Helicobacter pylori infects the human stomach and causes a spectrum of disease that includes gastritis, peptic ulcers, and gastric adenocarcinoma. A chronic, neutrophil-rich inflammatory response characterizes this infection. It is established that H. pylori stimulates neutrophil chemotaxis and a robust respiratory burst, but other aspects of this interaction are incompletely defined. We demonstrate here that H. pylori induces N1-like subtype differentiation of human neutrophils as indicated by profound nuclear hypersegmentation, a CD62Ldim, CD16bright, CD11bbright, CD66bbright, CD63bright surface phenotype, proinflammatory cytokine secretion, and cytotoxicity. Hypersegmentation requires direct neutrophil-H. pylori contact as well as transcription and both host and bacterial protein synthesis, but not urease, NapA, VacA, CagA or CagT. The concept of neutrophil plasticity is new, and to our knowledge these data are the first evidence that neutrophils can undergo subtype differentiation in vitro in response to bacterial pathogen infection. We hypothesize that these changes favor H. pylori persistence and disease.

Polymorphonuclear leukocytes (PMN) achieve an intermediate or “primed” state of activation following stimulation with certain agonists. Primed PMN have enhanced responsiveness to subsequent stimuli, which can be beneficial in eliminating microbes but may cause host tissue damage in certain disease contexts including sepsis. As PMN priming by TLR4 agonists is well-described, we hypothesized that ligation of TLR2/1 or TLR2/6 would prime PMN. Surprisingly, PMN from only a subset of donors were primed in response to the TLR2/1 agonist, Pam3CSK4, although PMN from all donors were primed by the TLR2/6 agonist, FSL-1. Priming responses included generation of intracellular and extracellular reactive oxygen species (ROS), MAPK phosphorylation, integrin activation, secondary granule exocytosis, and cytokine secretion. Genotyping studies revealed that PMN responsiveness to Pam3CSK4 was enhanced by a common single nucleotide polymorphism (SNP) in TLR1 (rs5743618). Notably, PMN from donors with the SNP had higher surface levels of TLR1, and were demonstrated to have enhanced association of TLR1 with the ER chaperone gp96. We analyzed TLR1 genotypes in a pediatric sepsis database and found that patients with sepsis or septic shock who had a positive blood culture and were homozygous for the SNP associated with neutrophil priming had prolonged pediatric intensive care unit (PICU) length of stay. We conclude that this TLR1 SNP leads to excessive PMN priming in response to cell stimulation. Based on our finding that septic children with this SNP had longer PICU stays, we speculate that this SNP results in hyperinflammation in diseases such as sepsis.

Francisella tularensis infects several cell types including neutrophils, and aberrant neutrophil accumulation contributes to tissue destruction during tularaemia. We demonstrated previously that F. tularensis strains Schu S4 and live vaccine strain markedly delay human neutrophil apoptosis and thereby prolong cell lifespan, but the bacterial factors that mediate this aspect of virulence are undefined. Herein, we demonstrate that bacterial conditioned medium (CM) can delay apoptosis in the absence of direct infection. Biochemical analyses show that CM contained F. tularensis surface factors as well as outer membrane components. Our previous studies excluded roles for lipopolysaccharide and capsule in apoptosis inhibition, and current studies of [14C] acetate‐labelled bacteria argue against a role for other bacterial lipids in this process. At the same time, studies of isogenic mutants indicate that TolC and virulence factors whose expression requires FevR or MglA were also dispensable, demonstrating that apoptosis inhibition does not require Type I or Type VI secretion. Instead, we identified bacterial lipoproteins (BLPs) as active factors in CM. Additional studies of isolated BLPs demonstrated dose‐dependent neutrophil apoptosis inhibition via a TLR2‐dependent mechanism that is significantly influenced by a common polymorphism, rs5743618, in human TLR1. These data provide fundamental new insight into pathogen manipulation of neutrophil lifespan and BLP function.

The systemic inflammatory response syndrome (SIRS) is a clinical condition occurring in intensive care unit patients as a consequence of both infectious and noninfectious insults. The mechanisms underlying resolution of SIRS are not well characterized. NOX2 (NADPH oxidase 2)-derived reactive oxygen species are critical for killing of certain pathogens by polymorphonuclear leukocytes (PMN). Patients with chronic granulomatous disease who lack functional NOX2 are not only prone to serious infections, they also exhibit chronic inflammatory conditions, suggesting a local anti-inflammatory role for NOX2. We hypothesized that NOX2 is required for the resolution of sterile systemic inflammation. Using a murine model of sterile generalized inflammation, we observed dramatically increased mortality of gp91^phox-/y (NOX2-deficient) as compared to wild-type (WT) mice. Both genotypes developed robust SIRS with hypothermia, hypotension, and leukopenia; however, WT mice recovered within 48 h whereas NOX2-deficient mice did not. Although both groups displayed rapid peritoneal PMN recruitment, the recruited NOX2-deficient PMN demonstrated an enhanced inflammatory phenotype. Moreover, NOX2-deficient mice exhibited a hemorrhagic inflammatory response in the lungs with rapid and persistent recruitment of neutrophils to the alveolar space, whereas WT mice had minimal lung pathology. Several proinflammatory cytokines remained elevated in NOX2-deficient mice. The persistent inflammatory environment observed in NOX2-deficient mice resulted from continued peritoneal chemokine secretion and not delayed apoptosis of PMN. These data suggest a requirement for NOX2 in the resolution of systemic inflammation.

Systemic inflammatory response syndrome (SIRS) is a common clinical condition in patients in intensive care units that can lead to complications, including multiple organ dysfunction syndrome (MODS). MODS carries a high mortality rate, and it is unclear why some patients resolve SIRS, whereas others develop MODS. Although oxidant stress has been implicated in the development of MODS, several recent studies have demonstrated a requirement for NADPH oxidase 2 (NOX2)-derived oxidants in limiting inflammation. We recently demonstrated that NOX2 protects against lung injury and mortality in a murine model of SIRS. In the present study, we investigated the role of NOX2-derived oxidants in the progression from SIRS to MODS. Using a murine model of sterile systemic inflammation, we observed significantly greater illness and subacute mortality in gp91(phox-/y) (NOX2-deficient) mice compared with wild-type mice. Cellular analysis revealed continued neutrophil recruitment to the peritoneum and lungs of the NOX2-deficient mice and altered activation states of both neutrophils and macrophages. Histological examination showed multiple organ pathology indicative of MODS in the NOX2-deficient mice, and several inflammatory cytokines were elevated in lungs of the NOX2-deficient mice. Overall, these data suggest that NOX2 function protects against the development of MODS and is required for normal resolution of systemic inflammation.