The Inflammasome and the Epidermal Growth Factor Receptor (EGFR) Are Involved in the Staphylococcus aureus-Mediated Induction of IL-1alpha and IL-1beta in Human Keratinocytes

Staphylococcus (S.) aureus is an important pathogen causing various infections including those of the skin. Keratinocytes are able to sense invading S. aureus and to initiate a fast defense reaction by the rapid release of innate defense mediators such as antimicrobial peptides and cytokines. There is increasing evidence that the cytokines IL-1alpha and IL-1beta, which both signal through the IL-1 receptor, play an important role in cutaneous defense against S. aureus. The aim of this study was to gain more insight into the underlying mechanisms leading to the S. aureus-induced IL-1alpha and IL-1beta expression in keratinocytes. Infection of human primary keratinocytes with S. aureus led to the induction of gene expression and protein secretion of IL-1alpha and IL-1beta. Full S. aureus-induced IL-1 protein release required the inflammasome components caspase-1 and ASC (apoptosis-associated speck-like protein containing a CARD) whereas gene induction of IL-1alpha and IL-beta by S. aureus was not dependent on caspase-1 and ASC. Since patients receiving anti-cancer therapy by inhibition of the epidermal growth factor receptor (EGFR) often suffer from skin infections caused by S. aureus we additionally evaluated whether the EGFR pathway may be involved in the IL-1alpha and IL-1beta induction by S. aureus. Inactivation of the EGFR with a blocking antibody decreased the S. aureus-mediated IL-1alpha and IL-1beta induction in primary keratinocytes. Moreover, the use of siRNA experiments revealed that ADAM17 (A Disintegrin and A Metalloprotease 17), a metalloproteinase known to mediate the shedding and release of EGFR ligands, was required for full induction of IL-1alpha and IL-1beta in keratinocytes infected with S. aureus. A failure of keratinocytes to adequately upregulate IL-1alpha and IL-1beta may promote S. aureus skin infections.     

Prointerleukin-18 is activated by meprin beta in vitro and in vivo in intestinal inflammation

Interleukin-18 (IL-18), a pro-inflammatory cytokine, is a key factor in inflammatory bowel disease (IBD). Caspase-1 activates this cytokine, but other proteases are likely involved in maturation. Because meprin metalloproteinases have been implicated in IBD, the interaction of these proteases with proIL-18 was studied. The results demonstrate that the meprin beta subunit of meprins A and B cleaves proIL-18 into a smaller 17-kDa product. The cleavage is at the Asn51-Asp52 bond, a site C-terminal to caspase-1 cleavage. The cleavage occurred in vitro with a Km of 1.3 microm and in Madin-Darby canine kidney cells transfected with meprin beta when proIL-18 was added to the culture medium. The product of meprin B cleavage of proIL-18 activated NF-kappaB in EL-4 cells, indicating that it was biologically active. To determine the physiological significance of the interactions of meprins with proIL-18, an experimental model of IBD was produced by administering dextran sulfate sodium (DSS) to wild-type and meprin beta knock-out (betaKO) mice, and the serum levels of active IL-18 were determined. DSS-treated meprin betaKO mice had lower levels of the active cytokine in the serum compared with wild-type mice. Furthermore, in meprin alphaKO mice, which express meprin beta but not alpha, active IL-18 was elevated in the serum of DSS-treated mice compared with wild-type mice, indicating that the meprin isoforms have opposing effects on the IL-18 levels in vivo. This study identifies proIL-18 as a biologically important substrate for meprin beta and implicates meprins in the modulation of inflammation.     

Immediate and carryover effects of Gram-negative and Gram-positive toxin-induced mastitis on follicular function in dairy cows

This study compared immediate and carryover effects of mastitis induced by Gram-negative endotoxin (E. coli LPS) and Gram-positive exosecretions (Staph. aureus ex.) on preovulatory follicle function. Synchronized, uninfected cyclic lactating Holstein cows were treated with PGF_2α on day 6 of the cycle and 36 h later, a dose of either E. coli LPS (n = 8), S. aureus ex. (n = 10), or saline (n = 9) was administered into the mammary gland. Follicular fluids and granulosa cells were aspirated 6 h later from the preovulatory follicles and cows were treated with GnRH. This (cycle 1; immediate effect) was repeated three times (excluding the mammary injections) to induce three 7 d cycles (cycles 2, 3, and 4; carryover effect). E. coli LPS increased body temperature, plasma cortisol concentration, and somatic cell count (SCC), whereas S. aureus ex. induced a minor, subclinical elevation of SCC and slight rise (NS) in body temperature and cortisol concentration. Follicular estradiol, androstenedione, and progesterone concentrations in the E. coli LPS group decreased (P < 0.05) in cycle 1 to about 40%, 13%, and 35%, respectively, of control levels, whereas in the S. aureus ex. group, only estradiol decreased (P < 0.05), to 56% of control concentrations. In cycles 3 and 4, follicular steroids in the E. coli LPS group returned to control concentrations, whereas in the S. aureus ex. group, follicular concentrations of estradiol and androstenedione were lower (P < 0.10) than in controls. In the control group, the concentrations of all follicular and circulating steroids remained stable (P > 0.05) throughout the study. Follicle size was similar in all groups, but the S. aureus ex. treatment caused a decrease (P < 0.02) in the number of follicles developed in cycles 3 and 4. The mRNA expression of steroidogenic genes and LHCGR in the granulosa cells was not affected (P > 0.05) by either treatment during the study, except for a tendency toward lower (P < 0.1) expression in cycle 1 and lower (P < 0.05) expression in cycle 4 of the latter in the S. aureus ex. group. Strain levels, such as SCC and body temperature, following toxin injection correlated well with the magnitude of the immediate decline in follicular steroids. As is typical for Gram-negative clinical events, E. coli LPS-induced acute mastitis caused immediate, short-term, but not long-term impairment of follicular responses, whereas the Gram-positive S. aureus ex.-induced subclinical mastitis exhibited both immediate and carryover disruptive effects on preovulatory follicle function.     

Virus infection activates IL-1 beta and IL-18 production in human macrophages by a caspase-1-dependent pathway.

Monocytes and macrophages play a significant role in host's defense system, since they produce a number of cytokines in response to microbial infections. We have studied IL-1 beta, IL-18, IFN-alpha/beta, and TNF-alpha gene expression and protein production in human primary monocytes and GM-CSF-differentiated macrophages during influenza A and Sendai virus infections. Virus-infected monocytes released only small amounts of IL-1 beta or IL-18 protein, whereas 7- and 14-day-old GM-CSF-differentiated macrophages readily produced these cytokines. Constitutive expression of proIL-18 was seen in monocytes and macrophages, and the expression of it was enhanced during monocyte/macrophage differentiation. Expression of IL-18 mRNA was clearly induced only by Sendai virus, whereas both influenza A and Sendai viruses induced IL-1 beta mRNA expression. Since caspase-1 is known to cleave proIL-1 beta and proIL-18 into their mature, active forms, we analyzed the effect of a specific caspase-1 inhibitor on virus-induced IL-1 beta and IL-18 production. The release of IL-1 beta and IL-18, but not that of IFN-alpha/beta or TNF-alpha, was clearly blocked by the inhibitor. Our results suggest that the cellular differentiation is a crucial factor that affects the capacity of monocytes/macrophages to produce IL-1 beta and IL-18 in response to virus infections. Furthermore, the virus-induced activation of caspase-1 is required for the efficient production of biologically active IL-1 beta and IL-18.     

Lactobacillus rhamnosus GR-1 Ameliorates Escherichia coli-Induced Inflammation and Cell Damage via Attenuation of ASC-Independent NLRP3 Inflammasome Activation

Escherichia coli is a major environmental pathogen causing bovine mastitis, which leads to mammary tissue damage and cell death. We explored the effects of the probiotic Lactobacillus rhamnosus GR-1 on ameliorating E. coli-induced inflammation and cell damage in primary bovine mammary epithelial cells (BMECs). Increased Toll-like receptor 4 (TLR4), NOD1, and NOD2 mRNA expression was observed following E. coli challenge, but this increase was attenuated by L. rhamnosus GR-1 pretreatment. Immunofluorescence and Western blot analyses revealed that L. rhamnosus GR-1 pretreatment decreased the E. coli-induced increases in the expression of the NOD-like receptor family member pyrin domain-containing protein 3 (NLRP3) and the serine protease caspase 1. However, expression of the adaptor protein apoptosis-associated speck-like protein (ASC, encoded by the Pycard gene) was decreased during E. coli infection, even with L. rhamnosus GR-1 pretreatment. Pretreatment with L. rhamnosus GR-1 counteracted the E. coli-induced increases in interleukin-1β (IL-1β), -6, -8, and -18 and tumor necrosis factor alpha mRNA expression but upregulated IL-10 mRNA expression. Our data indicate that L. rhamnosus GR-1 reduces the adhesion of E. coli to BMECs, subsequently ameliorating E. coli-induced disruption of cellular morphology and ultrastructure and limiting detrimental inflammatory responses, partly via promoting TLR2 and NOD1 synergism and attenuating ASC-independent NLRP3 inflammasome activation. Although the residual pathogenic activity of L. rhamnosus, the dosage regimen, and the means of probiotic supplementation in cattle remain undefined, our data enhance our understanding of the mechanism of action of this candidate probiotic, allowing for development of specific probiotic-based therapies and strategies for preventing pathogenic infection of the bovine mammary gland.     

IL-1RI (interleukin-1 receptor type I) signalling is essential for host defence and hemichannel activity during acute central nervous system bacterial infection

Staphylococcus aureus is a common aetiological agent of bacterial brain abscesses. We have previously established that a considerable IL-1 (interleukin-1) response is elicited immediately following S. aureus infection, where the cytokine can exert pleiotropic effects on glial activation and blood–brain barrier permeability. To assess the combined actions of IL-1α and IL-1β during CNS (central nervous system) infection, host defence responses were evaluated in IL-1RI (IL-1 receptor type I) KO (knockout) animals. IL-1RI KO mice were exquisitely sensitive to intracerebral S. aureus infection, as demonstrated by enhanced mortality rates and bacterial burdens within the first 24 h following pathogen exposure compared with WT (wild-type) animals. Loss of IL-1RI signalling also dampened the expression of select cytokines and chemokines, concomitant with significant reductions in neutrophil and macrophage infiltrates into the brain. In addition, the opening of astrocyte hemichannels during acute infection was shown to be dependent on IL-1RI activity. Collectively, these results demonstrate that IL-1RI signalling plays a pivotal role in the genesis of immune responses during the acute stage of brain abscess development through S. aureus containment, inflammatory mediator production, peripheral immune cell recruitment, and regulation of astrocyte hemichannel activity. Taken in the context of previous studies with MyD88 (myeloid differentiation primary response gene 88) and TLR2 (Toll-like receptor 2) KO animals, the current report advances our understanding of MyD88-dependent cascades and implicates IL-1RI signalling as a major antimicrobial effector pathway during acute brain-abscess formation.     

Febrile temperatures attenuate IL-1 beta release by inhibiting proteolytic processing of the proform and influence Th1/Th2 balance by favoring Th2 cytokines

We investigated possible feedback mechanisms of febrile temperatures on LPS- and staphylococcal enterotoxin B (SEB)-induced cytokine release in human whole blood. LPS-induced IL-1beta release was inhibited at temperatures >38 degrees C, whereas intracellular proIL-1beta formation as well as the release of other cytokines except IL-18 were only attenuated above 42 degrees C, indicating that febrile temperatures impair the proteolytic processing of proIL-1beta. This attenuated processing is not due to either heat inactivation of caspase-1 or structural changes in proIL-1beta produced at higher temperatures. Instead, we propose that febrile conditions change cytosolic compartmentation or trafficking, so that synthesized proIL-1beta cannot encounter caspase-1. Febrile temperatures also influenced Th1/Th2 cytokine balance. We observed a 3-fold increase in the Th2-cytokines IL-5 and IL-13 and a reduction to 15% of the Th1-cytokine IL-2 when SEB-stimulated whole blood was incubated at 40 degrees C compared with 37 degrees C. These results indicate that fever limits the production of the fever-inducing IL-1beta and also influences the adaptive immune response, favoring Th2 cytokine production.     

Inflammasome components caspase-1 and adaptor protein apoptosis-associated speck-like proteins are important in resistance to Cryptosporidium parvum

Cryptosporidium spp. are opportunistic protozoan parasites that infect epithelial cells in the intestinal tract and cause a flu-like diarrheal illness. Innate immunity is key to limiting the expansion of parasitic stages early in infection. One mechanism in which it does this is through the generation of early cytokines, such as IL-18. The processing and secretion of mature IL-18 (and IL-1β) is mediated by caspase-1 which is activated within an inflammasome following the engagement of inflammasome-initiating sensors. We examined how the absence of caspase-1 and caspase-11, the adapter protein Asc, and other inflammasome components affects susceptibility to cryptosporidial infection by these and other key cytokines in the gut. We found that Casp-11^?/?Casp-1^?/? knockout mice have increased susceptibility to Cryptosporidium parvum infection as demonstrated by the 35-fold higher oocyst production (at peak infection) compared to wild-type mice. Susceptibility correlated with a lack of IL-18 in caspase-1 and caspase1/11 knockout mice, whereas IL-18 is significantly elevated in wildtype mice. IL-1β was not generated in any significant amount following infection nor was any increased susceptibility observed in IL-1β knockout mice. We also show that the adapter protein Asc is important to susceptibility, and that the caspase-1 canonical inflammasome signaling pathway is the dominant pathway in C. parvum resistance.     

Endotoxin-stimulated monocytes release multiple forms of IL-1 beta, including a proIL-1 beta form whose detection is affected by export

The processing and release of 31-kDa proIL-1 beta to the mature 17-kDa form of IL-1 beta are still poorly understood. To help elucidate the mechanisms involved in IL-1 beta processing and release, we measured IL-1 beta forms released from endotoxin-stimulated monocytes by immunoprecipitation of [35S]methionine-labeled protein, by Western blots, and by our recently developed ELISA specific for proIL-1 beta. Our studies demonstrate that in addition to the 17-kDa mature IL-1 beta, IL-1 beta is also released as 31-, 28-, and 3-kDa molecules. The 31-kDa-released form of proIL-1 beta represented 20-40% of the total released IL-1 beta, as measured by SDS-PAGE with densitometry. This released proIL-1 beta was susceptible to ICE processing; however, this proIL-1 beta was not detectable by either a mature or proIL-1 beta-specific ELISA, suggesting that release induces a conformational change. The ELISA inability to detect proIL-1 beta was not due to inadequate sensitivity or subsequent degradation in the ELISA. Furthermore, while immunoaffinity-purified cytosolic proIL-1 beta could complex the type II IL-1R, released proIL-1 beta did not. Finally, the absence of a band shift in nondenaturing gel electrophoresis excluded proIL-1 beta binding to another protein. These findings imply that IL-1 beta is exported from monocytes as 3-, 17-, 28-, and 31-kDa forms and that the released 31-kDa form differs from cytosolic proIL-1 beta.     

The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta

Generation of Interleukin (IL)-1beta via cleavage of its proform requires the activity of caspase-1 (and caspase-11 in mice), but the mechanism involved in the activation of the proinflammatory caspases remains elusive. Here we report the identification of a caspase-activating complex that we call the inflammasome. The inflammasome comprises caspase-1, caspase-5, Pycard/Asc, and NALP1, a Pyrin domain-containing protein sharing structural homology with NODs. Using a cell-free system, we show that proinflammatory caspase activation and proIL-1beta processing is lost upon prior immunodepletion of Pycard. Moreover, expression of a dominant-negative form of Pycard in differentiated THP-1 cells blocks proIL-1beta maturation and activation of inflammatory caspases induced by LPS in vivo. Thus, the inflammasome constitutes an important arm of the innate immunity.     

Gene Expression-Based Classifiers Identify Staphylococcus aureus Infection in Mice and Humans

Staphylococcus aureus causes a spectrum of human infection. Diagnostic delays and uncertainty lead to treatment delays and inappropriate antibiotic use. A growing literature suggests the host’s inflammatory response to the pathogen represents a potential tool to improve upon current diagnostics. The hypothesis of this study is that the host responds differently to S. aureus than to E. coli infection in a quantifiable way, providing a new diagnostic avenue. This study uses Bayesian sparse factor modeling and penalized binary regression to define peripheral blood gene-expression classifiers of murine and human S. aureus infection. The murine-derived classifier distinguished S. aureus infection from healthy controls and Escherichia coli-infected mice across a range of conditions (mouse and bacterial strain, time post infection) and was validated in outbred mice (AUC>0.97). A S. aureus classifier derived from a cohort of 94 human subjects distinguished S. aureus blood stream infection (BSI) from healthy subjects (AUC 0.99) and E. coli BSI (AUC 0.84). Murine and human responses to S. aureus infection share common biological pathways, allowing the murine model to classify S. aureus BSI in humans (AUC 0.84). Both murine and human S. aureus classifiers were validated in an independent human cohort (AUC 0.95 and 0.92, respectively). The approach described here lends insight into the conserved and disparate pathways utilized by mice and humans in response to these infections. Furthermore, this study advances our understanding of S. aureus infection; the host response to it; and identifies new diagnostic and therapeutic avenues.     

Saikosaponin A alleviates Staphylococcus aureus-induced mastitis in mice by inhibiting ferroptosis via SIRT1/Nrf2 pathway

Mastitis is a common and serious bacterial infection of the mammary gland. Saikosaponin A (SSA) is a triterpenoid saponin isolated from Bupleurum falcatum that has the ability to treat various diseases. However, little is known about the role of SSA in achieving mastitis remission. Here, we found that SSA alleviated Staphylococcus aureus (S. aureus)-induced mastitis by attenuating inflammation and maintaining blood-milk barrier integrity. Furthermore, S. aureus activated nuclear factor kappa B (NF-κB) pathway by upregulated p-p65 and p-IκB. S. aureus also induced ferroptosis in mammary gland in mice, mainly characterized by excessive iron accumulation, mitochondrial morphological changes and impaired antioxidant production. However, S. aureus-induced NF-κB activation and ferroptosis were prevented by SSA. Moreover, SAA could upregulate the expression of SIRT1, Nrf2, HO-1 and GPX4. And the inhibitory effects of SAA on inflammation and ferroptosis were reversed by SIRT1 inhibitor EX-527. In conclusion, SAA protected S. aureus-induced mastitis through suppressing inflammation and ferroptosis by activating SIRT1/Nrf2 pathway.     

Interleukin-18 induces expression and release of cytokines from murine glial cells: interactions with interleukin-1 beta

Interleukin (IL)-18, a member of the IL-1 cytokine family, is an important mediator of peripheral inflammation and host defence responses. IL-1 is a key proinflammatory cytokine in the brain, but the role of IL-18 in the CNS is not yet clear. The objective of this study was to investigate the actions of IL-18 on mouse glial cells. IL-18 induced intracellular expression of IL-1 alpha and proIL-1 beta, and release of IL-6 from mixed glia. Treatment of lipopolysaccharide-primed microglia with adenosine triphosphate (ATP), an endogenous secondary stimulus, induced IL-1 beta and IL-18 release. Although deletion of the IL-18 gene did not affect IL-1 beta expression or release in this experimental paradigm, IL-1 beta knockout microglia released significantly less IL-18 compared to wild-type microglia. In addition, ATP induced release of mature IL-1 beta from IL-18-primed microglia. These data suggest that IL-18 may contribute to inflammatory responses in the brain, and demonstrate that, in spite of several common features, IL-18 and IL-1 beta differ in their regulation and actions.     

Gas6 negatively regulates the Staphylococcus aureus-induced inflammatory response via TLR signaling in the mouse mammary gland

Staphylococcus aureus (S. aureus)-induced mastitis is the most frequent, pathogenic, and prevalent infection of the mammary gland. The ligand growth arrest-specific 6 (Gas6) is a secretory protein that binds to and activates Tyro3, Axl, and MerTK receptors. This study explored the role of Gas6 in S. aureus-induced mastitis. Our results revealed that TLR receptors initiate the innate immune response in mammary gland tissues and epithelial cells and that introducing S. aureus activates TLR2 and TLR6 to drive multiple intracellular mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) pathways. Moreover, S. aureus also induces Gas6, which then activates the TAM receptor kinase pathway, which is related to the inhibition of TLR2- and TLR6-mediated inflammatory pathways through SOCS1 and SOCS3 induction. Gas6 absence alone was found to be involved in the downregulation of TAM receptor-mediated anti-inflammatory effects by inducing significantly prominent expression of TRAF6 and low protein and messenger RNA expression of SOCS1 and SOCS3. S. aureus-induced MAPK and NF-ĸB p65 phosphorylation were also dependent on Gas6, which negatively regulated the production of Pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in S. aureus-treated mammary tissues and mammary epithelial cells. Our in vivo and in vitro study uncovered the Gas6-mediated negative feedback mechanism, which inhibits TLR2- and TLR6-mediated MAPK and NF-ĸB signaling by activating TAM receptor kinase (MerTK, Axl, and Tyro3) through the induction of SOCS1/SOCS3 proteins.     

Differential regulation of CCL-11/eotaxin-1 and CXCL-8/IL-8 by Gram-positive and Gram-negative bacteria in human airway smooth muscle cells

Background

Bacterial infections are a cause of exacerbation of airway disease. Airway smooth muscle cells (ASMC) are a source of inflammatory cytokines/chemokines that may propagate local airway inflammatory responses. We hypothesize that bacteria and bacterial products could induce cytokine/chemokine release from ASMC.

Methods

Human ASMC were grown in culture and treated with whole bacteria or pathogen associated molecular patterns (PAMPs) for 24 or 48 h. The release of eotaxin-1, CXCL-8 or GMCSF was measured by ELISA.

Results

Gram-negative E. coli or Gram-positive S. aureus increased the release of CXCL-8, as did IL-1β, LPS, FSL-1 and Pam₃CSK4, whereas FK565, MODLys18 or Poly I:C did not. E. coli inhibited eotaxin-1 release under control conditions and after stimulation with IL-1β. S. aureus tended to inhibit eotaxin-1 release stimulated with IL-1β. E. coli or LPS, but not S. aureus, induced the release of GMCSF.

Conclusion

Gram-positive or Gram-negative bacteria activate human ASMC to release CXCL-8. By contrast Gram-negative bacteria inhibited the release of eotaxin-1 from human ASMCs. E. coli, but not S. aureus induced GMCSF release from cells.Our findings that ASMC can respond directly to Gram-negative and Gram-positive bacteria by releasing the neutrophil selective chemokine, CXCL-8, is consistent with what we know about the role of neutrophil recruitment in bacterial infections in the lung. Our findings that bacteria inhibit the release of the eosinophil selective chemokine, eotaxin-1 may help to explain the mechanisms by which bacterial immunotherapy reduces allergic inflammation in the lung.     

Biological activity and receptor binding of human prointerleukin-1 beta and subpeptides

We report here that the human interleukin-1 beta precursor (proIL-1 beta) protein as well as several interleukin-1 beta (IL-1 beta) subpeptides bind cellular receptors specifically and exhibit biological activity by stimulating proliferation of helper T-cells. IL-1 beta polypeptides have been synthesized by in vitro translation of mRNAs transcribed from plasmid vectors containing the bacteriophage SP6 promoter joined to the complete IL-1 beta cDNA or to deletion constructs. The quantity of IL-1 beta in vitro translation products was increased significantly by replacing the cognate IL-1 beta untranslated leader sequence with a 37-nucleotide plant viral untranslated leader. Translation of chimeric mRNAs followed by direct bioactivity assay demonstrated that mature IL-1 beta-(117-269), proIL-1 beta-(1-269), and peptide IL-1-(71-269) were all biologically active. Specific binding to cellular receptors was observed with these three IL-1 beta molecules; moreover, several peptides with minimal biological activity also bound receptor specifically. The biological activity and receptor binding properties of the IL-1 beta proteins reported here contrast with those described by Mosley et al. (Mosley, B., Urdal, D. L., Prickett, K. S., Larsen, A., Cosman, D., Conlon, P. J., Gillis, S., and Dower, S. K. (1987) J. Biol. Chem. 262, 2941-2944; Mosley, B., Dower, S. K., Gillis, S., and Cosman, D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4572-4576), who reported that proIL-1 beta-(1-269) had no biological activity and does not bind receptor. Our results indicate that proIL-1 beta is active at a relatively high concentration, and analysis of the proIL-1 beta-(1-269) and IL-1-(71-269) bioactivity data suggests a possible relationship with membrane-bound IL-1.     

NF-kappaB- and C/EBPbeta-driven interleukin-1beta gene expression and PAK1-mediated caspase-1 activation play essential roles in interleukin-1beta release from Helicobacter pylori lipopolysaccharide-stimulated macrophages

Helicobacter pylori is a Gram-negative microaerophilic bacterium that causes chronic gastritis, peptic ulcer, and gastric carcinoma. Interleukin-1beta (IL-1beta) is one of the potent proinflammatory cytokines elicited by H. pylori infection. We have evaluated the role of H. pylori lipopolysaccharide (LPS) as one of the mediators of IL-1beta release and dissected the signaling pathways leading to LPS-induced IL-1beta secretion. We demonstrate that both the NF-kappaB and the C/EBPbeta-binding elements of the IL-1beta promoter drive LPS-induced IL-1beta gene expression. NF-kappaB activation requires the classical TLR4-initiated signaling cascade leading to IkappaB phosphorylation as well as PI-3K/Rac1/p21-activated kinase (PAK) 1 signaling, whereas C/EBPbeta activation requires PI-3K/Akt/p38 mitogen-activated protein (MAP) kinase signaling. We observed a direct interaction between activated p38 MAP kinase and C/EBPbeta, suggesting that p38 MAPK is the immediate upstream kinase responsible for activating C/EBPbeta. Most important, we observed a role of Rac1/PAK1 signaling in activation of caspase-1, which is necessary for maturation of pro-IL-1beta. H. pylori LPS induced direct interaction between PAK1 and caspase-1, which was inhibited in cells transfected with dominant-negative Rac1. PAK1 immunoprecipitated from lysates of H. pylori LPS-challenged cells was able to phosphorylate recombinant caspase-1, but not its S376A mutant. LPS-induced caspase-1 activation was abrogated in cells transfected with caspase-1(S376A). Taken together, these results suggested a role of PAK1-induced phosphorylation of caspase-1 at Ser376 in activation of caspase-1. To the best of our knowledge our studies show for the first time that LPS-induced Rac1/PAK1 signaling leading to caspase-1 phosphorylation is crucial for caspase-1 activation. These studies also provide detailed insight into the regulation of IL-1beta gene expression by H. pylori LPS and are particularly important in the light of the observations that IL-1beta gene polymorphisms are associated with increased risk of H. pylori-associated gastric cancer.