Here our ex vivo systemic analysis of the impact of

Here, our ex vivo systemic analysis of the impact of extracellular Hb and LTA on whole WBC populations demonstrates that neutrophils are as sensitive as monocytes to metHb. Interestingly other leukocytes in the whole blood system seemed to dampen the synergistic effect of metHb+LTA on the neutrophils. For example, the presence of other blood cell types suppressed further increase of ROS production induced by metHb+LTA compared to metHb in neutrophils; otherwise the metHb+LTA caused synergistic increase in ROS production in isolated neutrophils (Fig. 3b, d). Plausibly, the clearance of metHb by scavenging receptors such as CD163 and (SR)-B1, which are known to be present on macrophages and monocytes, partially relieves the impact of metHb+LTA on the neutrophils. However, metHb- or (metHb+LTA)-induced increase in the expressions of activation markers and cytokines in the other leukocytes indicates that the other leukocytes are more actively engaged in response to the effects of metHb on neutrophils. Particularly, the increase in cell adhesion molecules caused by metHb, LTA or metHb+LTA, in various types of leukocytes suggests that upon encountering metHb and LTA (e.g. in a Gram-positive haemolytic infection), the neutrophils probably interact with other leukocytes through cell–cell contact, and they secrete cytokines and chemokines leading to the modulation of effector cell function at a focal site of inflammation (Figs. 6, 7). The regulation of neutrophil survival and death is critical to resolve inflammation efficiently. TLR agonists and inflammatory cytokines have been reported to delay apoptosis of neutrophils, which is associated with the expression of anti-apoptotic molecules such as survivin protein and myeloid leukaemia cell differentiation protein (Francois et al., 2005; Baumann et al., 2003; Altznauer et al., 2004). Furthermore, activated NK formyl and T cells drive IFNγ and GM-CSF to prolong the human neutrophil survival (Costantini et al., 2010; Pelletier et al., 2010). We found that metHb does not increase TNFα production in isolated neutrophils (Fig. 4), whereas metHb+LTA does. However, metHb induces TNFα in whole WBC, resulting in more severe apoptosis of neutrophils compared to the effect in isolated neutrophils (Fig. 5). It was reported that low concentrations of TNFα enhance the survival of neutrophils, but high concentrations induce apoptosis (Cross et al., 2008). Our results suggest that apoptosis of neutrophils induced by metHb and metHb+LTA, when in the presence of other leukocytes, may be caused by both: (i) TNFα produced from the other types of leukocytes and (ii) ROS produced in the neutrophils. Overall, our findings provide mechanistic insights on how metHb, together with LTA, released during a systemic Gram-positive haemolytic infection triggers neutrophil response and how the other leukocytes might collaborate to orchestrate cellular defence as illustrated in Fig. 8. Future characterization of the proposed unconventional TLR pathway could provide deeper understanding on how our blood system circumvents the danger of the redox-active metHb, and PAMPs to restore homeostasis. The following are the supplementary data related to this article.
Declaration of Interests
Author Contributions
Funding This study was supported by the Ministry of Education, Singapore (grant: MOE2013-T2-2-007) and the NUS Graduate School for Integrative Sciences and Engineering (NGS Grant reference C-154-000-017-091). The funding bodies do not play any role in the study design, data collection, data analysis, interpretation or writing of the manuscript.
Introduction Neisseria meningitidis, a Gram negative diplococcal bacterium, is normally a commensal resident of the oropharynx of a high percentage (10–30%) of the human population, very occasionally, causing life-threatening meningitis and septicaemia (Caugant and Maiden, 2009). The only well-established virulence factor of N. meningitidis is the polysaccharide capsule, which mediates resistance to complement-mediated lysis and opsonophagocytosis. Based on biochemical composition as well as genetic analysis, 12 serogroups have been described of which 6 (serogroups A, B, C, W, Y and, X) are associated with most disease worldwide (Harrison et al., 2013). Capsule polysaccharide conjugate vaccines have been successfully used to induce protective immunity against N. meningitidis serogroups A, C, W and, Y. However, due to similarities between the serogroup B polysaccharide and human glycoprotein structures, no such vaccine targeting this serogroup is available.