The older the faster: aged neutrophils in inflammation

In this issue of Blood, Uhl et al provide evidence that aged neutrophils arrive at sites of inflammation prior to nonaged neutrophils, dominate the inflammatory focus in terms of numbers, and phagocytize bacteria more efficiently.¹ 

Until now, aged neutrophils were considered end-stage cells whose fate was limited to disposal in specialized organs. Moreover, it was believed that aged neutrophils might migrate poorly to sites of inflammation as their expression of CXCR2, the receptor for several major neutrophil-targeted chemokines, such as CXCL8/interleukin-8, is decreased.²  The discovery that these “age-wise” cells are the first and dominant subtype of neutrophil to be recruited to the sites of infection sheds new light on efficiency and adaptability of the immune system.

Although the life span of neutrophils in circulation in vivo might be somewhat longer than was commonly believed,³  their half-life can still be expressed in hours rather than days. In the absence of infection, where neutrophils are recruited to and die within inflammatory sites, neutrophils in circulation do not die in the bloodstream but rather are eliminated in bone marrow (predominant site), spleen, or liver by specialized macrophages.²  Recently, it has been shown that the aging of neutrophils is microbiota-driven and depends on Toll-like receptors (TLRs), including TLR-2 and TLR-4, in a Myd88-dependent manner.⁴  Uhl et al extend this observation by reporting that these TLRs (TLR-4 in particular) are also critical for recruitment of aged neutrophils to the inflammatory focus, and this recruitment process depends on a p38 MAPK pathway (see figure).

For many years, it has been recognized that aged neutrophils acquire higher expression of CXCR4, a receptor that helps to redirect these cells to bone marrow, a tissue that constitutively produces its ligand, CXCL12/stromal cell-derived factor 1 (SDF-1). Furthermore, aged neutrophils have lower expression of selectin L (CD62L), a molecule that is used during first steps of the transmigration cascade to the inflamed tissues.¹  This marker, however, cannot be used to discriminate aged and nonaged neutrophils during endotoxemia as nonaged neutrophils lose much of their CD62L signal upon LPS challenge, most probably due to the L-selectin shedding, a phenomenon commonly observed in sepsis.⁵  Interestingly, despite higher expression of CXCR4, the redirection of aged neutrophils to bone marrow is halted during endotoxemia. It still remains to be resolved what chemokines/chemokine receptors are responsible for the recruitment of the aged neutrophils to the inflammatory sites. As the expression of CXCR1 is unaltered between the 2 populations, aged and not-aged,⁶  this receptor may be an important candidate molecule mediating recruitment of aged neutrophils. Furthermore, it cannot be excluded that ligands of CXCR1, or the other putative chemokine receptors operating in this system, desensitize CXCR4, preventing cell responsiveness to SDF-1 signal and homing to bone marrow.

Leukocyte recruitment depends on presence of adhesion molecules, and in the case of integrins, on their affinity for respective ligands. Increased expression of numerous adhesion molecules, including lymphocyte function–associated antigen-1 (CD11a/β2), Mac-1 (CD11b/β2), and their ligand ICAM-1, was previously described on aged neutrophils⁴  but Uhl et al report that Mac-1 expression is further increased during LPS-induced inflammation. This molecule is primarily involved in events leading to adherence and crawling of neutrophils during diapedesis. Importantly, Uhl et al showed that ICAM-1 more strongly associated with aged neutrophils and β2 integrins on the aged neutrophil existed in their higher affinity confirmation, allowing for better association with ICAM-1. These findings might provide an explanation for the stronger recruitment of aged neutrophils to inflamed organs even if observed chemokine receptors are not upregulated on the cells. Interestingly, among other molecules involved in adhesion, CD44 levels were also increased on aged neutrophils. This molecule is required for neutrophil accumulation in the systemically inflamed liver and thus might rationalize the absolute dominance of aged vs nonaged neutrophils in this organ during LPS challenge.⁷ 

Probably the most important question resulting from the study by Uhl et al is whether the early, and dominant, infiltration of inflamed tissues and organs by aged neutrophils is a good thing. In the study, the aged cells were shown to exhibit increased capacity to phagocytize both gram-negative and gram-positive bacteria when stimulated with LPS; however, neither respiratory burst (measured also in vivo) nor cytokine production was observed to increase in these aged cells. Considering that the excessive generation of ROS and cytokine storm are deleterious in systemic inflammation, this functional phenotype suggests that aged neutrophils play a positive role in inflammation. In contrast, Zhang et al reported that in response to LPS in vitro, aged neutrophils produce more neutrophil extracellular traps (NETs) and ROS. Considering that NETs themselves can be harmful to bystander cells,⁸  strong accumulation and activity of nonaged neutrophils at the sites of inflammation might also have side effects. Importantly, in line with these latter observations, in some conditions, preventing the recruitment of aged neutrophils has been shown to be protective against tissue damage.⁴ 

It remains to be resolved what consequences result from the recruitment of aged neutrophils prior to the recruitment of the more classical nonaged cells and the dominancy of this aged population at the inflammatory focus. This is especially important given this behavior/phenotype of aged neutrophils was observed not only following LPS challenge but also following both lipoteichoic acid challenge, a key pathogen-associated molecule pattern of gram-positive bacteria, and in response to HMGB-1, a representative damage-associated molecular pattern.¹  Therefore, this early and robust response by aged neutrophils appears to be a universal phenomenon. The upregulated expression of receptors required for pathogen recognition and recruitment certainly naturally predispose these cells to this process, but is it simply the consequence of the aged phenotype or is it designed that these “experienced” neutrophils are called upon as the very first line of defense? These questions remain open and their answers will be key to our understanding of inflammation, tissue damage, and repair.