InVivoMAb anti-mouse Ly6G/Ly6C

It is well known that neutrophils are rapidly recruited to a site of injury or infection and perform a critical role in pathogen clearance and inflammation. However, they are also able to interact with and regulate innate and adaptive immune cells and some stimuli induce the migration of neutrophils to lymph nodes (LNs). Previously, we demonstrated that the immune complex (IC) generated by injecting OVA into the footpad of OVA/CFA immunized mice induced the migration of OVA(+) neutrophils to draining LNs (dLNs). Here we investigate the effects of these neutrophils which reach dLNs on CD4(+) T cell response. Our findings here strongly support a dual role for neutrophils in dLNs regarding CD4(+) T cell response modulation. On the one hand, the CD4(+) T cell population expands after the influx of OVA(+) neutrophils to dLNs. These CD4(+) T cells enlarge their proliferative response, activation markers and IL-17 and IFN-gamma cytokine production. On the other hand, these neutrophils also restrict CD4(+) T cell expansion. The neutrophils in the dLNs upregulate PD-L1 molecules and are capable of suppressing CD4(+) T cell proliferation. These results indicate that neutrophils migration to dLNs have an important role in the homeostasis of adaptive immunity. This report describes for the first time that the influx of neutrophils to dLNs dependent on IC presence improves CD4(+) T cell response, at the same time controlling CD4(+) T cell proliferation through a PD-L1 dependent mechanism.

Although extracellular ATP is abundant at sites of inflammation, its role in activating inflammasome signalling in neutrophils is not well characterized. In the current study, we demonstrate that human and murine neutrophils express functional cell-surface P2X7R, which leads to ATP-induced loss of intracellular K(+), NLRP3 inflammasome activation and IL-1beta secretion. ATP-induced P2X7R activation caused a sustained increase in intracellular [Ca(2+)], which is indicative of P2X7R channel opening. Although there are multiple polymorphic variants of P2X7R, we found that neutrophils from multiple donors express P2X7R, but with differential efficacies in ATP-induced increase in cytosolic [Ca(2+)]. Neutrophils were also the predominant P2X7R-expressing cells during Streptococcus pneumoniae corneal infection, and P2X7R was required for bacterial clearance. Given the ubiquitous presence of neutrophils and extracellular ATP in multiple inflammatory conditions, ATP-induced P2X7R activation and IL-1beta secretion by neutrophils likely has a significant, wide ranging clinical impact.

To examine the role of caspase-1 and the NLRC4 inflammasome during bacterial infection, C57BL/6, IL-1beta(-/-), caspase-1(-/-), and NLRC4(-/-) mouse corneas were infected with ExoS/T- or ExoU-expressing Pseudomonas aeruginosa. We found that IL-1beta was essential for neutrophil recruitment and bacterial clearance and was produced by myeloid cells rather than resident cells. In addition, neutrophils were found to be the primary source of mature IL-1beta during infection, and there was no significant difference in IL-1beta processing between C57BL/6 and caspase-1(-/-) or NLRC4(-/-) infected corneas. IL-1beta cleavage by human and mouse neutrophils was blocked by serine protease inhibitors and was impaired in infected neutrophil elastase (NE)(-/-) corneas. NE(-/-) mice also had an impaired ability to clear the infection. Together, these results demonstrate that during P. aeruginosa infection, neutrophils are the primary source of mature IL-1beta and that IL-1beta processing is dependent on serine proteases and not NLRC4 or caspase-1.