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    • Neutrophils are one of the

    2018-11-09

    Neutrophils are one of the first responding immune cells to an infection. The migration of neutrophils into the site of infection–inflammation is mediated by PAMPs from microbes or DAMPs derived from disrupted host cells. At the site of infection, neutrophils rapidly combat pathogens by unleashing ROS and proteases. Furthermore, antimicrobial proteins stored in their cytoplasmic granules are released (Nathan, 2006). The azurophil granules contain myeloperoxidase, defensins, cathepsin G, and elastase, which are released upon activation and degranulation of the neutrophils. Amongst these, elastase which is a major serine protease is involved in various inflammatory responses (Pham, 2006). Neutrophils are known to interact with other leukocytes through cell–cell contact, and they secrete cytokines and chemokines. They modulate dendritic cell maturation and trafficking and are able to cross-present MDV3100 manufacturer to memory CD4+ T cells as well as to naïve CD8+ T cells, which subsequently amplify CD8+ T cell response to the antigen (Beauvillain et al., 2007). Direct interaction between neutrophils and T cells has been shown in the regression of cancer as well as infectious diseases (Stoppacciaro et al., 1993; Ma et al., 2009). ROS, produced by activated neutrophils, inhibits the effector functions of NK cells, while cytokines such as GM-CSF and IFN-γ released from activated NK cells, prolong the survival of neutrophils in an in vitro system (Costantini and Cassatella, 2011). Moreover, depletion of neutrophils impairs the recruitment of monocytes and lymphocytes to the inflammatory site. On the other hand, the immune suppressive capacity of neutrophils in T cell proliferation during acute systemic inflammation has also been reported (Pillay et al., 2012). Considering the diverse functions of neutrophils in inflammation, we envisaged that neutrophils would play a significant role in response to plasma metHb during a haemolytic condition. Therefore, we investigated the response of neutrophils and the other types of blood cells, to metHb and LTA. Here, we show that metHb is an endogenous DAMP ligand for TLR2, and that neutrophils are one of the most sensitive cell types responding to (metHb+LTA)-induced production of ROS. Interestingly, this effect is diminished by the presence of other leukocytes, indicating that the white blood cells communicate with each other to modulate cellular response during a haemolytic infection.
    Materials and Methods
    Results
    Discussion One of the underlying mechanisms of the cytotoxicity of metHb is its ability to generate ROS and scavenge NO; the latter is known to play a protective role in vascular homeostasis. Recent studies in our lab have shown that the POX activity of metHb is triggered synergistically by microbial proteases and PAMPs such as LTA or LPS to produce ROS (O2) which was confirmed in this study (Jiang et al., 2007). Binding of LTA or LPS to metHb causes a conformational change of metHb which may explain the increase in metHb-POX activity. But we found, in this study that ODN2395 dramatically reduced the POX activity of metHb as well as that of metHb+LTA complex (Fig. 1b). The decreased POX activity appears to be due to the direct binding of ODN2395 to metHb, which probably caused allosteric inhibition rather than a direct catalytic site competition against LTA. These results suggest differential modulation of the metHb-POX activity by different PAMPs during a haemolytic infection. By further investigations on the effects of interaction between metHb and LTA on live human leukocytes, we discovered that metHb is an endogenous ligand for TLR2 (Fig. 2a). As confirmed in our experiment, a typical TLR2 activation by LTA has been known to inhibit apoptosis of neutrophils (Lotz et al., 2004). However, we observed metHb to induce apoptosis of neutrophils, which was significantly increased by metHb+LTA complex. As anticipated, blocking TLR2 in isolated neutrophils reduced the LTA-induced ROS production to basal level compared to mock control (buffer treatment). Interestingly, higher levels of ROS were produced by metHb and metHb+LTA compared to mock control, even in the presence of TLR2 antibody. These results suggest that there are other receptors on the neutrophils that may interact with metHb and metHb+LTA complex, besides TLR2. Since innate immune responses are mediated multifariously, it is conceivable that metHb-PAMPs modulate the functions of TLRs to stimulate distinct signalling pathways in diverse immune cells, which warrants future investigation at the systemic level. Supporting this is our earlier demonstration of the co-operation of monocytes with endothelial cells in the endocytosis of extracellular metHb4. Chronic haemolytic conditions, which prevail in sickle cell anaemia, are accompanied by systemic endothelial activation resulting in more adhesion of blood cells (Chen et al., 2011).