Concurrent Proinflammatory and Apoptotic Activity of a Helicobacter pylori Protein (HP986) Points to Its Role in Chronic Persistence

Helicobacter pylori induces cytokine mediated changes in gastroduodenal pathophysiology, wherein, the activated macrophages at the sub-mucosal space play a central role in mounting innate immune response against the antigens. The bacterium gains niche through persistent inflammation and local immune-suppression causing peptic ulcer disease or chronic gastritis; the latter being a significant risk factor for the development of gastric adenocarcinoma. What favors persistence of H. pylori in the gastric niches is not clearly understood. We report detailed characterization of a functionally unknown gene (HP986), which was detected in patient isolates associated with peptic ulcer and gastric carcinoma. Expression and purification of recombinant HP986 (rHP986) revealed a novel, ∼29 kDa protein in biologically active form which associates with significant levels of humoral immune responses in diseased individuals (p<0.001). Also, it induced significant levels of TNF-α and Interleukin-8 in cultured human macrophages concurrent to the translocation of nuclear transcription factor-κB (NF-κB). Further, the rHP986 induced apoptosis of cultured macrophages through a Fas mediated pathway. Dissection of the underlying signaling mechanism revealed that rHP986 induces both TNFR1 and Fas expression to lead to apoptosis. We further demonstrated interaction of HP986 with TNFR1 through computational and experimental approaches. Independent proinflammatory and apoptotic responses triggered by rHP986 as shown in this study point to its role, possibly as a survival strategy to gain niche through inflammation and to counter the activated macrophages to avoid clearance

Neutrophil Apoptosis in Neutropenic Patients With Hepatitis C Infection: Role of Caspases 3, 10, and GM-CSF

Patients with chronic HCV infection are prone to increased susceptibility bacterial infection due to neutropenia complicating the course of this disease. Neutropenia in those patients may stem from enhanced neutrophil apoptosis. However, the molecular mechanism of neutrophil apoptosis has not been clearly defined. Neutrophils harvested from 26 neutropenic patients with hepatitis C infection and nine age and sex-matched healthy control subjects were examined for the degree of apoptosis. Neutrophil apoptosis was quantified by flow cytometry through determination of annexin-V expression at 0 time (fresh neutrophil), and 24 h culture. Neutrophils from healthy subjects were also incubated with either 10% heterologous normal or neutropenic sera, with and without 10 µg GM-CSF. Caspases 3, 10 were assessed colormetrically in neutrophils at 0 times and after 24 h culture. At 0 time culture the neutrophil apoptosis of the HCV patients was in significantly higher as compared to that of normal control (P = 0.059). At 24 h culture patients neutrophils cultured with neutropenic patients own sera showed neutrophil apoptosis significantly increased as compared to that at 0 time culture and this effect was significantly attenuated in similar culture with addition of GM-CSF (P < 0.001). On the other hand patient’s neutrophil cultured with normal sera showed insignificantly increased neutrophil apoptosis at 24 h culture as compared to that at 0 time culture. Caspases 3 and 10 activities were significantly higher in patients neutrophil after 24 h cultured with patients own sera as compared to 0 time culture (P < 0.001 for both). Addition of GM-CSF to the neutrophil culture down regulates the caspases 3 and 10 activities. The correlation study between annexin-V expression and caspases activities revealed a borderline positive correlation between annexin-V and caspase 3 (r = 0.376, P = 0.058), and significant positive correlation with caspase 10 activity (r = 0.494, P = 0.01). In conclusion, these findings suggest that enhanced neutrophil apoptosis demonstrated in neutropenic patients with HCV infection might be induced through activation of caspase 10 and is attenuated by GM-CSF