AIM:To investigate whether caspase-1 activation/intracellular processing of pro-interleukin-1β(pro-IL-1β) and extracellular release of mature IL-1β from activated monocytes are separable events.METHODS:All experime...AIM:To investigate whether caspase-1 activation/intracellular processing of pro-interleukin-1β(pro-IL-1β) and extracellular release of mature IL-1β from activated monocytes are separable events.METHODS:All experiments were performed on fresh or overnight cultured human peripheral blood monocytes(PBMCs) that were isolated from healthy donors.PBMCs were activated by lipopolysaccharide(LPS) stimulation before being treated with Adenosine triphosphate(ATP,1 mmol/L),human α-defensin-5(HD-5,50 μg/mL),and/or nigericin(Nig,30 μmol/L).For each experiment,the culture supernatants were collected separately from the cells.Cell lysates and supernatants were both subject to immunoprecipitation with anti-IL1β antibodies followed by western blot analysis with anti-caspase-1 and anti-IL-1β antibodies.RESULTS:We found that pro-IL-1β was processed to mature IL-1β in LPS-activated fresh and overnight cultured human monocytes in response to ATP stimulation.In the presence of HD-5,this release of IL-1β,but not the processing of pro-IL-1β to IL-1β,was completely inhibited.Similarly,in the presence of HD-5,the release of IL-1β,but not the processing of IL-1β,was significantly inhibited from LPS-activated monocytes stimulated with Nig.Finally,we treated LPS-activated monocytes with ATP and Nig and collected the supernatants.We found that both ATP and Nig stimulation could activate and release cleaved caspase-1 from the monocytes.Interestingly,and contrary to IL-1β processing and release,caspase-1 cleavage and release was not blocked by HD-5.All images are representative of three independent experiments.CONCLUSION:These data suggest that caspase-1 activation/processing of pro-IL-1β by caspase-1 and the release of mature IL-1β from human monocytes are distinct and separable events.展开更多
Attaran et al[1] have recently shown that decreased susceptibility of established Helicobacter pylori(H. pylori) biofilms to specific antibiotics,was associated with the overtly enhanced transcription of two efflux pu...Attaran et al[1] have recently shown that decreased susceptibility of established Helicobacter pylori(H. pylori) biofilms to specific antibiotics,was associated with the overtly enhanced transcription of two efflux pump genes,hp1165 and hef A,involved in specific resistance to tetracycline and multiple antibiotics,respectively. Apart from antibiotic exposure,secretion of multiple antimicrobial peptides,such as human β-defensins(hβDs),by the gastric epithelium upon Hp challenge,may act as early triggering events that positively impact biofilm formation and thus,antibiotic resistance. In this regard,we undertook genomic transcriptional studies using Hp 26695 strain following exposure to sublethal,similar to those present in the gastric niche,concentrations of hβDs in an attempt to provide preliminary data regarding possible mechanisms of immune evasion and selective sensitivity of Hp. Our preliminary results indicate that hβD exposure ignites a rapid response that is largely due to the activation of several,possibly interconnected transcriptional regulatory networks – origons-that ultimately coordinate cellular processes needed to maintain homeostasis and successful adaptation of the bacterium in the gastric environment. In addition,we have shown that both antibiotic and hβD resistance are mediated by dedicated periplasmic transporters,including the aforementioned efflux pump genes hp1165 and hef A,involved in active export of antibiotics from the cell membrane and/or,as recently suggested,substrate sensing and signalling. Furthermore,itappears that sublethal doses of hβDs may enhance biofilm formation by the sustained expression of,mainly,quorum sensing-related genes. In conclusion,we provide additional data regarding the role of specific innate immune molecules in antibiotic cross-resistance mechanisms that may deepen our understanding in the context of the development of novel eradication regimens.展开更多
基金Supported by NIH R21 AI085416 (to Shi J)NIH NCRR P20-RR017686 (to PI:Daniel MarcusShi J)
文摘AIM:To investigate whether caspase-1 activation/intracellular processing of pro-interleukin-1β(pro-IL-1β) and extracellular release of mature IL-1β from activated monocytes are separable events.METHODS:All experiments were performed on fresh or overnight cultured human peripheral blood monocytes(PBMCs) that were isolated from healthy donors.PBMCs were activated by lipopolysaccharide(LPS) stimulation before being treated with Adenosine triphosphate(ATP,1 mmol/L),human α-defensin-5(HD-5,50 μg/mL),and/or nigericin(Nig,30 μmol/L).For each experiment,the culture supernatants were collected separately from the cells.Cell lysates and supernatants were both subject to immunoprecipitation with anti-IL1β antibodies followed by western blot analysis with anti-caspase-1 and anti-IL-1β antibodies.RESULTS:We found that pro-IL-1β was processed to mature IL-1β in LPS-activated fresh and overnight cultured human monocytes in response to ATP stimulation.In the presence of HD-5,this release of IL-1β,but not the processing of pro-IL-1β to IL-1β,was completely inhibited.Similarly,in the presence of HD-5,the release of IL-1β,but not the processing of IL-1β,was significantly inhibited from LPS-activated monocytes stimulated with Nig.Finally,we treated LPS-activated monocytes with ATP and Nig and collected the supernatants.We found that both ATP and Nig stimulation could activate and release cleaved caspase-1 from the monocytes.Interestingly,and contrary to IL-1β processing and release,caspase-1 cleavage and release was not blocked by HD-5.All images are representative of three independent experiments.CONCLUSION:These data suggest that caspase-1 activation/processing of pro-IL-1β by caspase-1 and the release of mature IL-1β from human monocytes are distinct and separable events.
文摘Attaran et al[1] have recently shown that decreased susceptibility of established Helicobacter pylori(H. pylori) biofilms to specific antibiotics,was associated with the overtly enhanced transcription of two efflux pump genes,hp1165 and hef A,involved in specific resistance to tetracycline and multiple antibiotics,respectively. Apart from antibiotic exposure,secretion of multiple antimicrobial peptides,such as human β-defensins(hβDs),by the gastric epithelium upon Hp challenge,may act as early triggering events that positively impact biofilm formation and thus,antibiotic resistance. In this regard,we undertook genomic transcriptional studies using Hp 26695 strain following exposure to sublethal,similar to those present in the gastric niche,concentrations of hβDs in an attempt to provide preliminary data regarding possible mechanisms of immune evasion and selective sensitivity of Hp. Our preliminary results indicate that hβD exposure ignites a rapid response that is largely due to the activation of several,possibly interconnected transcriptional regulatory networks – origons-that ultimately coordinate cellular processes needed to maintain homeostasis and successful adaptation of the bacterium in the gastric environment. In addition,we have shown that both antibiotic and hβD resistance are mediated by dedicated periplasmic transporters,including the aforementioned efflux pump genes hp1165 and hef A,involved in active export of antibiotics from the cell membrane and/or,as recently suggested,substrate sensing and signalling. Furthermore,itappears that sublethal doses of hβDs may enhance biofilm formation by the sustained expression of,mainly,quorum sensing-related genes. In conclusion,we provide additional data regarding the role of specific innate immune molecules in antibiotic cross-resistance mechanisms that may deepen our understanding in the context of the development of novel eradication regimens.
