Osteopontin plays diverse cellular functions in innate immune system starting from promoting T helper cell type 1 (Th1) skewing and extended through the differentiation and proliferation of immune reactive cells [
31,
36,
37]. OPN also functions as a proinflammatory cytokine and recruits inflammatory cells to exaggerate tissue damage [
11]. Considering its above features, we aim to reveal its expression, function and therapeutic implication in murine experimental sepsis. Our current study demonstrates an increased expression of OPN in lungs and spleen tissues, as well as in the plasma of septic animals. A strategy utilizing the neutralizing Ab against OPN dramatically reduced systemic inflammatory responses, organ injuries, and neutrophil infiltration into the lungs, leading to the improvement of the integrity of lung architecture and downregulation of MPO activity after sepsis. It is reasonable to think that the overall improvement of sepsis by neutralizing OPN could be due to the inhibition of systemic inflammatory and injury markers in general. Correspondingly, the decreased levels of MIP-2, a potent chemoattractant for neutrophils in plasma as well as in lungs after sepsis in animals treated with anti-OPN Ab might also indirectly regulate excess neutrophil migration at the infectious foci as well as in remote organs. Interestingly, our
in vivo finding demonstrating excessive neutrophil migration in lungs as a result of intratracheal rmOPN injection provided direct involvement of OPN-mediated induction of ALI (Figure
6A,B), which might in turn be reversed by administrating neutralizing Ab against OPN to attenuate lung neutrophil migration in sepsis-induced ALI. Moreover, our
in vitro approaches clearly confirmed OPN as a potent chemoattractant for neutrophil migration directly by upregulating MAP kinase pathway molecules, p38 and ERK with the fact that utilizing their inhibitors greatly diminished the OPN-mediated neutrophil migration. OPN can recognize several integrins, such as α
vβ
3, α
vβ
5, α
vβ
1, α
8β
1 and others as its receptors for signal transduction [
38]. Since these integrins are known to activate FAK [
39-
41], it is therefore meaningful to examine the effect of OPN for FAK activation in neutrophil cells.
OPN is strikingly upregulated at the sites of inflammation and during tissue remodeling [
42,
43]. As a proinflammatory cytokine, increased levels of OPN have been demonstrated in a variety of chronic inflammatory diseases including Crohn’s disease, cancer, atherosclerosis, aortic abdominal aneurysms, and autoimmune diseases [
11-
17]. In contrast, a few studies were performed to focus on its expression during acute inflammatory disease conditions. Studies using the human subjects, Vaschetto
et al. have recently reported increased concentration of plasma OPN in sepsis patients [
27]. In our study, we have also noticed significant upregulation of OPN in plasma as well as in lung and spleen tissues at 20 h after sepsis. Sepsis is characterized by a biphasic immunological phenomenon where the early acute hyperinflammatory phase is accompanied by the immunosuppressive phase at the late stage of sepsis [
1]. In our current study, we have emphasized the early acute phase of sepsis to demonstrate the effects of anti-OPN Ab as a preventive approach. However, the setting up of an anti-OPN Ab-based treatment strategy in sepsis considering its pathophysiological window period in mice would reflect its clinical relevancy. Recently, Vaschetto
et al. measured the levels of OPN for fifteen days in patients with high risk of developing septic shock and noticed increased levels of OPN started at four days before shock development [
44]. Thus, OPN can be considered as the prognostic marker of shock and the therapeutic measures targeting OPN should be implemented based on this window period. However, a recent article strongly suggests that the mouse models to study sepsis differ from what is seen clinically in humans [
45]. This is due to the fact that rodents are highly resistant to most types of induced inflammation compared with humans. Moreover, compared with what is seen in sepsis syndrome in humans, mice within a CLP model tend to have a shorter duration of the disease, which is often terminated with a quite sudden death and less organ failure [
45]. As a result of the discrepancies between human and animal models of sepsis, the actual window period of the disease can vary and the treatment strategy may not be truly transferable to the clinical practice. Considering the aberrant expression of OPN in lungs, we next attempted to delineate whether or not it plays a deleterious role in lungs during sepsis. OPN’s role as a chemoattractant to promote the migration of immune cells to the site of inflammation has been demonstrated in earlier studies [
11,
23]. Nonetheless, the majority of the reports were mainly based on the role of OPN on macrophage migration. Correspondingly, Bruemmer
et al. also have shown that acute macrophage infiltration was dramatically diminished in OPN-null mice compared to wild-type mice in a thioglycollate-induced peritonitis model [
46]. In our current study, we for the first time revealed the role of OPN for neutrophil migration into the lungs during polymicrobial sepsis in mice. Consistent with our findings, OPN was shown to be upregulated and associated with neutrophil and macrophage infiltration in glioblastoma, the most invasive type of brain tumors/glioma [
47]. In our study, administration of the neutralizing Ab against OPN in mice during sepsis greatly ameliorated the contents of infiltrating neutrophils in the lungs, thereby protecting mice from developing ALI induced by sepsis. Similar to our approach, a recent study has also shown that the antibody-mediated neutralization of OPN significantly reduced the obesity-induced inflammation and insulin resistance in mice [
48]. Beside this report, Fortis
et al. have recently demonstrated reduced levels of plasma cytokines and chemokines in OPN knockout mice in a murine model of sepsis [
49], which indirectly validated our approach for attenuating sepsis-associated ALI in mice by the treatment of anti-OPN Ab. Although the above study revealed a deleterious role of OPN in sepsis, the mechanism remained unexplored. In our current study, we therefore not only demonstrated the beneficial outcomes of OPN-neutralizing Ab in sepsis-induced ALI, but also elucidated intracellular signaling events involving the activation of ERK and P38 MAP kinases to govern OPN-induced neutrophil migration. Hence, our approach for utilizing anti-OPN Ab as a potential therapeutic tool for treating ALI caused by excessive neutrophil infiltration is permissible. It has previously been demonstrated that the MAP kinases play a pivotal role in cell migration [
34]. The involvements of ERK and P38 MAP kinases for neutrophil migration have been well characterized in recent studies [
50,
51]. In contrast, the stress-activated protein kinases (SAPK)/Jun amino-terminal kinases (JNK) are best known for their pivotal roles in cellular growth, differentiation, survival, and apoptosis [
52] and are less focused on neutrophil migration. We therefore emphasized evaluating the phosphorylation status of ERK and P38 for OPN-mediated neutrophil migration. Beside this, FAK and its signaling pathways are also involved in cell migration [
53]. In an
in vitro system we have noticed considerable upregulation of ERK, p38 and FAK in human neutrophil cell line treated with rmOPN. Besides, we also confirmed the involvement of ERK and p38 for OPN-mediated neutrophil migration by using their inhibitors, which showed dramatic decrease in OPN-mediated neutrophil migration. Moreover, our study showing remarkable upregulation of pFAK by rmOPN treatment not only indicated the pFAK’s role in cellular migration, but also clarified its upstream signaling event that could be mediated through the interaction of its N-terminal RGD motif and α
vβ
3-integrin. Recently, a bidirectional regulatory role of MAP kinase for neutrophil migration has been demonstrated where the activation of p38 molecule enhanced the chemokine-mediated neutrophil migration, while the activation of ERK pathway inhibited their migration [
50]. In this study, they have utilized a potent chemokine, fMLP, to show the bidirectional regulatory roles of MAP kinases that mediated through the G-protein coupled receptor kinase-2 (GRK-2). On the other hand, we have utilized a different chemoattractive molecule, OPN, which could promote the downstream signaling for neutrophil migration via recognizing its integrin receptor without involving the GRKs.