Myometrial immune cells contribute to term parturition,preterm labour and post‐partum involution in mice

This study aimed to determine the mechanism of uterine activation during labour, both term (TL) and preterm (PTL). We hypothesized that the peripheral leucocytes are recruited to uterine tissues by locally produced cytokines where they contribute to the initiation of parturition. Mouse uteri were collected (i) during gestation, TL and post‐partum (PP), (ii) during PTL initiated by intrauterine infusion of LPS (125 μg) or (iii) injection of the progesterone receptor antagonist RU486 and analysed for multiple cytokine expression levels by real‐time polymerase chain reaction (RT‐PCR) and 23‐plex Cytokine assay or enzymatically dispersed for assessment of immune cell populations. Markers of myeloid cell differentiation (Gr1, Neu7/4 and F4/80) were evaluated by FACS to define tissue macrophages (Macs), monocytes (M) and neutrophils (N) and by immunohistochemistry to detect tissue Macs and N. Our results indicate that: (1) Macs were elevated in mouse myometrium before TL (P < 0.05) followed by an increase in M and N; these changes were accompanied by an increase in multiple pro‐inflammatory cytokines/chemokines genes. The expression of corresponding proteins increased PP. (2) TL and RU486‐PTL models showed similar gene/protein expression profiles, (3) LPS‐PTL was characterized by strong pro‐inflammatory response and massive influx of N in myometrial tissues showing a pattern different from TL and RU486‐PTL, (4) The PP period appears similar in all three models, with elevated myometrial cytokine levels and high infiltration of immune cells. We concluded that leucocytes infiltrate myometrium around the time of parturition implicating their potential role in labour activation (both term and preterm) and major role in PP uterine involution.     

Adiponectin promotes human jaw bone marrow mesenchymal stem cell chemotaxis via CXCL1 and CXCL8

Adiponectin (APN) is known to promote the osteogenic differentiation of human jaw bone marrow mesenchymal stem cells (h‐JBMMSCs). However, the underlying mechanism has not been fully elucidated. Previously, we showed that APN could promote h‐JBMMSC osteogenesis via APPL1‐p38 by up‐regulating osteogenesis‐related genes. Here, we aimed to determine whether APN could promote h‐JBMMSC chemotaxis through CXCL1/CXCL8. The CCK‐8, wound healing and transwell assays were used to evaluate the proliferation, migration and chemotaxis of h‐JBMMSCs with or without APN treatment. Chemotaxis‐related genes were screened using RNA‐seq, and the results were validated using real‐time PCR and ELISA. We also performed Western blot using the AMPK inhibitor, WZ4003, and the p38 MAPK inhibitor, SB203580, to identify the signalling pathway involved. We found that APN could promote h‐JBMMSC chemotaxis in the co‐culture transwell system. CXCL1 and CXCL8 were screened and confirmed as the up‐regulated target genes. The APN‐induced CXCL1/8 up‐regulation to promote chemotaxis could be blocked by CXCR2 inhibitor SB225002. Western blot revealed that the phosphorylation of AMPK and p38 MAPK increased in a time‐dependent manner with APN treatment. Additionally, WZ4003 and SB203580 could suppress the APN‐induced overexpression of CXCL1 and CXCL8. The results of the transwell chemotaxis assay also supported the above results. Our data suggest that APN can promote h‐JBMMSC chemotaxis by up‐regulating CXCL1 and CXCL8.     

Commensal and pathogenic biofilms differently modulate peri‐implant oral mucosa in an organotypic model

The impact of oral commensal and pathogenic bacteria on peri‐implant mucosa is not well understood, despite the high prevalence of peri‐implant infections. Hence, we investigated responses of the peri‐implant mucosa to Streptococcus oralis or Aggregatibacter actinomycetemcomitans biofilms using a novel in vitro peri‐implant mucosa‐biofilm model. Our 3D model combined three components, organotypic oral mucosa, implant material, and oral biofilm, with structural assembly close to native situation. S. oralis induced a protective stress response in the peri‐implant mucosa through upregulation of heat shock protein (HSP70) genes. Attenuated inflammatory response was indicated by reduced cytokine levels of interleukin‐6 (IL‐6), interleukin‐8 (CXCL8), and monocyte chemoattractant protein‐1 (CCL2). The inflammatory balance was preserved through increased levels of tumor necrosis factor‐alpha (TNF‐α). A. actinomycetemcomitans induced downregulation of genes important for cell survival and host inflammatory response. The reduced cytokine levels of chemokine ligand 1 (CXCL1), CXCL8, and CCL2 also indicated a diminished inflammatory response. The induced immune balance by S. oralis may support oral health, whereas the reduced inflammatory response to A. actinomycetemcomitans may provide colonisation advantage and facilitate later tissue invasion. The comprehensive characterisation of peri‐implant mucosa‐biofilm interactions using our 3D model can provide new knowledge to improve strategies for prevention and therapy of peri‐implant disease.     

YB‐1 orchestrates onset and resolution of renal inflammation via IL10 gene regulation

The Y‐box‐binding protein (YB)‐1 plays a non‐redundant role in both systemic and local inflammatory response. We analysed YB‐1‐mediated expression of the immune regulatory cytokine IL‐10 in both LPS and sterile inflammation induced by unilateral renal ischaemia–reperfusion (I/R) and found an important role of YB‐1 not only in the onset but also in the resolution of inflammation in kidneys. Within a decisive cis‐regulatory region of the IL10 gene locus, the fourth intron, we identified and characterized an operative YB‐1 binding site via gel shift experiments and reporter assays in immune and different renal cells. In vivo, YB‐1 phosphorylated at serine 102 localized to the fourth intron, which was paralleled by enhanced IL‐10 mRNA expression in mice following LPS challenge and in I/R. Mice with half‐maximal expression of YB‐1 (Yb1^+/?) had diminished IL‐10 expression upon LPS challenge. In I/R, Yb1^+/? mice exhibited ameliorated kidney injury/inflammation in the early‐phase (days 1 and 5), however showed aggravated long‐term damage (day 21) with increased expression of IL‐10 and other known mediators of renal injury and inflammation. In conclusion, these data support the notion that there are context‐specific decisions concerning YB‐1 function and that a fine‐tuning of YB‐1, for example, via a post‐translational modification regulates its activity and/or localization that is crucial for systemic processes such as inflammation.     

Progesterone and calcitriol attenuate inflammatory cytokines CXCL1 and CXCL2 in ovarian and endometrial cancer cells

Cytokines/chemokines are key players in cancer‐related inflammation. Increasing evidence suggests that chemokines produced by tumor cells are the mediators of metastasis. Thus, agents that can downregulate chemokines expression have potential against cancer metastasis. We have previously shown inhibition of ovarian and endometrial cancer cell growth with progesterone and calcitriol. In the present study, we evaluated the effect of these two agents on the expression of inflammatory genes. Using a RT‐PCR array of inflammatory cytokines/chemokines and their receptors, we found a marked attenuation of CXCL1 and CXCL2 (GRO‐α and ‐β) in cancer cells by both treatments. Knockdown of NFκB resulted in a reduced expression of CXCL1 and CXCL2 and the inhibitory effect of progesterone and calcitriol on the expression of chemokines was abrogated in NFκB‐silenced cancer cells. Silencing of IκBα increased the expression of CXCL1 and CXCL2 in cancer cells, which can be attributed to the increased activation of NFκB‐p65, caused by the lack of its inhibitor. Progesterone and calcitriol‐induced inhibition was abolished in IκBα‐knockdown cells. Our results demonstrate that suppression of IκBα phosphorylation by progesterone and calcitriol contributes to the reduced expression of CXCL1 and CXCL2. Downregulation of CXCL1 and CXCL2 was associated with a marked inhibition of metastasis‐promoting genes. Overall, our results indicate that progesterone and calcitriol inhibit IκBα phosphorylation, NFκB activation, and the expression of NFκB regulated metastasis promoting genes. These results provide attractive data for the possible use of progesterone and calcitriol in the management of endometrial and ovarian tumors. J. Cell. Biochem. 113: 3143–3152, 2012. © 2012 Wiley Periodicals, Inc.     

The role of EGF‐EGFR signalling pathway in hepatocellular carcinoma inflammatory microenvironment

Epidermal growth factor (EGF) and their receptor (EGFR) play an important role in the development of cancer proliferation, and metastasis, although the mechanism remains unclear. The present study aimed at investigating the role of EGF‐EGFR signalling pathway in the development of human hepatocellular carcinoma (HCC) inflammatory environment. Gene profiles of inflammatory cytokines from HCC were measured. Cell bio‐behaviours of HCC with low or high metastasis were detected by the live cell monitoring system. Cell proliferation was measured by CCK8. The protein level of CXCL5 and CXCL8 was measured by ELISA. The phosphorylation of PI3K, ERK, MAPK was measured by western blot. EGF significantly induced cell proliferation in HepG2 cells, but not in HCCLM3 cells. EGF prompted the cell movement in both HepG2 and HCCLM3 and regulated the production of CXCL5 and CXCL8 from HCC, which were inhibited by EGFR inhibitor, Erk inhibitor (U0126), or PI3K inhibitors (BEZ‐235 and SHBM1009). HCC proliferation, metastasis and production of inflammatory cytokines were regulated via EGF‐EGFR signal pathways. CXCL5 could interact with CXCL8, possibly by CXCR2 or the cross‐talk between CXCR2 and EGFR. EGF‐EGFR signaling pathway can be the potential target of therapies for HCC.     

Direct interaction of surfactant protein A with myometrial binding sites: signaling and modulation by bacterial lipopolysaccharide

Surfactant protein A (SFTPA1), a member of the collagenous lectin (collectin) family, was first described as a major constituent of lung surfactant, but has recently also been found in the female genital tract. Various microorganisms colonize this area and may cause intrauterine infection or trigger preterm labor. We found that SFTPA1 was not produced in the uterus. Instead, it was immunodetected transiently in rat myometrium at the end (Days 19 and 21) of gestation, but not postpartum, and in cultured myometrial cells. Fluorescence microscopy showed that Texas Red-labeled SFTPA1 bound to myometrial cells. This result was confirmed by biochemical approaches. [(125)I]-SFTPA1 bound to two myometrial cell proteins (55 and 210 kDa). This interaction was dependent on the integrity of the collagenlike domain of SFTPA1. SFTPA1 rapidly activated mitogen-activated protein kinase 1/3 (MAPK1/3) in myometrial cells. Bacterial lipopolysaccharide (LPS), an agent known to trigger uterine contractions and preterm birth, also activated MAPK1/3. The prolonged treatment of myometrial cells with LPS or SFTPA1 upregulated PTGS2 (COX2) protein levels. The addition of rough-type LPS to SFTPA1 blocked the interaction of SFTPA1 with its binding sites and the activation of MAPK1/3 and PTGS2 by SFTPA1. Our data provide the first demonstration of a direct effect of SFTPA1 on rat myometrial cells and inhibitory cross talk between SFTPA1 and LPS signals, providing new insight into the mechanisms of normal and preterm parturition.     

Loss‐of‐function of ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (APN1) in Lotus japonicus restricts efficient nitrogen‐fixing symbiosis with specific Mesorhizobium loti strains

The nitrogen‐fixing symbiosis of legumes and Rhizobium bacteria is established by complex interactions between the two symbiotic partners. Legume Fix^– mutants form apparently normal nodules with endosymbiotic rhizobia but fail to induce rhizobial nitrogen fixation. These mutants are useful for identifying the legume genes involved in the interactions essential for symbiotic nitrogen fixation. We describe here a Fix^– mutant of Lotus japonicus, apn1, which showed a very specific symbiotic phenotype. It formed ineffective nodules when inoculated with the Mesorhizobium loti strain TONO. In these nodules, infected cells disintegrated and successively became necrotic, indicating premature senescence typical of Fix^– mutants. However, it formed effective nodules when inoculated with the M. loti strain MAFF303099. Among nine different M. loti strains tested, four formed ineffective nodules and five formed effective nodules on apn1 roots. The identified causal gene, ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (LjAPN1), encodes a nepenthesin‐type aspartic peptidase. The well characterized Arabidopsis aspartic peptidase CDR1 could complement the strain‐specific Fix^– phenotype of apn1. LjAPN1 is a typical late nodulin; its gene expression was exclusively induced during nodule development. LjAPN1 was most abundantly expressed in the infected cells in the nodules. Our findings indicate that LjAPN1 is required for the development and persistence of functional (nitrogen‐fixing) symbiosis in a rhizobial strain‐dependent manner, and thus determines compatibility between M. loti and L. japonicus at the level of nitrogen fixation.     

Porphyromonas gingivalis induces the production of interleukin‐31 by human mast cells,resulting in dysfunction of the gingival epithelial barrier

Interleukin (IL)‐31 is important for innate immunity in mucosal tissues and skin, and increased IL‐31 expression participates in the pathogenesis of chronic inflammatory diseases affecting the skin, airways, lungs, and intestines. We investigated the contribution of mast cells to the induction of IL‐31 production following infection with the periodontal pathogen, Porphyromonas gingivalis. We found that oral infection with P. gingivalis increased IL‐31 expression in the gingival tissues of wild‐type mice but not in those of mast cell‐deficient mice. The P. gingivalis‐induced IL‐31 production by human mast cells occurred through the activation of the JNK and NF‐κB signalling pathways and was dependent on the P. gingivalis lysine‐specific protease gingipain‐K. P. gingivalis infection induced IL‐31 receptor α and oncostatin M receptor β expression in human gingival epithelial cells. Notably, the P. gingivalis‐induced IL‐31 production by mast cells led to the downregulation of claudin‐1, a tight junction molecule, in gingival epithelial cells, resulting in an IL‐31‐dependent increase in the paracellular permeability of the gingival epithelial barrier. These findings suggest that IL‐31 produced by mast cells in response to P. gingivalis infection causes gingival epithelial barrier dysfunction, which may contribute to the chronic inflammation observed in periodontitis.     

Prostaglandin E2 Represses Interleukin 1 Beta-Induced Inflammatory Mediator Output from Pregnant Human Myometrial Cells Through the EP2 and EP4 Receptors

Inflammatory mediators, including prostaglandins, cytokines, and chemokines, are strongly implicated in the mechanism of human labor, though their precise roles remain unknown. Here we demonstrate that interleukin 1 beta (IL-1beta) significantly increased the expression and release of interleukin-8 (CXCL8), monocyte chemotactic protein-1 (CCL2), and granulocyte macrophage colony-stimulating factor (CSF2) by primary human myometrial cells. However, this effect was repressed by prostaglandin E(2) (PGE(2)). As PGE(2) can activate four distinct PGE(2) receptors (EP(1), EP(2), EP(3), and EP(4)) to elicit various responses, we sought to define the EP receptor(s) responsible for this repression. Using selective EP receptor agonists and a selective EP(4) antagonist, we show that PGE(2) mediates the repression of IL-1beta-induced release of CXCL8, CCL2, and CSF2 via activation of the EP(2) and EP(4) receptors. The use of siRNA gene-specific knockdown further confirmed a role for both receptors. Real-time RT-PCR demonstrated that EP(2) was the most highly expressed of all four EP receptors at the mRNA level in human myometrial cells, and immunocytochemistry showed that EP(2) protein is abundantly present throughout the cells. Interestingly, PGE(2) does not appear to reduce mRNA expression of CXCL8, CCL2, and CSF2. Our results demonstrate that PGE(2) can elicit anti-inflammatory responses via activation of the EP(2) and EP(4) receptors in lower segment term pregnant human myometrial cells. Further elucidation of the EP receptor-mediated signaling pathways in the pregnant human uterus may be beneficial for optimizing the maintenance of pregnancy, induction of labor or indeed treatment of preterm labor.     

CD1d‐mediated activation of group 3 innate lymphoid cells drives IL‐22 production

Innate lymphoid cells (ILCs) are a heterogeneous family of immune cells that play a critical role in a variety of immune processes including host defence against infection, wound healing and tissue repair. Whether these cells are involved in lipid‐dependent immunity remains unexplored. Here we show that murine ILCs from a variety of tissues express the lipid‐presenting molecule CD1d, with group 3 ILCs (ILC3s) showing the highest level of expression. Within the ILC3 family, natural cytotoxicity triggering receptor (NCR)^?CCR6⁺ cells displayed the highest levels of CD1d. Expression of CD1d on ILCs is functionally relevant as ILC3s can acquire lipids in vitro and in vivo and load lipids on CD1d to mediate presentation to the T‐cell receptor of invariant natural killer T (iNKT) cells. Conversely, engagement of CD1d in vitro and administration of lipid antigen in vivo induce ILC3 activation and production of IL‐22. Taken together, our data expose a previously unappreciated role for ILCs in CD1d‐mediated immunity, which can modulate tissue homeostasis and inflammatory responses.     

Valsartan independent of AT1 receptor inhibits tissue factor,TLR‐2 and ‐4 expression by regulation of Egr‐1 through activation of AMPK in diabetic conditions

Patients suffering from diabetes mellitus (DM) are at a severe risk of atherothrombosis. Early growth response (Egr)‐1 is well characterized as a central mediator in vascular pathophysiology. We tested whether valsartan independent of Ang II type 1 receptor (AT₁R) can reduce tissue factor (TF) and toll‐like receptor (TLR)‐2 and ‐4 by regulating Egr‐1 in THP‐1 cells and aorta in streptozotocin‐induced diabetic mice. High glucose (HG, 15 mM) increased expressions of Egr‐1, TF, TLR‐2 and ‐4 which were significantly reduced by valsartan. HG increased Egr‐1 expression by activation of PKC and ERK1/2 in THP‐1 cells. Valsartan increased AMPK phosphorylation in a concentration and time‐dependent manner via activation of LKB1. Valsartan inhibited Egr‐1 without activation of PKC or ERK1/2. The reduced expression of Egr‐1 by valsartan was reversed by either silencing Egr‐1, or compound C, or DN‐AMPK‐transfected cells. Valsartan inhibited binding of NF‐κB and Egr‐1 to TF promoter in HG condition. Furthermore, valsartan reduced inflammatory cytokine (TNF‐α, IL‐6 and IL‐1β) production and NF‐κB activity in HG‐activated THP‐1 cells. Interestingly, these effects of valsartan were not affected by either silencing AT₁R in THP‐1 cells or CHO cells, which were devoid of AT₁R. Importantly, administration of valsartan (20 mg/kg, i.p) for 8 weeks significantly reduced plasma TF activity, expression of Egr‐1, TLR‐2, ‐4 and TF in thoracic aorta and improved glucose tolerance of streptozotocin‐induced diabetic mice. Taken together, we concluded that valsartan may reduce atherothrombosis in diabetic conditions through AMPK/Egr‐1 regulation.     

Processing and secretion of guanylate binding protein‐1 depend on inflammatory caspase activity

Human guanylate binding protein‐1 (GBP‐1) belongs to the family of large GTPases. The expression of GBP‐1 is inducible by inflammatory cytokines, and the protein is involved in inflammatory processes and host defence against cellular pathogens. GBP‐1 is the first GTPase which was described to be secreted by eukaryotic cells. Here, we report that precipitation of GBP‐1 with GMP‐agarose from cell culture supernatants co‐purified a 47‐kD fragment of GBP‐1 (p47‐GBP‐1) in addition to the 67‐kD full‐length form. MALDI‐TOF sequencing revealed that p47‐GBP‐1 corresponds to the C‐terminal helical part of GBP‐1 and lacks most of the globular GTPase domain. In silico analyses of protease target sites, together with cleavage experiments in vitro and in vivo, showed that p67‐GBP‐1 is cleaved by the inflammatory caspases 1 and 5, leading to the formation of p47‐GBP‐1. Furthermore, the secretion of p47‐GBP‐1 was found to occur via a non‐classical secretion pathway and to be dependent on caspase‐1 activity but independent of inflammasome activation. Finally, we showed that p47‐GBP‐1 represents the predominant form of secreted GBP‐1, both in cell culture supernatants and, in vivo, in the cerebrospinal fluid of patients with bacterial meningitis, indicating that it may represent the biologically active form of extracellular GBP‐1. These findings confirm the involvement of caspase‐1 in non‐classical secretion mechanisms and open novel perspectives for the extracellular function of secreted GBP‐1.     

Cordycepin activates AMP‐activated protein kinase (AMPK) via interaction with the γ1 subunit

Cordycepin is a bioactive component of the fungus Cordyceps militaris. Previously, we showed that cordycepin can alleviate hyperlipidemia through enhancing the phosphorylation of AMP‐activated protein kinase (AMPK), but the mechanism of this stimulation is unknown. Here, we investigated the potential mechanisms of cordycepin‐induced AMPK activation in HepG2 cells. Treatment with cordycepin largely reduced oleic acid (OA)‐elicited intracellular lipid accumulation and increased AMPK activity in a dose‐dependent manner. Cordycepin‐induced AMPK activation was not accompanied by changes in either the intracellular levels of AMP or the AMP/ATP ratio, nor was it influenced by calmodulin‐dependent protein kinase kinase (CaMKK) inhibition; however, this activation was significantly suppressed by liver kinase B1 (LKB1) knockdown. Molecular docking, fluorescent and circular dichroism measurements showed that cordycepin interacted with the γ1 subunit of AMPK. Knockdown of AMPKγ1 by siRNA substantially abolished the effects of cordycepin on AMPK activation and lipid regulation. The modulating effects of cordycepin on the mRNA levels of key lipid regulatory genes were also largely reversed when AMPKγ1 expression was inhibited. Together, these data suggest that cordycepin may inhibit intracellular lipid accumulation through activation of AMPK via interaction with the γ1 subunit.