Comparison of the effect of recombinant bovine wild and mutant lipopolysaccharide-binding protein in lipopolysaccharide-challenged bovine mammary epithelial cells

Lipopolysaccharide (LPS)-binding protein (LBP) plays a crucial role in the recognition of bacterial components, such as LPS that causes an immune response. The aim of this study was to compare the different effects of recombinant bovine wild LBP and mutant LBP (67 Ala?→?Thr) on the LPS-induced inflammatory response of bovine mammary epithelial cells (BMECs). When BMECs were treated with various concentrations of recombinant bovine lipopolysaccharide-binding protein (RBLBP) (1, 5, 10, and 15 μg/mL) for 12 h, RBLBP of 5 μg/mL increased the apoptosis of BMECs induced by LPS without cytotoxicity, and mutant LBP resulted in a higher cell apoptosis than wild LBP did. By gene-chip microarray and bioinformatics, the data identified 2306 differentially expressed genes that were changed significantly between the LPS-induced inflamed BMECs treated with 5 μg/mL of mutant LBP and the BMECs only treated with 10 μg/mL of LPS (fold change ≥2). Meanwhile, 1585 genes were differently expressed between the inflamed BMECs treated with 5 μg/mL of wild LBP and 10 μg/mL of LPS-treated BMECs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that these differentially expressed genes were involved in different pathways that regulate the inflammation response. It predicted that carriers of this mutation increase the risk for a more severe inflammatory response. Our study provides an overview of the gene expression profile between wild LBP and mutant LBP on the LPS-induced inflammatory response of BMECs, which will lead to further understanding of the potential effects of LBP mutations on bovine mammary glands.     

Heat stress prevents lipopolysaccharide-induced apoptosis in pulmonary microvascular endothelial cells by blocking calpain/p38 MAPK signalling

Pulmonary microvascular endothelial cells (PMECs) injury including apoptosis plays an important role in the pathogenesis of acute lung injury during sepsis. Our recent study has demonstrated that calpain activation contributes to apoptosis in PMECs under septic conditions. This study investigated how calpain activation mediated apoptosis and whether heat stress regulated calpain activation in lipopolysaccharides (LPS)-stimulated PMECs. In cultured mouse primary PMECs, incubation with LPS (1 μg/ml, 24 h) increased active caspase-3 fragments and DNA fragmentation, indicative of apoptosis. These effects of LPS were abrogated by pre-treatment with heat stress (43 °C for 2 h). LPS also induced calpain activation and increased phosphorylation of p38 MAPK. Inhibition of calpain and p38 MAPK prevented apoptosis induced by LPS. Furthermore, inhibition of calpain blocked p38 MAPK phosphorylation in LPS-stimulated PMECs. Notably, heat stress decreased the protein levels of calpain-1/2 and calpain activities, and blocked p38 MAPK phosphorylation in response to LPS. Additionally, forced up-regulation of calpain-1 or calpain-2 sufficiently induced p38 MAPK phosphorylation and apoptosis in PMECs, both of which were inhibited by heat stress. In conclusion, heat stress prevents LPS-induced apoptosis in PMECs. This effect of heat stress is associated with down-regulation of calpain expression and activation, and subsequent blockage of p38 MAPK activation in response to LPS. Thus, blocking calpain/p38 MAPK pathway may be a novel mechanism underlying heat stress-mediated inhibition of apoptosis in LPS-stimulated endothelial cells.     

Effect of N-salicyloyltryptamine (STP), a novel tryptamine analogue, on parameters of cell viability, oxidative stress, and immunomodulation in RAW 264.7 macrophages

Immunomodulatory actions exerted by some classes of tryptamines, such as benzoyltryptamine analogues, suggest these molecules as promising candidates to develop new therapies to treat conditions associated to acute and chronic pain and inflammation. N-salicyloyltryptamine (STP) was observed to act as an anticonvulsive agent and exert antinociceptive effects in mouse. In the present work, we performed a screening of cytotoxic, cytoprotective, immunomodulatory, and redox properties of STP in RAW 264.7 macrophages challenged with hydrogen peroxide and LPS. Our results show that STP presents no cytotoxicity in the range of 0.001 to 1 μg/mL, but doses of 50 and 100 μg/mL caused loss of cell viability (IC₅₀?=?22.75 μg/mL). Similarly, STP at 0.001 to 1 μg/mL did not cause oxidative stress to RAW 264.7 cells, although it did not prevent cell death induced by H₂O₂ 0.5 mM. At 1 μg/mL, STP reversed some redox and inflammatory parameters induced by LPS. These include thiol (sulfhydryl) oxidation, superoxide dismutase activation, and morphological changes associated to macrophage activation. Besides, STP significantly inhibited LPS-induced TNF-α and IL-1β release, as well as CD40 and TNF-α protein upregulation. Signaling events induced by LPS, such as phosphorylation of ERK 1/2 and IκBα and p65 nuclear translocation (NF-kB activation) were also inhibited by STP. These data indicate that STP is able to modulate inflammatory parameters at doses that do not interfere in cell viability.     

Humanin Protects Cortical Neurons from Ischemia and Reperfusion Injury by the Increased Activity of Superoxide Dismutase

The neuroprotective effects of superoxide dismutase (SOD) against hypoxia/reperfusion (I/R) injury and of humanin (HN) against toxicity by familial amyotrophic lateral sclerosis (ALS)-related mutant SOD led us to hypothesize that HN might have a role to increase the activity of SOD, which might be involved in the protective effects of HN on neuron against Alzheimer’s disease-unrelated neurotoxicities. In the present study, we found that 4 h ischemia and 24 h reperfusion induced a significant increase in lactate dehydrogenase (LDH) release, malondialdehyde (MDA) formation and the number of karyopyknotic nuclei (4′,6-diamidino-2-phenylindole dihydrochloride nuclear dyeing) and a decrease in the number of Calcein-AM-positive living cells and cell viability. Pretreatment of the cells with HN led to a significant decrease in LDH release, MDA formation and the number of karyopyknotic nuclei, and an increase in the number of Calcein-AM-positive living cells and cell viability in neurons treated with I/R. We also found a significant decrease in SOD activity in neurons treated with I/R only, while pre-treatment with HN before I/R induced a significant increase in the activity of SOD as compared with the I/R group. Our findings implied that HN protects cortical neurons from I/R injury by the increased SOD activity and that the protective effect of HN on neurons against I/R is concentration-dependent.     

Lipopolysaccharide-Induced Apoptosis of Astrocytes: Therapeutic Intervention by Minocycline

Astrocytes are most abundant glial cell type in the brain and play a main defensive role in central nervous system against glutamate-induced toxicity by virtue of numerous transporters residing in their membranes and an astrocyte-specific enzyme glutamine synthetase (GS). In view of that, a dysregulation in the astrocytic activity following an insult may result in glutamate-mediated toxicity accompanied with astrocyte and microglial activation. The present study suggests that the lipopolysaccharide (LPS)-induced inflammation results in significant astrocytic apoptosis compared to other cell types in hippocampus and minocycline could not efficiently restrict the glutamate-mediated toxicity and apoptosis of astrocytes. Upon LPS exposure 76 % astrocytes undergo degeneration followed by 44 % oligodendrocytes, 26 % neurons and 10 % microglia. The pronounced astrocytic apoptosis resulted from the LPS-induced glutamate excitotoxicity leading to their hyperactivation as evident from their hypertrophied morphology, glutamate transporter 1 upregulation and downregulation of GS. Therapeutic minocycline treatment to LPS-infused rats efficiently restricted the inflammatory response and degeneration of other cell types but could not significantly combat with the apoptosis of astrocytes. Our study demonstrates a novel finding on cellular degeneration in the hippocampus revealing more of astrocytic death and suggests a more careful consideration on the protective efficacy of minocycline.     

The activation of HMGB1 as a progression factor on inflammation response in normal human bronchial epithelial cells through RAGE/JNK/NF-κB pathway

Extracellular high-mobility group box-1 (HMGB-1) has been implicated in the inflammation response leading to the precancerous lesions of non-small cell lung cancer (NSCLC). However, the role of HMGB-1 in the inflammation response in normal human bronchial epithelial (NHBE) cells and its underlying mechanisms were still not fully understood. In this study, the inflammation response in NHBE cells was stimulated by 2.5, 5, and 10 μg/ml HMGB-1. However, the receptor for advanced glycation end products (RAGE) blocker RAGE-Ab (5 μg/ml) or 10 μM c-Jun N-terminal kinases (JNK) inhibitor SP600125 could inhibit HMGB1-induced the release of inflammation cytokines including TNF-α, IL-8, IL-10, and MCP-1 in a dose-dependent manner. Furthermore, HMGB1-induced RAGE protein expression, JNK and NF-κB activation were attenuated by the pretreatment with RAGE-Ab or JNK inhibitor SP600125 in Western blot analysis. Our data indicated that HMGB-1 induced inflammation response in NHBE cells through activating RAGE/JNK/NF-κB pathway. HMGB-1 could act as a therapeutic target for inflammation leading NHBE cells to the precancerous lesions of NSCLC.     

Sphingosine kinase 1 deficiency exacerbates LPS-induced neuroinflammation

The pathogenesis of inflammation in the central nervous system (CNS), which contributes to numerous neurodegenerative diseases and results in encephalopathy and neuroinflammation, is poorly understood. Sphingolipid metabolism plays a crucial role in maintaining cellular processes in the CNS, and thus mediates the various pathological consequences of inflammation. For a better understanding of the role of sphingosine kinase activation during neuroinflammation, we developed a bacterial lipopolysaccharide (LPS)-induced brain injury model. The onset of the inflammatory response was observed beginning 4 hours after intracerebral injection of LPS into the lateral ventricles of the brain. A comparison of established neuroinflammatory parameters such as white matter rarefactions, development of cytotoxic edema, astrogliosis, loss of oligodendrocytes, and major cytokines levels in wild type and knockout mice suggested that the neuroinflammatory response in SphK1-/- mice was significantly upregulated. At 6 hours after intracerebroventricular injection of LPS in SphK1-/- mice, the immunoreactivity of the microglia markers and astrocyte marker glial fibrillary acidic protein (GFAP) were significantly increased, while the oligodendrocyte marker O4 was decreased compared to WT mice. Furthermore, western blotting data showed increased levels of GFAP. These results suggest that SphK1 activation is involved in the regulation of LPS induced brain injury. RESEARCH HIGHLIGHTS: ? Lipopolysaccharide (LPS) intracerebral injection induces severe neuroinflammation. ? Sphingosine kinase 1 deletion worsens the effect of the LPS. ? Overexpression of SphK1 might be a potential new treatment approach to neuroinflammation.     

Serum amyloid A promotes LPS clearance and suppresses LPS‐induced inflammation and tissue injury

Lipopolysaccharide (LPS) is a major microbial mediator for tissue injury and sepsis resulting from Gram‐negative bacterial infection. LPS is an external factor that induces robust expression of serum amyloid A (SAA), a major constituent of the acute‐phase proteins, but the relationship between SAA expression and LPS‐induced tissue injury remains unclear. Here, we report that mice with inducible transgenic expression of human SAA1 are partially protected against inflammatory response and lung injury caused by LPS and cecal ligation and puncture (CLP). In comparison, transgenic SAA1 does not attenuate TNFα‐induced lung inflammation and injury. The SAA1 expression level correlates inversely with the endotoxin concentrations in serum and lung tissues since SAA1 binds directly to LPS to form a complex that promotes LPS uptake by macrophages. Disruption of the SAA1‐LPS interaction with a SAA1‐derived peptide partially reduces the protective effect and exacerbates inflammation. These findings demonstrate that acute‐phase SAA provides innate feedback protection against LPS‐induced inflammation and tissue injury.     

ST2 protein induced by inflammatory stimuli can modulate acute lung inflammation

We have investigated gene and protein expression of ST2/ST2L in a murine alveolar macrophage (AM) cell line, MH-S, reacting to inflammatory stimuli in vitro and in the lung tissue of an acute lung injury model in vivo. We have also analyzed the effect of soluble ST2 protein on inflammatory response of MH-S cells. Lipopolysaccharide (LPS) and proinflammatory cytokines such as IL-1beta, IL-6, and TNF-alpha induced ST2 mRNA expression in MH-S cells. In an acute lung injury model, protein and mRNA expression levels of ST2 increased to the maximal level at 24-72h after the LPS challenge. Furthermore, pretreatment with ST2 protein significantly reduced the protein production and gene expression of IL-1alpha, IL-6, and TNF-alpha in LPS-stimulated MH-S cells in vitro. These results suggest that increases in endogenous ST2 protein in AM, which is induced by inflammatory stimuli, such as LPS and proinflammatory cytokines, may modulate acute lung inflammation.     

Adrenaline stimulates the proliferation and migration of mesenchymal stem cells towards the LPS‐induced lung injury

Bone marrow‐derived mesenchymal stem cells (BMSCs) could modulate inflammation in experimental lung injury. On the other hand, adrenergic receptor agonists could increase DNA synthesis of stem cells. Therefore, we investigated the therapeutic role of adrenaline‐stimulated BMSCs on lipopolysaccharide (LPS)‐induced lung injury. BMSCs were cultured with adrenergic receptor agonists or antagonists. Suspensions of lung cells or sliced lung tissue from animals with or without LPS‐induced injury were co‐cultured with BMSCs. LPS‐stimulated alveolar macrophages were co‐cultured with BMSCs (with adrenaline stimulation or not) in Transwell for 6 hrs. A preliminary animal experiment was conducted to validate the findings in ex vivo study. We found that adrenaline at 10 μM enhanced proliferation of BMSCs through both α‐ and β‐adrenergic receptors. Adrenaline promoted the migration of BMSCs towards LPS‐injured lung cells or lung tissue. Adrenaline‐stimulated BMSCs decreased the inflammation of LPS‐stimulated macrophages, probably through the expression and secretion of several paracrine factors. Adrenaline reduced the extent of injury in LPS‐injured rats. Our data indicate that adrenaline‐stimulated BMSCs might contribute to the prevention from acute lung injury through the activation of adrenergic receptors, promotion of proliferation and migration towards injured lung, and modulation of inflammation.     

Map kinase phosphatase 5 protects against sepsis-induced acute lung injury

Mitogen-activated protein kinases (MAPKs) play a critical role in inflammation. Although activation of MAPK in inflammatory cells has been studied extensively, much less is known about the inactivation of these kinases. MAPK phosphatase 5 (MKP5) is a member of the dual-specificity phosphatase family that dephosphorylates activated MAPKs. Here we report that MKP5 protects sepsis-induced acute lung injury. Mice lacking MKP5 displayed severe lung tissue damage following LPS challenge, characterized with increased neutrophil infiltration and edema compared with wild-type (WT) controls. In response to LPS, MKP5-deficient macrophages produced significantly more inflammatory factors including inflammatory cytokines, nitric oxide, and superoxide. Phosphorylation of p38 MAPK, JNK, and ERK were enhanced in MKP5-deficient macrophages upon LPS stimulation. Adoptive transfer of MKP5-deficient macrophages led to more severe lung inflammation than transfer of WT macrophages, suggesting that MKP5-deficient macrophages directly contribute to acute lung injury. Taken together, these results suggest that MKP5 is crucial to homeostatic regulation of MAPK activation in inflammatory responses.     

Species-specific microRNA roles elucidated following astrocyte activation

MicroRNAs (miRNAs) are short non-coding RNAs that play a central role in regulation of gene expression by binding to target genes. Many miRNAs were associated with the function of the central nervous system (CNS) in health and disease. Astrocytes are the CNS most abundant glia cells, providing support by maintaining homeostasis and by regulating neuronal signaling, survival and synaptic plasticity. Astrocytes play a key role in repair of brain insults, as part of local immune reactivity triggered by inflammatory or pathological conditions. Thus, astrocyte activation, or astrogliosis, is an important outcome of the innate immune response, which can be elicited by endotoxins such as lipopolysaccharide (LPS) and cytokines such as interferon-gamma (IFN-γ). The involvement of miRNAs in inflammation and stress led us to hypothesize that astrogliosis is mediated by miRNA function. In this study, we compared the miRNA regulatory layer expressed in primary cultured astrocyte derived from rodents (mice) and primates (marmosets) brains upon exposure to LPS and IFN-γ. We identified subsets of differentially expressed miRNAs some of which are shared with other immunological related systems while others, surprisingly, are mouse and rat specific. Of interest, these specific miRNAs regulate genes involved in the tumor necrosis factor-alpha (TNF-α) signaling pathway, indicating a miRNA-based species-specific regulation. Our data suggests that miRNA function is more significant in the mechanisms governing astrocyte activation in rodents compared to primates.     

MiR‐643 inhibits lipopolysaccharide‐induced endometritis progression by targeting TRAF6

Endometritis is a prevalent disease with inflammation of uterus endangering women reproductive health. MicroRNAs (miRNAs) play important roles in inflammatory disorders, including endometritis. However, the role and mechanism of miR‐643 in endometritis development remain unclear. This study aimed to investigate the effect of miR‐643 on lipopolysaccharide (LPS)‐induced inflammatory response and clarify the potential mechanism. LPS‐treated human endometrial epithelial cells (HEECs) were cultured to investigate the role of miR‐643 in vitro. The expression levels of miR‐643 and tumor necrosis factor receptor‐associated factor 6 (TRAF6) were measured via quantitative real‐time polymerase chain reaction and western blot, respectively. LPS‐induced inflammatory response was assessed by inflammatory cytokines secretion via enzyme‐linked immunosorbent assay. The activation of nuclear factor‐κB (NF‐κB) pathway was investigated by western blot. The interaction between miR‐643 and TRAF6 was validated by bioinformatics analysis, luciferase reporter assay, and RNA immunoprecipitation. The expression of miR‐643 was decreased and TRAF6 protein level was enhanced in LPS‐treated HEECs. The overexpression of miR‐643 suppressed LPS‐induced secretion of inflammatory cytokines (tumor necrosis factor‐α, interleukin‐1β [IL‐1β], and IL‐6) and activation of NF‐κB pathway. The knockdown of TRAF6 inhibited LPS‐induced inflammatory response in HEECs. TRAF6 was validated as a target of miR‐643 and TRAF6 restoration reversed the effect of miR‐643 on inflammation response in LPS‐treated HEECs. Collectively, miR‐643 attenuated LPS‐induced inflammatory response by targeting TRAF6, indicating a novel avenue for the treatment of endometritis.     

Airway epithelium controls lung inflammation and injury through the NF-kappa B pathway

Although airway epithelial cells provide important barrier and host defense functions, a crucial role for these cells in development of acute lung inflammation and injury has not been elucidated. We investigated whether NF-kappaB pathway signaling in airway epithelium could decisively impact inflammatory phenotypes in the lungs by using a tetracycline-inducible system to achieve selective NF-kappaB activation or inhibition in vivo. In transgenic mice that express a constitutively active form of IkappaB kinase 2 under control of the epithelial-specific CC10 promoter, treatment with doxycycline induced NF-kappaB activation with consequent production of a variety of proinflammatory cytokines, high-protein pulmonary edema, and neutrophilic lung inflammation. Continued treatment with doxycycline caused progressive lung injury and hypoxemia with a high mortality rate. In contrast, inducible expression of a dominant inhibitor of NF-kappaB in airway epithelium prevented lung inflammation and injury resulting from expression of constitutively active form of IkappaB kinase 2 or Escherichia coli LPS delivered directly to the airways or systemically via an osmotic pump implanted in the peritoneal cavity. Our findings indicate that the NF-kappaB pathway in airway epithelial cells is critical for generation of lung inflammation and injury in response to local and systemic stimuli; therefore, targeting inflammatory pathways in airway epithelium could prove to be an effective therapeutic strategy for inflammatory lung diseases.     

Natural IgM anti-leukocyte autoantibodies attenuate excess inflammation mediated by innate and adaptive immune mechanisms involving Th-17

Little is known about the function of natural IgM autoantibodies, especially that of IgM anti-leukocyte autoantibodies (IgM-ALA). Natural IgM-ALA are present at birth and characteristically increase during inflammatory and infective conditions. Our prior clinical observations and those of other investigators showing fewer rejections in renal and cardiac allografts transplanted into recipients with high levels of IgM-ALA led us to investigate whether IgM-ALA regulate the inflammatory response. In this article, we show that IgM, in physiologic doses, inhibit proinflammatory cells from proliferating and producing IFN-γ and IL-17 in response to alloantigens (MLR), anti-CD3, and the glycolipid α-galactosyl ceramide. We showed in an IgM knockout murine model, with intact B cells and regulatory T cells, that there was more severe inflammation and loss of function in the absence of IgM after renal ischemia reperfusion injury and cardiac allograft rejection. Replenishing IgM in IgM knockout mice or increasing the levels of IgM-ALA in wild-type B6 mice significantly attenuated the inflammation in both of these inflammatory models that involve IFN-γ and IL-17. The protective effect on renal ischemia reperfusion injury was not observed using IgM preadsorbed with leukocytes to remove IgM-ALA. We provide data to show that the anti-inflammatory effect of IgM is mediated, in part, by inhibiting TLR-4-induced NF-κB translocation into the nucleus and inhibiting differentiation of activated T cells into Th-1 and Th-17 cells. These observations highlight the importance of IgM-ALA in regulating excess inflammation mediated by both innate and adaptive immune mechanisms and where the inflammatory response involves Th-17 cells that are not effectively regulated by regulatory T cells.     

Tumor necrosis factor-alpha production by astrocytes. Induction by lipopolysaccharide, IFN-gamma, and IL-1 beta

Astrocytes have the capacity to secrete or respond to a variety of cytokines including IL-1, IL-6, IL-3, and TNF-alpha. In this study, we have examined the capacity of astrocytes to secrete TNF-alpha in response to a variety of biologic stimuli, particularly cytokines such as IL-1 and IFN-gamma, which are known to be present in the central nervous system during neurologic diseases associated with inflammation. Rat astrocytes do not constitutively produce TNF-alpha, but have the ability to secrete TNF-alpha in response to LPS, and can be primed by IFN-gamma to respond to a suboptimal dose of LPS. IFN-gamma and IL-1 beta alone do not induce TNF-alpha production, however, the combined treatment of IFN-gamma and IL-1 beta results in a striking synergistic effect on astrocyte TNF-alpha production. Astrocyte TNF-alpha protein production induced by a combined treatment of either IFN-gamma/LPS or IFN-gamma/IL-1 beta occurs in a dose- and time-dependent manner, and appears to require a "priming signal" initiated by IFN-gamma, which then renders the astrocyte responsive to either a suboptimal dose of LPS or IL-1 beta. Astrocyte TNF-alpha production by IFN-gamma/LPS stimulation can be inhibited by the addition of anti-rat IFN-gamma antibody, whereas IFN-gamma/IL-1-induced TNF-alpha production is inhibited by antibody to either IFN-gamma or IL-1 beta. Polyclonal antisera reactive with mouse macrophage-derived TNF-alpha neutralized the cytotoxicity of IFN-gamma/LPS and IFN-gamma/IL-1 beta-induced astrocyte TNF-alpha, demonstrating similarities between these two sources of TNF-alpha. We propose that astrocyte-produced TNF-alpha may have a pivotal role in augmenting intracerebral immune responses and inflammatory demyelination due to its diverse functional effects on glial cells such as oligodendrocytes and astrocytes themselves.     

Repeated Stress Exaggerates Lipopolysaccharide-Induced Inflammatory Response in the Rat Spleen

Spleen is an immune organ innervated with sympathetic nerves which together with adrenomedullary system control splenic immune functions. However, the mechanism by which prior stress exposure modulates the immune response induced by immunogenic challenge is not sufficiently clarified. Thus, the aim of this study was to investigate the effect of a single (2 h) and repeated (2 h daily for 7 days) immobilization stress (IMO) on the innate immune response in the spleen induced by lipopolysaccharide (LPS, 100 µg/kg). LPS elevated splenic levels of norepinephrine and epinephrine, while prior IMO prevented this response. LPS did not alter de novo production of catecholamines, however, prior IMO attenuated phenylethanolamine N-methyltransferase gene expression. Particularly repeated IMO exacerbated LPS-induced down-regulation of α1B- and β1-adrenergic receptors (ARs), while enhanced α2A- and β2-AR mRNAs. Elevated expression of inflammatory mediators (iNOS2, IL-1β, IL-6, TNF-α, IL-10) was observed following LPS and repeated IMO again potentiated this effect. These changes were associated with enhanced Ly6C gene expression, a monocyte marker, and elevated MCP-1, GM-CSF, and CXCL1 mRNAs suggesting an increased recruitment of monocytes and neutrophils into the spleen. Additionally, we observed increased Bax/Bcl-1 mRNA ratio together with reduced B cell numbers in rats exposed to repeated IMO and treated with LPS but not in acutely stressed rats. Altogether, these data indicate that repeated stress via changes in CA levels and specific α- and β-AR subtypes exaggerates the inflammatory response likely by recruiting peripheral monocytes and neutrophils to the spleen, resulting in the induction of apoptosis within this tissue, particularly in B cells. These changes may alter the splenic immune functions with potentially pathological consequences.     

Dehydrozingerone ameliorates Lipopolysaccharide induced acute respiratory distress syndrome by inhibiting cytokine storm,oxidative stress via modulating the MAPK/NF-κB pathway

BackgroundInflammation-mediated lung injury is a major cause of health problems in many countries and has been the leading cause of morbidity/mortality in intensive care units. In the current COVID-19 pandemic, the majority of the patients experienced serious pneumonia resulting from inflammation (Acute respiratory distress syndrome/ARDS). Pathogenic infections cause cytokine release syndrome (CRS) by hyperactivation of immune cells, which in turn release excessive cytokines causing ARDS. Currently, there are no standard therapies for viral, bacterial or pathogen-mediated CRS.PurposeThis study aimed to investigate and validate the protective effects of Dehydrozingerone (DHZ) against LPS induced lung cell injury by in-vitro and in-vivo models and to gain insights into the molecular mechanisms that mediate these therapeutic effects.MethodsThe therapeutic activity of DHZ was determined in in-vitro models by pre-treating the cells with DHZ and exposed to LPS to stimulate the inflammatory cascade of events. We analysed the effect of DHZ on LPS induced inflammatory cytokines, chemokines and cell damage markers expression/levels using various cell lines. We performed gene expression, ELISA, and western blot analysis to elucidate the effect of DHZ on inflammation and its modulation of MAPK and NF-κB pathways. Further, the prophylactic and therapeutic effect of DHZ was evaluated against the LPS induced ARDS model in rats.ResultsDHZ significantly (p < 0.01) attenuated the LPS induced ROS, inflammatory cytokine, chemokine gene expression and protein release in macrophages. Similarly, DHZ treatment protected the lung epithelial and endothelial cells by mitigating the LPS induced inflammatory events in a dose-dependent manner. In vivo analysis showed that DHZ treatment significantly (p < 0.001) mitigated the LPS induced ARDS pathophysiology of increase in the inflammatory cells in BALF, inflammatory cytokine and chemokines in lung tissues. LPS stimulated neutrophil-mediated events, apoptosis, alveolar wall thickening and alveolar inflammation were profoundly reduced by DHZ treatment in a rat model.ConclusionThis study demonstrates for the first time that DHZ has the potential to ameliorate LPS induced ARDS by inhibiting cytokine storm and oxidative through modulating the MAPK and NF-κB pathways. This data provides pre-clinical support to develop DHZ as a potential therapeutic agent against ARDS.     

Extracellular Hsp70 induces inflammation and modulates LPS/LTA-stimulated inflammatory response in THP-1 cells

Extracellular Hsp70 (eHsp70) can act as damage-associated molecular pattern (DAMP) via Toll-like receptors TLR2 and TLR4, and stimulate immune and inflammatory responses leading to sterile inflammation and propagation of already existing inflammation. It was found elevated in the blood of patients with chronic obstructive pulmonary disease (COPD), who might suffer occasional bacterial colonizations and infections. We used a monocytic THP-1 cell line as a cellular model of systemic compartment of COPD to assess inflammatory effects of eHsp70 when present alone or together with bacterial products lypopolysaccharide (LPS) and lypoteichoic acid (LTA). THP-1 cells were differentiated into macrophage-like cells and treated with various concentrations of recombinant human Hsp70 protein (rhHsp70), LPS (TLR4 agonist), LTA (TLR2 agonist), and their combinations for 4, 12, 24, and 48 h. Concentrations of IL-1α, IL-6, IL-8, and TNF-α were determined by ELISA. Cell viability was assessed by MTS assay, and mode of cell death by luminometric measurements of caspases-3/7, -8, and -9 activities. rhHsp70 showed cell protecting effect by suppressing caspases-3/7 activation, while LPS provoked cytotoxicity through caspases-8 and -3/7 pathway. Regarding inflammatory processes, rhHsp70 alone induced secretion of IL-1α and IL-8, but had modulatory effects on release of all four cytokines when applied together with LPS or LTA. Combined effect with LPS was mainly synergistic, and with LTA mainly antagonistic, although it was cytokine- and time-dependent. Our results confirmed pro-inflammatory function of extracellular Hsp70, and suggest its possible implication in COPD exacerbations caused by bacterial infection through desensitization or inappropriate activation of TLR2 and TLR4 receptors.