Opisthorchis viverrini (OV) infection is a major public health problem in South East Asia and is endemic in the Lower Mekong Basin, with 8–10 million residents infected with OV in Thailand and Lao PDR alone (Sripa et al., 2010). Infection with this liver fluke results in hepatobiliary abnormalities such as cholangitis, periductal fibrosis, cholecystitis, and cholelithiasis. Importantly, OV is a known risk factor for the onset of cholangiocarcinoma (CCA), a bile duct cancer with a high rate of mortality (Sripa et al., 2017). Ultrasonography assessment of 3359 participants in Khon Kaen province who were positive for OV after analysis of stool samples, found that around 25 % of OV-infected individuals had developed advanced periductal fibrosis, an inflammatory abnormality of the bile duct that is a risk factor for CCA development, and approx. 1 % of those infected had CCA (Mairiang et al., 2012).

Recently, OV has been shown to be a reservoir for Helicobacter pylori, so individuals may be co-infected with both this bacterium and OV (Deenonpoe et al., 2015). H. pylori is a gram-negative, microaerophilic bacterium that can induce gastric and extra-gastric diseases and is classified as a Class I biological carcinogen (Bouvard et al., 2009). H. pylori was identified in 64.6 % of OV-infected individuals by PCR using primers for ureA in stool samples compared to 29.6 % in an uninfected group (Deenonpoe et al., 2017). In addition, the levels of H. pylori per gram of stool samples matched the intensity of OV infection (Deenonpoe et al., 2017). After coinfection with OV and H. pylori, Syrian hamsters developed more severe hepatobiliary abnormalities and had a lower survival rate than single pathogen-infected animals or in uninfected groups, and in the dual-infected group, high-levels of expression of TNFα, IL-1, and IL-6 were observed (Dangtakot et al., 2017) indicating high degrees of inflammation.

Despite the clearance of OV infection by praziquantel as part of an effective drug treatment and control program, the prevalence of CCA remains high in many regions, especially in Northeast Thailand, and CCA incidence in the Lower Mekong region ranges from 93.8 to 317.6 per 100,000 people (Steele et al., 2018). In addition, two year follow-up studies after treatment with praziquantel showed that H. pylori levels, especially cagA+ strains, were significantly higher in those with relapsed or persistent advanced periductal fibrosis after OV clearance (Phung et al., 2022). Therefore, there is much to still be discovered about OV pathogenesis, including how H. pylori coinfection with OV can contribute to inflammation, liver disease and the development of CCA.

A key virulence factor of H. pylori is, cagA, an oncoprotein encoded by the cagA gene localized on the cag pathogenicity island (cagPAI) and is delivered into host cells by a type-4 secretory system (T4SS) to induce cellular alterations that can lead to pathological changes (Tohidpour, 2016). cagA+ H. pylori induces the infiltration of neutrophils and peripheral mononuclear cells into tissues with the secretion of pro-inflammatory cytokines such as IL-8, IL-10, and TNFα by epithelial cells (Yamaoka et al., 1996), and secretion of IL-8 attracts neutrophils and causes mucosal tissue damage (Crabtree et al., 1994). Therefore, cagA+ H. pylori strains are associated with strong inflammatory responses and severe clinical outcomes (Tohidpour, 2016). cagA+ H. pylori are frequently detected in CCA (36.2 %) but are less common in those with cholelithiasis (9.1 %); moreover, infected individuals with grade 2 and grade 3 liver fibrosis had a higher association with cagA+ H. pylori than those OV- infected individuals without liver fibrosis (Deenonpoe et al., 2017).

Neutrophils are the most dominant white blood cells which play a crucial role in innate immunity. Our group has reported that these innate immune cells may be involved in carcinogenesis due to the release of their cytotoxic products causing damaging bystander effects (Edwards et al., 2018), but the mechanisms by which these cells are activated in OV infection, especially in those co-infected with H. pylori, are largely unknown (Salao et al., 2019). Recently, our group has found that patients with hepatobiliary diseases after chronic OV infection have enhanced neutrophil functions compared to healthy controls, but the mechanism of activation was not defined (Salao et al., 2020). Moreover, the precise role and functions of neutrophils activated by cagA+ H. pylori, and how these contribute to hepatobiliary disease are largely unknown.

Therefore, the aim of this study was to characterize the activation of human neutrophils by H. pylori, and in particular to define the role of cagA in these activation processes. We show that cagA+ and cagA- strains induce neutrophil activation and promote rapid (within 30 minutes) neutrophil death within a sub-population of cells, but promote cell survival in those cells that survive this initial death event. Importantly, these bacteria induce the rapid formation of late-apoptotic/necrotic neutrophils (cells with a leaky plasma membrane) that could promote inflammation and tissue damage, which may help explain the mechanisms that lead to liver disease in individuals co-infected with liver fluke and H. pylori.