Age-related decline in the resistance of mice to bacterial infection and in LPS/TLR4 pathway-dependent neutrophil responses

Abstract

Host defense against bacterial and fungal infections diminishes with age. In humans, impaired neutrophil responses are thought to contribute to this decline. However, it remains unclear whether neutrophil responses are also impaired in old mice. Here, we investigated neutrophil function in old mice, focusing on responses primed by lipopolysaccharide (LPS), an endotoxin released by gram-negative bacteria like E. coli, which signals through toll-like receptor (TLR) 4. We show that old mice have a reduced capacity to clear pathogenic E. coli during septic peritonitis. Neutrophil recruitment was elevated during LPS-induced but not aseptic peritonitis. Neutrophils from old mice showed reduced killing of E. coli. Their reactive oxygen species (ROS) production was impaired upon priming with LPS but not with GM-CSF/TNFα. Phagocytosis and degranulation were reduced in a partially LPS-dependent manner, whereas impairment of NET release in response to S. aureus was independent of LPS. Unexpectedly, chemotaxis was normal, as were Rac1 and Rac2 GTPase activities. LPS-primed activation of Erk and p38 Mapk was defective. PIP3 production was reduced upon priming with LPS but not with GM-CSF/TNFα, whereas PIP2 levels were constitutively low. The expression of 5% of neutrophil proteins was dysregulated in old age. Granule proteins, particularly cathepsins and serpins, as well as TLR-pathway proteins and membrane receptors were upregulated, whereas chromatin and RNA regulators were downregulated. The upregulation of CD180 and downregulation of MyD88 likely contribute to the impaired LPS signaling. In summary, all major neutrophil responses except chemotaxis decline with age in mice, particularly upon LPS priming. This LPS/TLR4 pathway dependence resolves previous controversy regarding effects of age on murine neutrophils and confirms that mice are an appropriate model for the decline in human neutrophil function.

Aging modulates homeostatic leukocyte trafficking to the peritoneal cavity in a sex-specific manner

“…Peritonitis is a well-established and widely used model to study leukocyte trafficking during acute inflammation, in which the effects of age 36 , 37 and sex 15 , 38 , 39 on leukocyte recruitment to the peritoneal cavity in this model have been investigated. But studies investigating age-related changes to peritoneal leukocyte populations in the steady state are often limited to male mice.…”

Dock2 generates characteristic spatiotemporal patterns of Rac activity to regulate neutrophil polarisation, migration and phagocytosis

“…Mature primary neutrophils were freshly isolated each day from mouse bone marrow by Percoll PLUS gradient at 4°C using endotoxin-free reagents throughout, essentially as previously described ( 23 , 46 ). Briefly, mouse bone-marrow was flushed from femurs, tibias and pelvic bones with ice-cold Hank’s Balanced Salt Solution (HBSS) without Ca 2+ or Mg 2+ (HBSS – , Sigma H6648) supplemented with 15 mM HEPES, pH 7.4 (RT) (Sigma, H3784) and 0.25% fatty acid-free (FAF) BSA (Sigma, A8806) (HBSS –++ ), triturated and filtered through a 40 μm cell strainer.…”

“…Chemokinesis assays were performed essentially as described ( 46 ), combined with TIRF-FRET imaging of Rac actvity. Isolated neutrophils were resuspended at 4.5 x 10 6 /ml in DPBS ++++ and primed with 50 ng/ml GM-CSF and 20 ng/ml TNFα for 45 min at 37°C.…”

Functions and mechanisms of the GPCR adaptor protein Norbin

“…Neutrophils are tightly regulated, their excessive responses can exacerbate inflammatory diseases such as rheumatoid arthritis [ 37 ], but despite Norbin functioning as an immune-suppressor, autoinflammation was not detected in Ncdn Δmye mice [ 24 ]. Of note, our recent evaluation of the neutrophil proteomes from young and old mice showed no age-related difference in Norbin expression, suggesting no obvious role in inflamm-aging, a state of chronic, low-grade sterile inflammation associated with aging [ 38 ]. Furthermore, in lymphoma-derived MSB-1 cells, Norbin was shown to indirectly affect the phosphorylation of nuclear factor of activated T cells (NFAT), and may thus play a role in transcription [ 39 ].…”