Contribution of Lipocalin-2 from Both Myeloid Cells and Epithelium/Liver Is Required for Optimal Resistance to K. Pneumonia Infection
Authors: Præstekjær Cramer Elisabeth , Dahl Sara Louise , Rozell Björn , Jermiin Knudsen Kasper , Thomsen Kim , Moser Claus , Borregaard Niels , Cowland Jack B. 
Published: October 2019

Abstract
Introduction
NGAL/lipocalin-2 is a siderophore-binding protein stored in high amounts in specific granules of neutrophils. In addition, expression and constitutive secretion of lipocalin-2 can be induced in macrophages and epithelial cells under inflammatory conditions. In mice, lipocalin-2 is furthermore an acute phase-protein. Siderophores are the strongest iron chelators known and are produced by certain microorganisms to retrieve soluble iron from the host. By preventing uptake of siderophore bound iron, lipocalin-2 is bacteriostatic to bacteria that are dependent on this mechanism for uptake of iron. In accordance, lipocalin-2 knock-out mice are susceptible to infection by such bacteria. It is, however, not known whether it is the induced production of lipocalin-2 in epithelial cells and liver or the delivery of lipocalin-2 from infiltrating myeloid cells (neutrophils and macrophages) that is most important for these mechanisms of host defense against invading pathogens.
Methods
To study the contributions of lipocalin-2 from epithelial cells and liver compared to infiltrating myeloid cells, we used a Klebsiella pneumoniae lung infection model in C57BL/6 mice with chimeric expression of lipocalin-2. Bone marrow transplantation of lethally irradiated mice generated wild type-mice with a lipocalin-2 knock-out bone marrow (WT/KO) expressing lipocalin-2 in epithelium and liver but not in myeloid cells, and conversely knock out-mice with wild-type bone marrow (KO/WT) expressing lipocalin-2 in myeloid cells and not in epithelium and liver. Wild-type mice transplanted with wild-type bone marrow (WT/WT) and knock-out mice transplanted with knock-out bone marrow (KO/KO) were also generated. After 7 weeks of reconstitution, mice were nasally challenged with K. pneumoniae for induction of pneumonia and potential dissemination of the infection. The mice were sacrificed twenty-four hours after inoculation and examined.
Results
Lipocalin-2 levels in broncho alveolar lavage (BAL) fluid were comparable between WT/KO and KO/WT mice. Consistent with this, no difference in bacterial counts (CFU) in BAL fluid was seen. No differences in spleen CFUs were evident between the two chimeric subgroups WT/KO and KO/WT despite a quantitatively larger mean lipocalin-2 plasma level in WT/KO mice (almost 50 times) derived from epithelium and liver compared to the contribution from myeloid cells in KO/WT mice. However, mean CFU in spleen homogenates from KO/KO mice were more than 870 times higher compared to WT/WT mice. Both the lipocalin-2 contribution from myeloid cells and from epithelium and liver appeared to be indispensable judged by the higher spleen CFUs in mice lacking lipocalin-2 from either of the two compartments. Lipocalin-2 mRNA in the liver was present in equal amounts in mice with wild-type background despite the presence or absence of lipocalin-2 in the myeloid cells. No differences in neutrophil influx to the lungs were seen between groups as determined by MPO ELISA on lung homogenates.
We conclude that lipocalin-2 derived both from myeloid cells and from epithelium and liver is required for full resistance to a siderophore-producing pathogen. Despite the higher levels of plasma lipocalin-2 in WT/KO mice compared to KO/WT mice, their bacteriostatic capacity is equal. The induction of lipocalin-2 in the liver is not dependent on the presence of lipocalin-2 in the myeloid cells, just as the migration of neutrophils to the infected lung is not, thus refuting a recent report that lipocalin-2 affects neutrophil migration.

Disclosures
No relevant conflicts of interest to declare.