Bacterial lipopolysaccharides form procollagen-endotoxin complexes that trigger cartilage inflammation and degeneration: implications for the development of rheumatoid arthritis

Introduction

We have previously reported that bacterial toxins, especially endotoxins such as lipopolysaccharides (LPS), might be important causative agents in the pathogenesis of rheumatoid arthritis (RA) in an in vitro model that simulates the potential effects of residing in damp buildings. Since numerous inflammatory processes are linked with the nuclear factor-κB (NF-κB), we investigated in detail the effects of LPS on the NF-κB pathway and the postulated formation of procollagen-endotoxin complexes.

Methods

An in vitro model of human chondrocytes was used to investigate LPS-mediated inflammatory signaling.

Results

Immunoelectron microscopy revealed that LPS physically interact with collagen type II in the extracellular matrix (ECM) and anti-collagen type II significantly reduced this interaction. BMS-345541 (a specific inhibitor of IκB kinase (IKK)) or wortmannin (a specific inhibitor of phosphatidylinositol 3-kinase (PI-3K)) inhibited the LPS-induced degradation of the ECM and apoptosis in chondrocytes. This effect was completely inhibited by combining BMS-345541 and wortmannin. Furthermore, BMS-345541 and/or wortmannin suppressed the LPS-induced upregulation of catabolic enzymes that mediate ECM degradation (matrix metalloproteinases-9, -13), cyclooxygenase-2 and apoptosis (activated caspase-3). These proteins are regulated by NF-κB, suggesting that the NF-κB and PI-3K pathways are involved in LPS-induced cartilage degradation. The induction of NF-κB correlated with activation of IκBα kinase, IκBα phosphorylation, IκBα degradation, p65 phosphorylation and p65 nuclear translocation. Further upstream, LPS induced the expression of Toll-like receptor 4 (TLR4) and bound with TLR4, indicating that LPS acts through TLR4.

Conclusion

These results suggest that molecular associations between LPS/TLR4/collagen type II in chondrocytes upregulate the NF-κB and PI-3K signaling pathways and activate proinflammatory activity.     

Cyanidin-3-O-β-glucoside inhibits lipopolysaccharide-induced inflammatory response in mouse mastitis model

Cyanidin-3-O-β-glucoside (C3G) (CAS number 7084-24-4), a typical anthocyanin pigment that exists in the human diet, has been reported to have anti-inflammatory properties. However, the effect of C3G on lipopolysaccharide (LPS)-induced mastitis and the molecular mechanisms have not been investigated. In this study, we detected the protective effects of C3G on a LPS-induced mouse mastitis model and investigated the molecular mechanisms in LPS-stimulated mouse mammary epithelial cells (MMECs). Our results showed that C3G could attenuate mammary histopathologic changes and myeloperoxidase activity, and inhibit TNF-α, interleukin (IL)-1β, and IL-6 production caused by LPS. Meanwhile, C3G dose-dependently inhibited TNF-α and IL-6 in LPS-stimulated MMECs. C3G suppressed LPS-induced nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3) activation. Furthermore, C3G disrupted the formation of lipid rafts by depleting cholesterol. Moreover, C3G activated liver X receptor (LXR)-ABCG1-dependent cholesterol efflux. Knockdown of LXRα abrogated the anti-inflammatory effects of C3G. In conclusion, C3G has a protective effect on LPS-induced mastitis. The promising anti-inflammatory mechanisms of C3G are associated with upregulation of the LXRα-ABCG1 pathway which result in disrupting lipid rafts by depleting cholesterol, thereby suppressing toll-like receptor 4-mediated NF-κB and IRF3 signaling pathways induced by LPS.     

Overexpression of miR-125a in Myelodysplastic Syndrome CD34+ Cells Modulates NF-κB Activation and Enhances Erythroid Differentiation Arrest

Myelodysplastic syndromes (MDS) are characterized by impaired proliferation and differentiation of hematopoietic stem cells. The participation of toll-like receptor (TLR)-mediated signaling in MDS is well documented. Increased TLR signaling leads to the constitutive activation of NF-κB, which mediates inflammation, cell proliferation and apoptosis. In addition, the TLR pathway induces the expression of miRNAs which participate in the fine-tuning of the inflammatory response. miRNAs also regulate other biological processes, including hematopoiesis. miR-125a and miR-125b are known modulators of hematopoiesis and are abnormally expressed in several hematologic malignancies. However, little is known about their role in MDS. NF-κB-activating ability has been described for both miRNAs. We studied the role of miR-125a/miR-125b in MDS and their relationship with TLR signaling and hematopoietic differentiation. Our results indicate that miR-125a is significantly overexpressed in MDS patients and correlates negatively with patient survival. Expression of miR-99b, which is clustered with miR-125a, is also directly correlated with prognosis of MDS. Both miR-125a and miR-99b activated NF-κB in vitro; however, we observed a negative correlation between miR-99b expression and the levels of TLR2, TLR7 and two downstream genes, suggesting that NF-κB activation by the miRNA cluster occurs in the absence of TLR signaling. We also show that TLR7 is negatively correlated with patient survival in MDS. In addition, our data suggest that miR-125a may act as an NF-κB inhibitor upon TLR stimulation. These results indicate that miR-125a is involved in the fine-tuning of NF-κB activity and that its effects may depend on the status of the TLR pathway. Furthermore, we observed that miR-125a inhibits erythroid differentiation in leukemia and MDS cell lines. Therefore, this miRNA could serve as a prognostic marker and a potential therapeutic target in MDS.     

Regulation of NF-κB signaling by oxidized glycerophospholipid and IL-1β induced miRs-21-3p and -27a-5p in human aortic endothelial cells

Exposure of endothelial cells (ECs) to agents such as oxidized glycerophospholipids (oxGPs) and cytokines, known to accumulate in atherosclerotic lesions, perturbs the expression of hundreds of genes in ECs involved in inflammatory and other biological processes. We hypothesized that microRNAs (miRNAs) are involved in regulating the inflammatory response in human aortic endothelial cells (HAECs) in response to oxGPs and interleukin 1β (IL-1β). Using next-generation sequencing and RT-quantitative PCR, we characterized the profile of expressed miRNAs in HAECs pre- and postexposure to oxGPs. Using this data, we identified miR-21-3p and miR-27a-5p to be induced 3- to 4-fold in response to oxGP and IL-1β treatment compared with control treatment. Transient overexpression of miR-21-3p and miR-27a-5p resulted in the downregulation of 1,253 genes with 922 genes overlapping between the two miRNAs. Gene Ontology functional enrichment analysis predicted that the two miRNAs were involved in the regulation of nuclear factor κB (NF-κB) signaling. Overexpression of these two miRNAs leads to changes in p65 nuclear translocation. Using 3′ untranslated region luciferase assay, we identified 20 genes within the NF-κB signaling cascade as putative targets of miRs-21-3p and -27a-5p, implicating these two miRNAs as modulators of NF-κB signaling in ECs.     

Anisomycin protects against sepsis by attenuating IκB kinase-dependent NF-κB activation and inflammatory gene expression

Anisomycin is known to inhibit eukaryotic protein synthesis and has been established as an antibiotic and anticancer drug. However, the molecular targets of anisomycin and its mechanism of action have not been explained in macrophages. Here, we demonstrated the anti-inflammatory effects of anisomycin both in vivo and in vitro. We found that anisomycin decreased the mortality rate of macrophages in cecal ligation and puncture (CLP)- and lipopolysaccharide (LPS)-induced acute sepsis. It also declined the gene expression of proinflammatory mediators such as inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin-1β as well as the nitric oxide and proinflammatory cytokines production in macrophages subjected to LPS-induced acute sepsis. Furthermore, anisomycin attenuated nuclear factor (NF)-κB activation in LPS-induced macrophages, which correlated with the inhibition of phosphorylation of NF-κB-inducing kinase and IκB kinase, phosphorylation and IκBα proteolytic degradation, and NF-κB p65 subunit nuclear translocation. These results suggest that anisomycin prevented acute inflammation by inhibiting NF-κB-related inflammatory gene expression and could be a potential therapeutic candidate for sepsis.     

Yohimbine Enhances Protection of Berberine against LPS-Induced Mouse Lethality through Multiple Mechanisms

Sepsis remains a major cause of mortality in intensive care units, better therapies are urgently needed. Gram-negative bacterial lipopolysaccharide (LPS) is an important trigger of sepsis. We have demonstrated that berberine (Ber) protects against lethality induced by LPS, which is enhanced by yohimbine (Y) pretreatment, and Ber combined with Y also improves survival in septic mice. However, the precise mechanisms by which Y enhances protection of Ber against LPS - induced lethality remain unclear. The present study confirmed that simultaneously administered Y also enhanced protection of Ber against LPS-induced lethality. Ber or/and Y attenuated liver injury, but not renal injury in LPS-challenged mice. Ber or/and Y all inhibited LPS-stimulated IκBα, JNK and ERK phosphorylation, NF-κB activation as well as TNF-α production. Ber also increased IL-10 production in LPS-challenged mice, which was enhanced by Y. Furthermore, Ber or/and Y all suppressed LPS-induced IRF3, TyK2 and STAT1 phosphorylation, as well as IFN-β and IP-10 mRNA expression in spleen of mice at 1 h after LPS challenge. Especially, Y enhanced the inhibitory effect of Ber on LPS-induced IP-10 mRNA expression. In vitro experiments further demonstrated that Y significantly enhanced the inhibitory effect of Ber on TNF-α production in LPS-treated peritoneal macrophages, Ber combined with Y promoted LPS-induced IL-10 production and LPS-stimulated IκBα, JNK, ERK and IRF3 phosphorylation and NF-κB activation were also suppressed by Ber or/and Y pretreatment in peritoneal macrophages. Taken together, these results demonstrate that Y enhances the protection of Ber against LPS-induced lethality in mice via attenuating liver injury, upregulating IL-10 production and suppressing IκBα, JNK, ERK and IRF3 phosphorylation. Ber combined with Y may be an effective immunomodulator agent for the prevention of sepsis.     

Profiling the circulating miRNAs in mice exposed to gram-positive and gram-negative bacteria by Illumina small RNA deep sequencing

Background

We profiled the expression of circulating microRNAs (miRNAs) in mice using Illumina small RNA deep sequencing in order to identify the miRNAs that may potentially be used as biomarkers to distinguish between gram-negative and gram-positive bacterial infections.

Results

Recombinant-specific gram-negative pathogen Escherichia coli (Xen14) and gram-positive pathogen Staphylococcus aureus (Xen29) were used to induce bacterial infection in mice at a concentration of 1 × 10⁸ bacteria/100 μL of phosphate buffered saline (PBS). Small RNA libraries generated from the serum of mice after exposure to PBS, Xen14, Xen29, and Xen14 + Xen29 via the routes of subcutaneous injection (I), cut wound (C), or under grafted skin (S) were analyzed using an Illumina HiSeq2000 Sequencer. Following exposure to gram-negative bacteria alone, no differentially expressed miRNA was found in the injection, cut, or skin graft models. Exposure to mixed bacteria induced a similar expression pattern of the circulating miRNAs to that induced by gram-positive bacterial infection. Upon gram-positive bacterial infection, 9 miRNAs (mir-193b-3p, mir-133a-1-3p, mir-133a-2-3p, mir-133a-1-5p, mir-133b-3p, mir-434-3p, mir-127-3p, mir-676-3p, mir-215-5p) showed upregulation greater than 4-fold with a p-value < 0.01. Among them, mir-193b-3p, mir-133a-1-3p, and mir-133a-2-3p presented the most common miRNA targets expressed in the mice exposed to gram-positive bacterial infection.

Conclusions

This study identified mir-193b-3p, mir-133a-1-3p, and mir-133a-2-3p as potential circulating miRNAs for gram-positive bacterial infections.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0106-y) contains supplementary material, which is available to authorized users.