stress In Planta.

The lipopolysaccharides (LPS) of gram-negative bacteria trigger a nitrosative and oxidative burst in both animals and plants during pathogen invasion. Liberibacter crescens strain BT-1 is a surrogate for functional genomic studies of the uncultured pathogenic 'Candidatus Liberibacter' spp. that are associated with severe diseases such as citrus greening and potato zebra chip. Structural determination of L. crescens LPS revealed the presence of a very long chain fatty acid modification. L. crescens LPS pretreatment suppressed growth of Xanthomonas perforans on nonhost tobacco (Nicotiana benthamiana) and X. citri subsp. citri on host orange (Citrus sinensis), confirming bioactivity of L. crescens LPS in activation of systemic acquired resistance (SAR). L. crescens LPS elicited a rapid burst of nitric oxide (NO) in suspension cultured tobacco cells. Pharmacological inhibitor assays confirmed that arginine-utilizing NO synthase (NOS) activity was the primary source of NO generation elicited by L. crescens LPS. LPS treatment also resulted in biological markers of NO-mediated SAR activation, including an increase in the glutathione pool, callose deposition, and activation of the salicylic acid and azelaic acid (AzA) signaling networks. Transient expression of 'Ca. L. asiaticus' bacterioferritin comigratory protein (BCP) peroxiredoxin in tobacco compromised AzA signaling, a prerequisite for LPS-triggered SAR. Western blot analyses revealed that 'Ca. L. asiaticus' BCP peroxiredoxin prevented peroxynitrite-mediated tyrosine nitration in tobacco. 'Ca. L. asiaticus' BCP peroxiredoxin (i) attenuates NO-mediated SAR signaling and (ii) scavenges peroxynitrite radicals, which would facilitate repetitive cycles of 'Ca. L. asiaticus' acquisition and transmission by fecund psyllids throughout the limited flush period in citrus.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Possible Cytoprotection of Low Dose Lipopolysaccharide in Rat Thioacetamide-Induced Liver Lesions, Focusing on the Analyses of Hepatic Macrophages and Autophagy.

Lipopolysaccharide (LPS) may influence hepatic macrophages and autophagy. We evaluated the potential participation of macrophages and autophagosomes in thioacetamide (TAA)-induced rat liver injury under pretreatment of a low dose LPS (0.1 mg/kg BW, intraperitoneally; nonhepatotoxic dose). F344 rats were pretreated with LPS (LPS + TAA) or saline (TAA alone) at 24 hours before TAA injection (100 mg/kg BW, intraperitoneally); rats were examined on Days 0 (controls), 1, 2, and 3 after TAA injection. Data were compared between TAA alone and LPS + TAA rats. LPS pretreatment significantly reduced TAA-induced hepatic lesion (centrilobular necrosis with inflammation) on Days 1 and 2, being reflected by declined hepatic enzyme values and decreased number of apoptotic cells. LC3B-immunoreacting autophagosomes (as cytoplasmic fine granules) were significantly increased on Days 1 and 2 in hepatocytes of LPS + TAA rats. In LPS + TAA rats, hepatic macrophages reacting to CD68, CD163, and MHC class II mainly on Day 2 and mRNA levels of macrophage-related factors (MCP-1, IL-1β, and IL-4) on Day 1 were significantly decreased. Collectively, the low-dose LPS pretreatment might act as cytoprotection against TAA-induced hepatotoxicity through increased autophagosomes and decreased hepatic macrophages, although the dose/time-dependent cytoprotection of LPS should be further investigated at molecular levels.

Lipopolysaccharide-Preconditioned Dental Follicle Stem Cells Derived Small Extracellular Vesicles Treating Periodontitis via Reactive Oxygen Species/Mitogen-Activated Protein Kinase Signaling-Mediated Antioxidant Effect.

PurposeLipopolysaccharide (LPS) pretreatment can enhance the therapeutic effect of dental follicle stem cells-derived small extracellular vesicles (DFC-sEV) for periodontitis, and this study aimed to investigate the underlying mechanisms and clinical application Of LPS-preconditioned DFC-sEV in periodontitis.MethodsThe protein spectrum of DFC-sEV before and after LPS pretreatment was determined by liquid chromatography-tandem mass spectrometry and bioinformatic analysis. Their effects on inflammatory periodontal ligament stem cells (PDLSCs) and macrophages were investigated for cell proliferation, migration, type 2 macrophage (M2) polarization, and intracellular reactive oxygen species (ROS) levels separately. In addition, the regulation of ROS/Jun amino-terminal kinases (JNK) and ROS/extracellular signal-related kinases (ERK) signaling by LPS-preconditioned DFC-sEV was also studied to reveal the antioxidant mechanism. In vivo, two kinds of DFC-sEV loaded with 0.2% hyaluronic acid (HA) gel were applied for canine periodontitis to evaluate the therapeutic potential.ResultsThe proteomic analysis showed that thirty-eight proteins were differentially expressed in LPS-preconditioned DFC-sEV, and interestingly, the highly expressed proteins were mainly involved in antioxidant and enzyme-regulating activities. In addition to promoting PDLSCs and macrophage proliferation, LPS-preconditioned DFC-sEV inhibited intracellular ROS as an antioxidant. It reduced the RANKL/OPG ratio of PDLSCs by inhibiting ROS/JNK signaling under inflammatory conditions and promoted macrophages to polarize toward the M2 phenotype via ROS/ERK signaling. Furthermore, LPS-preconditioned DFC-sEV loaded with the HA injectable system could sustainably release sEV and enhance the therapeutic efficacy for periodontitis in canines.ConclusionLPS-preconditioned DFC-sEV could be effectively used as an auxiliary method for periodontitis treatment via antioxidant effects in a subgingival environment, and loading it with HA is feasible and effective for clinical applications.