Accumulating evidence indicates development of local extracellular acidosis in inflamed tissues in response to infection and tissue injury. Activation of infiltrating neutrophils contributes to a transient decrease in pH, which, in turn, triggers innate immunity. In this study, we investigated the impact of extracellular acidosis on neutrophil apoptosis, a critical determinant of the outcome of the inflammatory response and analyzed the underlying signaling pathways. Culture of human isolated neutrophils in mildly acidotic conditions (pH 6.5-7.0) resulted in activation of NF-κB; intracellular accumulation of cAMP; and phosphorylation of Akt, ERK, and p38 MAPK; and preservation of Mcl-1 expression. Consequently, extracellular acidosis prevented disruption of mitochondrial transmembrane potential and translocation of cytochrome and apoptosis-inducing factor from the mitochondria to cytoplasm and nuclei, respectively and inhibited caspase-3 activity. Pharmacological inhibition of ERK, PI3K, NF-κB, or PKA partially reversed survival cues by extracellular acidosis and redirected neutrophils to apoptosis. Conversely, dibutyryl cAMP (100-500 μM) delayed apoptosis of neutrophils cultured at pH 7.4. Extracellular acidosis-generated survival cues were additive to the potent prosurvival signals from bacterial DNA, LPS, modified C-reactive protein, and serum amyloid A. Acidosis increased CpG DNA uptake by neutrophils and augmented phosphorylation of ERK and Akt, leading to preservation of Mcl-1 expression. Our results identified extracellular acidosis as a survival signal for neutrophils by suppressing the constitutive apoptotic machinery and suggest that transient decreases in local pH can enhance neutrophil responses to inflammatory stimuli, thereby contributing to amplification or prolongation of the inflammatory response.

Neutrophil dysfunction, resulting in delayed apoptosis and inefficient bacterial clearance, is a characteristic feature of severe pathologies, including sepsis and cystic fibrosis. Human neutrophils detect and respond to bacterial DNA (CpG DNA) through TLR9. We investigated the impact of CpG DNA on phagocytosis, phagocytosis-induced neutrophil apoptosis and clearance of E coli. Culture of human neutrophils with CpG DNA (0.1–3.2 μg/ml) resulted in decreased phagocytosis of opsonized E. coli or yeast. CpG DNA upregulated C3R (CD11b) expression, downregulated C5aR (CD88) expression and induced release of neutrophil elastase. C5aR cleavage was prevented by a specific neutrophil elastase inhibitor and the broad-spectrum serine protease inhibitor PMSF. Consistently, CpG DNA reduced phagocytosis-induced NADPH oxidase-mediated activation of caspase8 and caspase-3. These actions of CpG DNA were blocked by the telomere-derived TLR9 inhibitory oligonucleotide 5′-TTT AGG GTT AGG GTT AGG G-3′. In mice, CpG DNA impaired pulmonary clearance of E coli, suppressed neutrophil apoptosis and delayed resolution of lung injury evoked by intratracheal instillation of live E. coli. Genetic deletion of TLR9 rendered mice unresponsive to CpG DNA. These results identify a novel mechanism, neutrophil elastase-mediated inactivation of C5aR-mediated phagocytosis, by which CpG DNA could contribute to neutrophil dysfunction and prolongation of tissue injury. Our findings also suggest a therapeutic potential for TLR9 antagonists or neutrophil elastase inhibitors for enhancing clearance of bacterial infections in an environment where bacterial DNA is abundantly present.

Emerging evidence indicates that local acidosis associated with infection and tissue injury triggers innate and adaptive immunity. Activation of infiltrating neutrophils contributes to transient drop in pH. We investigated the impact of extracellular acidosis on neutrophil apoptosis, one of the check points in the outcome of the inflammatory response, and on the survival signals generated by bacterial DNA and serum amyloid A. Culture of human isolated neutrophils under mild acidosis (pH 6.5–7.0) resulted in concurrent activation of NF-κB, adenyl cyclase and the ERK and PI3K/Akt signaling pathways, leading to preservation of Mcl-1. Consequently, extracellular acidosis prevented disruption of mitochondrial transmembrane potential and translocation of cytochrome c, endonuclease G and apoptosis-inducing factor (AIF) from the mitochondria to cytoplasm and nuclei, respectively and attenuated activation of caspase-3. Pharmacological inhibition of ERK, PI3K, NF-κB and adenylcyclase partially reversed the anti-apoptotic action of acidosis. Furthermore, mild acidosis enhanced the pro-survival signal from bacterial DNA and serum amyloid A by enhancing ERK and PI3K-mediated inhibition of Mcl-1 degradation. Our results identify mild acidosis as a survival signal for neutrophils by suppressing the constitutive apoptotic machinery and suggest that transient decreases in local pH could contribute to amplification of inflammation. Funding Support: Supported by grants from CIHR (MOP-97742).