Brucella abortus nitric oxide metabolite regulates inflammasome activation and IL‐1β secretion in murine macrophages

NLRP3 inflammasome is a protein complex crucial to caspase‐1 activation and IL‐1β and IL‐18 maturation. This receptor participates in innate immune responses to different pathogens, including the bacteria of genus Brucella. Our group recently demonstrated that Brucella abortus‐induced IL‐1β secretion involves NLRP3 inflammasome and it is partially dependent on mitochondrial ROS production. However, other factors could be involved, such as P2X7‐dependent potassium efflux, membrane destabilization, and cathepsin release. Moreover, there is increasing evidence that nitric oxide acts as a modulator of NLRP3 inflammasome. The aim of this study was to unravel the mechanism of NLRP3 inflammasome activation induced by B. abortus, as well as the involvement of bacterial nitric oxide (NO) as a modulator of this inflammasome pathway. We demonstrated that NO produced by B. abortus can be used by the bacteria to modulate IL‐1β secretion in infected murine macrophages. Additionally, our results suggest that B. abortus‐induced IL‐1β secretion depends on a P2X7‐independent potassium efflux, lysosomal acidification, cathepsin release, mechanisms clearly associated to NLRP3 inflammasome. In summary, our results help to elucidate the molecular mechanisms of NLRP3 activation and regulation during an intracellular bacterial infection.     

IL‐1β activation in response to Staphylococcus aureus lung infection requires inflammasome‐dependent and independent mechanisms

Maintaining balanced levels of IL‐1β is extremely important to avoid host tissue damage during infection. Our goal was to understand the mechanisms behind the reduced pathology and decreased bacterial burdens in Ifnlr1^?/? mice during lung infection with Staphylococcus aureus. Intranasal infection of Ifnlr1^?/? mice with S. aureus led to significantly improved bacterial clearance, survival and decrease of proinflammatory cytokines in the airway including IL‐1β. Ifnlr1^?/? mice treated with recombinant IL‐1β displayed increased bacterial burdens in the airway and lung. IL‐1β levels in neutrophils from Ifnlr1^?/? infected mice lungs were decreased when compared to neutrophils from WT mice. Mice lacking NLRP3 and caspase‐1 had reduced IL‐1β levels 4 h after infection, due to reductions or absence of active caspase‐1 respectively, but levels at 24 h were comparable to WT infected mice. Ifnlr1^?/? infected mice had decreases in both active caspase‐1 and neutrophil elastase indicating an important role for the neutrophil serine protease in IL‐1β processing. By inhibiting neutrophil elastase, we were able to decrease IL‐1β levels by 39% in Nlrp3^?/? infected mice when compared to WT mice. These results highlight the crucial role of both proteases in IL‐1β processing, via inflammasome‐dependent and ‐independent mechanisms.     

H. pylori CagA activates the NLRP3 inflammasome to promote gastric cancer cell migration and invasion

Objective

The CagA (cytotoxin-related gene A, CagA) protein is an important factor for the pathogenicity of Helicobacter pylori (H. pylori). Although H. pylori has previously been shown to activate the NLRP3 inflammasome, it remains unclear what role CagA plays in this process. In the current study, we aimed to investigate the effect of CagA on NLRP3 activation and how it is linked to gastric cancer cell migration and invasion.

Methods

CagA positive H. pylori strain (Hp/CagA⁺) and CagA gene knockout mutant (Hp/ΔCagA) infected and the pcDNA3.1/CagA plasmid transfected gastric epithelial cell lines, respectively. The morphological alterations of cells under a microscope; the NLRP3 inflammasome-related markers: NLRP3, caspase-1, and ASC protein levels were detected by Western blot, IL-1β and IL-18 levels were determined by ELISA; cell migration and invasion were determined by transwell assay; and the pyroptosis levels and intracellular ROS were determined by flow cytometry analysis. Then, pretreated with 5 mM NAC for 2 h and subsequently transfected with the pcDNA3.1/CagA plasmid for 48 h, the effects of NAC pretreatment on CagA-induced NLRP3 inflammasome-related markers expression and cell pyroptosis were examined, finally assessed the effect of CagA on migration and invasion in NLRP3-silenced cells.

Results

We found that Hp/CagA⁺ strain infection and pcDNA3.1/CagA vector transfection result in NLRP3 inflammasome activation, generation of intracellular ROS, and increased invasion and migration of gastric cancer cells. Moreover, we found that ROS inhibition via NAC effectively blocks NLRP3 activation and pyroptosis. Silencing of NLRP3 reduces the effects of CagA on gastric cancer cell migration and invasion.

Conclusion

Our study shows that CagA can promote the invasion and migration of gastric cancer cells by activating NLRP3 inflammasome pathway. These findings provide novel insights into the mechanism of gastric cancer induction by H. pylori.

     

The purinergic 2X7 receptor participates in renal inflammation and injury induced by high‐fat diet: possible role of NLRP3 inflammasome activation

Renal disease associated with type 2 diabetes and the metabolic syndrome is characterized by a distinct inflammatory phenotype. The purinergic 2X₇ receptor (P2X₇R) and the nucleotide‐binding and oligomerization domain‐like receptor containing a pyrin domain 3 (NLRP3) inflammasome have been separately shown to play a role in two models of non‐metabolic chronic kidney disease. Moreover, the NLRP3 inflammasome has been implicated in chronic low‐grade sterile inflammation characterizing metabolic disorders, though the mechanism(s) involved in inflammasome activation under these conditions are still unknown. We investigated the role of P2X₇R (through activation of the NLRP3 inflammasome) in renal inflammation and injury induced by a high‐fat diet, an established model of the metabolic syndrome. On a high‐fat diet, mice lacking P2X₇R developed attenuated renal functional and structural alterations as well as reduced inflammation, fibrosis, and oxidative/carbonyl stress, as compared with wild‐type animals, in the absence of significant differences in metabolic parameters. This was associated with blunted up‐regulation of the NLRP3 inflammasome components NLRP3, apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC), pro‐caspase 1, pro‐interleukin (IL)‐1β, and pro‐IL‐18, as well as reduced inflammasome activation, as evidenced by decreased formation of mature caspase 1, whereas mature IL‐1β and IL‐18 were not detected. Up‐regulated expression of NLRP3 and pro‐caspase 1, post‐translational processing of pro‐caspase‐1, and release of IL‐18 in response to lipopolysaccharide + 2′(3′)‐O‐(4‐benzoylbenzoyl)ATP were attenuated by P2X₇R silencing in cultured mouse podocytes. Protein and mRNA expression of P2X₇R, NLRP3, and ASC were also increased in kidneys from subjects with type 2 diabetes and the metabolic syndrome, showing histologically documented renal disease. These data provide evidence of a major role for the purinergic system, at least in part through activation of the NLRP3 inflammasome, in the process driving ‘metabolic’ renal inflammation and injury and identify P2X₇R and NLRP3 as novel therapeutic targets. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Expression and regulation of the NALP3 inflammasome complex in periodontal diseases

Periodontitis is an infectious process characterized by inflammation affecting the supporting structures of the teeth. Porphyromonas gingivalis is a major oral bacterial species implicated in the pathogenesis of periodontitis. Processing of interleukin (IL)‐1 family cytokines is regulated by an intracellular innate immune response system, known as the NALP3 [nacht domain‐, leucine‐rich repeat‐, and pyrin domain (PYD)‐containing protein 3] inflammasome complex. The aim of the present study was to investigate by quantitative real‐time polymerase chain reaction (PCR) the mRNA expression of NALP3, its effector molecule apoptosis associated speck‐like protein (ASC), its putative antagonist NLRP2 (NLR family, PYD‐containing protein 2), IL‐1β and IL‐18 (i) in gingival tissues from patients with gingivitis (n = 10), chronic periodontitis (n = 18), generalized aggressive periodontitis (n = 20), as well as in healthy subjects (n = 20), (ii) in vitro in a human monocytic cell line (Mono‐Mac‐6), in response to P. gingivalis challenge for 6 h. The clinical data indicate that NALP3 and NLRP2, but not ASC, are expressed at significantly higher levels in the three forms of inflammatory periodontal disease compared to health. Furthermore, a positive correlation was revealed between NALP3 and IL‐1β or IL‐18 expression levels in these tissues. The in vitro data demonstrate that P. gingivalis deregulates the NALP3 inflammasome complex in Mono‐Mac‐6 cells by enhancing NALP3 and down‐regulating NLRP2 and ASC expression. In conclusion, this study reveals a role for the NALP3 inflammasome complex in inflammatory periodontal disease, and provides a mechanistic insight to the host immune responses involved in the pathogenesis of the disease by demonstrating the modulation of this cytokine‐signalling pathway by bacterial challenge.     

NLRP3 inflammasome mediates interleukin‐1β production in immune cells in response to Acinetobacter baumannii and contributes to pulmonary inflammation in mice

Acinetobacter baumannii is a multi‐drug resistant, Gram‐negative bacteria and infection with this organism is one of the major causes of mortality in intensive care units. Inflammasomes are multiprotein oligomers that include caspase‐1, and their activation is required for maturation of interleukin‐1β (IL‐1β). Inflammasome signalling is involved in host defences against various microbial infections, but the precise mechanism by which A. baumannii activates inflammasomes and the roles of relevant signals in host defence against pulmonary A. baumannii infection are unknown. Our results showed that NLRP3, ASC and caspase‐1, but not NLRC4, are required for A. baumannii‐induced production of IL‐1β in macrophages. An inhibitor assay revealed that various pathways, including P2X7R, K⁺ efflux, reactive oxygen species production and release of cathepsins, are involved in IL‐1β production in macrophages in response to A. baumannii. Interleukin‐1β production in bronchoalveolar lavage (BAL) fluid was impaired in NLRP3‐deficient and caspase‐1/11‐deficient mice infected with A. baumannii, compared with that in wild‐type (WT) mice. However, the bacterial loads in BAL fluid and lungs were comparable between WT and NLRP3‐deficient or caspase‐1/11‐deficient mice. The severity of lung pathology was reduced in NLRP3‐ deficient, caspase‐1/11‐ deficient and IL‐1‐receptor‐deficient mice, although the recruitment of immune cells and production of inflammatory cytokines and chemokines were not altered in these mice. These findings indicate that A. baumannii leads to the activation of NLRP3 inflammasome, which mediates IL‐1β production and lung pathology.     

Effect of enterohaemorrhagic Escherichia coli O157:H7‐specific enterohaemolysin on interleukin‐1β production differs between human and mouse macrophages due to the different sensitivity of NLRP3 activation

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 infection in humans can cause acute haemorrhagic colitis and severe haemolytic uraemic syndrome. The role of enterohaemolysin (Ehx) in the pathogenesis of O157:H7‐mediated disease in humans remains undefined. Recent studies have revealed the importance of the inflammatory response in O157:H7 pathogenesis in humans. We previously reported that Ehx markedly induced interleukin‐1β (IL‐1β) production in human macrophages. Here, we investigated the disparity in Ehx‐induced IL‐1β production between human and mouse macrophages and explored the underlying mechanism regarding the activation of NOD‐like receptor family, pyrin domain containing 3 (NLRP3) inflammasomes. In contrast to the effects on human differentiated THP‐1 cells and peripheral blood mononuclear cells, Ehx exerted no effect on IL‐1β production in mouse macrophages and splenocytes because of a disparity in pro‐IL‐1β cleavage into mature IL‐1β upon caspase‐1 activation. Additionally, Ehx significantly contributed to O157:H7‐induced ATP release from THP‐1 cells, which was not detected in mouse macrophages. Confocal microscopy demonstrated that Ehx was a key inducer of cathepsin B release in THP‐1 cells but not in mouse IC‐21 cells upon O157:H7 challenge. Inhibitor experiments indicated that O157:H7‐induced IL‐1β production was largely dependent upon caspase‐1 activation and partially dependent upon ATP signalling and cathepsin B release, which were both involved in NLRP3 activation. Moreover, inhibition of K⁺ efflux drastically diminished O157:H7‐induced IL‐1β production and cytotoxicity. The findings in this study may shed light on whether and how the Ehx contributes to the development of haemolytic uraemic syndrome in human O157:H7 infection.     

Impaired NLRP3 inflammasome activity during fetal development regulates IL‐1β production in human monocytes

Interleukin‐1β (IL‐1β) production is impaired in cord blood monocytes. However, the mechanism underlying this developmental attenuation remains unclear. Here, we analyzed the extent of variability within the Toll‐like receptor (TLR)/NLRP3 inflammasome pathways in human neonates. We show that immature low CD14 expressing/CD16^pos monocytes predominate before 33 weeks of gestation, and that these cells lack production of the pro‐IL‐1β precursor protein upon LPS stimulation. In contrast, high levels of pro‐IL‐1β are produced within high CD14 expressing monocytes, although these cells are unable to secrete mature IL‐1β. The lack of secreted IL‐1β in these monocytes parallels a reduction of NLRP3 induction following TLR stimulation resulting in a lack of caspase‐1 activity before 29 weeks of gestation, whereas expression of the apoptosis‐associated speck‐like protein containing a CARD and function of the P2×7 receptor are preserved. Our analyses also reveal a strong inhibitory effect of placental infection on LPS/ATP‐induced caspase‐1 activity in cord blood monocytes. Lastly, secretion of IL‐1β in preterm neonates is restored to adult levels during the neonatal period, indicating rapid maturation of these responses after birth. Collectively, our data highlight important developmental mechanisms regulating IL‐1β responses early in gestation, in part due to a downregulation of TLR‐mediated NLRP3 expression. Such mechanisms may serve to limit potentially damaging inflammatory responses in a developing fetus.     

Secreted aspartic proteases of Candida albicans activate the NLRP3 inflammasome

In a recent report, we demonstrated that distinct members of the secreted aspartic protease (Sap) family of Candida albicans are able to induce secretion of proinflammatory cytokines by human monocytes, independently of their proteolytic activity and specific pH optima. In particular, C. albicans Sap2 and Sap6 potently induced IL‐1β, TNF‐α, and IL‐6 production. Here, we demonstrate that Sap2 and Sap6 proteins trigger IL‐1β and IL‐18 production through inflammasome activation. This occurs via NLRP3 and caspase‐1 activation, which cleaves pro‐IL‐1β into secreted bioactive IL‐1β, a cytokine that was induced by Saps in monocytes, in monocyte‐derived macrophages and in dendritic cells. Downregulation of NLRP3 by RNA interference strongly reduced the secretion of bioactive IL‐1β. Inflammasome activation required Sap internalization via a clathrin‐dependent mechanism, intracellular induction of K⁺ efflux, and ROS production. Inflammasome activation of monocytes induced by Sap2 and Sap6 differed from that induced by LPS‐ATP in several aspects. Our data reveal novel immunoregulatory mechanisms of C. albicans and suggest that Saps contribute to the pathogenesis of candidiasis by fostering rather than evading host immunity.     

PKC,ERK/p38 MAP kinases and NF‐κB targeted signalling play a role in the expression and release of IL‐1β and CXCL8 in Porphyromonas gingivalis‐infected THP1 cells

Porphyromonas gingivalis is a keystone pathogen in periodontitis and is gaining importance in cardiovascular pathogenesis. Protease‐activated receptors (PARs), toll‐like receptors (TLRs) and nucleotide‐binding oligomerization domain (NOD) on monocytes recognize the structural components on P. gingivalis, inducing inflammatory intermediates. Here, we elucidate the modulation of PARs, TLRs, NODs, and the role of MAPK and NF‐κB in IL‐1β and CXCL8 release. THP1 cells were stimulated with P. gingivalis wild‐type W50 and its isogenic gingipain mutants: Rgp mutant E8 and Kgp mutant K1A. We observed modulation of PARs, TLRs, NOD, IL‐1β and CXCL8 expression by P. gingivalis. Gingipains hydrolyse IL‐1β and CXCL8, which is more evident for IL‐1β accumulation at 24 h. Inhibition of PKC (protein kinase C), p38 and ERK (extracellular signal‐regulated kinases) partially reduced P. gingivalis‐induced IL‐1β at 6 h, whereas PKC and ERK reduced CXCL8 at both 6 and 24 h. Following NF‐κB inhibition, P. gingivalis‐induced IL‐1β and CXCL8 were completely suppressed to basal levels. Overall, TLRs, PARs and NOD possibly act in synergy with PKC, MAPK ERK/p38 and NF‐κB in P. gingivalis‐induced IL‐1β and CXCL8 release from THP1 cells. These pro‐inflammatory cytokines could affect leucocytes in circulation and exacerbate other vascular inflammatory conditions such as atherosclerosis.     

Stimulation of dendritic cells with Helicobacter pylori vacuolating cytotoxin negatively regulates their maturation via the restoration of E2F1

Helicobacter pylori induces an infiltration of dendritic cells (DCs) into the infected gastric mucosa. Although DCs play an important role in the regulation of inflammation, the effects of H. pylori vacuolating cytotoxin (VacA) on DC maturation process have not yet been elucidated. The role of VacA in DC maturation following co‐exposure to Escherichia coli lipopolysaccharide (LPS) was investigated. The treatment of immature DCs with LPS up‐regulated the expression of surface molecules [e.g. CD40, CD80, CD86 and major histocompatibility complex (MHC) class II], as well as the production of cytokines [e.g. interleukin (IL)‐1β, IL‐12p70 and tumour necrosis gactor (TNF)‐α] compared with those of unstimulated controls. Co‐stimulation with H. pylori VacA significantly reduced the up‐regulated DC maturation markers induced by LPS. In addition, VacA sustained the immature state of DCs with high endocytosis and low migratory capacity. The LPS‐induced down‐regulation of E2F1 expression in DCs was recovered by co‐stimulation with VacA. Moreover, suppression of E2F1 by small interfering RNA resulted in a significant recovery of the inhibited DC maturation by VacA. In contrast, VacA did not affect nuclear factor (NF)‐κB responses to LPS and the NF‐κB signal was not associated with VacA‐induced inhibition of DC maturation. These results suggest that the exposure of DCs to H. pylori VacA negatively regulates DC maturation via the restoration of E2F1. The immunomodulatory action of VacA on DCs may contribute to the ability of VacA‐producing H. pylori to establish a persistent infection in the gastric mucosa.     

Listeria monocytogenes is sensed by the NLRP3 and AIM2 inflammasome

The inflammasome pathway functions to regulate caspase‐1 activation in response to a broad range of stimuli. Caspase‐1 activation is required for the maturation of the pivotal pro‐inflammatory cytokines of the pro‐IL‐1β family. In addition, caspase‐1 activation leads to a certain type of cell death known as pyroptosis. Activation of the inflammasome has been shown to play a critical role in the recognition and containment of various microbial pathogens, including the intracellularly replicating Listeria monocytogenes; however, the inflammasome pathways activated during L. monocytogenes infection are only poorly defined. Here, we demonstrate that L. monocytogenes activates both the NLRP3 and the AIM2 inflammasome, with a predominant involvement of the AIM2 inflammasome. In addition, L. monocytogenes‐triggered cell death was diminished in the absence of both AIM2 and NLRP3, and is concomitant with increased intracellular replication of L. monocytogenes. Altogether, these data establish a role for DNA sensing through the AIM2 inflammasome in the detection of intracellularly replicating bacteria.     

Relevance of IL-1 and TNF gene polymorphisms on interleukin-1β and tumor necrosis factor-α gastric mucosal production

We aimed to evaluate the influence of Helicobacter pylori infection and IL-1/TNF gene polymorphisms on interleukin (IL)-1β and tumor necrosis factor (TNF)-α gastric mucosal production. IL-1β and TNF-α levels in homogenized biopsy specimens taken from the antrum and corpus of 81 patients were measured by enzyme-linked immunosorbent assay. Genomic DNA was typed for the IL1B-511, IL1B+3954, variable number of tandem repeat (VNTR) IL1RN, TNFA-308, TNFA-238, LTA NcoI, and LTA Bsi gene polymorphisms by polymerase chain reaction, restriction fragment length polymorphism, and TaqMan assays. H. pylori infection and CagA/VacA antibody status were determined by Western blot. IL-1β and TNF-α protein levels were significantly higher in the gastric antrum of patients infected with H. pylori compared with uninfected patients [9.54 (5.07–16.28) vs. 4.55 (3.69–8.28) pg IL-1β/mg protein, p = 0.004, and 1.5 (0.7–2.71) vs. 0.63 (0.3–1.26) pg TNF-α/mg protein, p = 0.001]. Among H. pylori-infected individuals, carriers of the IL1RN*2 allele had significantly higher antrum mucosal IL-1β levels than noncarriers [15.97 (9.59–26.6) vs. 10.08 (7.72–13.33), p = 0.008]. No association between gastric mucosal TNF-α levels and genotypes of the TNFA and LTA gene polymorphisms was reported. Our results indicate that the VNTR polymorphism of the IL1RN gene influences IL-1β gastric mucosal production in patients infected with H. pylori.     

The protein kinase PKR is critical for LPS‐induced iNOS production but dispensable for inflammasome activation in macrophages

Inflammasomes are multi‐protein platforms that drive the activation of caspase‐1 leading to the processing and secretion of biologically active IL‐1β and IL‐18. Different inflammasomes including NOD‐like receptor (NLR) family pyrin domain‐containing 3 (NLRP3), NLR caspase‐recruitment domain‐containing 4 (NLRC4) and absent in melanoma 2 (AIM2) are activated and assembled in response to distinct microbial or endogenous stimuli. However, the mechanisms by which upstream stimuli trigger inflammasome activation remain poorly understood. Double‐stranded RNA‐activated protein kinase (PKR), a protein kinase activated by viral infection, has been recently shown to be required for the activation of the inflammasomes. Using macrophages from two different mouse strains deficient in PKR, we found that PKR is important for the induction of the inducible nitric oxide synthase (iNOS). However, PKR was dispensable for caspase‐1 activation, processing of pro‐IL‐1β/IL‐18 and secretion of IL‐1β induced by stimuli that trigger the activation of NLRP3, NLRC4 and AIM2. These results indicate that PKR is not required for inflammasome activation in macrophages.     

NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase‐4 and caspase‐5

Humans encode two inflammatory caspases that detect cytoplasmic LPS, caspase‐4 and caspase‐5. When activated, these trigger pyroptotic cell death and caspase‐1‐dependent IL‐1β production; however the mechanism underlying this process is not yet confirmed. We now show that a specific NLRP3 inhibitor, MCC950, prevents caspase‐4/5‐dependent IL‐1β production elicited by transfected LPS. Given that both caspase‐4 and caspase‐5 can detect cytoplasmic LPS, it is possible that these proteins exhibit some degree of redundancy. Therefore, we generated human monocytic cell lines in which caspase‐4 and caspase‐5 were genetically deleted either individually or together. We found that the deletion of caspase‐4 suppressed cell death and IL‐1β production following transfection of LPS into the cytoplasm, or in response to infection with Salmonella typhimurium. Although deletion of caspase‐5 did not confer protection against transfected LPS, cell death and IL‐1β production were reduced after infection with Salmonella. Furthermore, double deletion of caspase‐4 and caspase‐5 had a synergistic effect in the context of Salmonella infection. Our results identify the NLRP3 inflammasome as the specific platform for IL‐1β maturation, downstream of cytoplasmic LPS detection by caspase‐4/5. We also show that both caspase‐4 and caspase‐5 are functionally important for appropriate responses to intracellular Gram‐negative bacteria.     

Regulation of cathepsin X overexpression in H. pylori‐infected gastric epithelial cells and macrophages

Cathepsin X (CTSX) is strongly up‐regulated in Helicobacter pylori‐infected gastric mucosa and intestinal‐type gastric cancer. The overexpression of CTSX is mediated predominantly by associated macrophages; depends on a functional type IV‐secretion system; and leads to increased migration of gastric epithelial cells. In the present study, we analysed the role of CagA in CTSX overexpression and identified H. pylori‐induced inflammatory factors and signalling pathways required for stimulating CTSX expression by H. pylori. Gastric epithelial cells were co‐cultured with macrophages in Transwell chambers of 0.4 µm pore size, enabling exchange of fluids but retracting H. pylori. N87 gastric epithelial cells were infected with H. pylori P1 wild‐type strain in the presence of inhibitors for p38, JNK, and ERK1/2 signal transduction pathways. Furthermore, cytokines and growth factors were tested for their regulatory function using inhibitory antibodies, and their gene expression was studied by quantitative RT‐PCRs and western blots. CTSX is strongly up‐regulated at both the mRNA and the protein levels by TNF‐α, IL‐1β, IL‐6, and IL‐8, depending on cell type. All these cytokines were found to be increased by five‐ to ten‐fold in macrophages by H. pylori infection of co‐cultured N87 gastric epithelial cells. In macrophages, H. pylori up‐regulated CTSX via ERK1/2 signalling pathways, and in N87 cells via JNK irrespective of p38 signalling. Our results suggest that H. pylori induced overexpression of CTSX in macrophages and epithelium through specific cytokines that are initiated by CagA‐dependent pathways in a cell type‐dependent manner. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.