Leptin Receptor q223r Polymorphism Influences Clostridioides difficile Infection-Induced Neutrophil CXCR2 Expression in an Interleukin-1β Dependent Manner

Abstract

Neutrophils are key first-responders in the innate immune response to C. difficile infection (CDI) and play a central role in disease pathogenesis. Studies have clearly shown that tissue neutrophil numbers need to be tightly regulated for optimal CDI outcomes: while excessive colonic neutrophilia is associated with severe CDI, neutrophil depletion also results in worse outcomes. However, the biological mechanisms that control CDI-induced neutrophilia remain poorly defined. C-X-C chemokine receptor 2 (CXCR2) is a chemotactic receptor that is critical in neutrophil mobilization from bone marrow to blood and tissue sites. We have previously reported that a single nucleotide polymorphism (SNP) in leptin receptor (LEPR), present in up to 50% of people, influenced CDI-induced neutrophil CXCR2 expression and tissue neutrophilia. Homozygosity for mutant LEPR (i.e. RR genotype) was associated with higher CXCR2 expression and more tissue neutrophils. Here, we investigated the biological mechanisms that regulate neutrophil CXCR2 expression after CDI, and the influence of host genetics on this process. Our data reveal that: a) CXCR2 plays a key role in CDI-induced neutrophil extravasation from blood to colonic tissue; b) plasma from C. difficile-infected mice upregulated CXCR2 on bone marrow neutrophils; c) plasma from C. difficile-infected RR mice induced a higher magnitude of CXCR2 upregulation and had more IL-1β; and d) IL-1β neutralization reduced CXCR2 expression on bone marrow and blood neutrophils and their subsequent accrual to colonic tissue. In sum, our data indicate that IL-1β is a key molecular mediator that communicates between gastro-intestinal tract (i.e. site of CDI) and bone marrow (i.e. primary neutrophil reservoir) and regulates the intensity of CDI-induced tissue neutrophilia by modulating CXCR2 expression. Further, our studies highlight the importance of host genetics in affecting these innate immune responses and provide novel insights into the mechanisms by which a common SNP influences CDI-induced neutrophilia.

Olfactomedin-4+neutrophils exacerbate intestinal epithelial damage and worsen host survival afterClostridioides difficileinfection

“…Bone marrow neutrophils were sorted from WT and OLFM4 -/mice using a MACS mouse neutrophil isolation kit (Miltenyi Biotech #130-097-658, Germany) or a Sony MA900 sorter as described previously (3). Neutrophil purity and percentage of cells expressing OLFM4 was evaluated by flow cytometry as described elsewhere (3,54).…”

“…Bone marrow neutrophils were sorted from WT and OLFM4 -/mice using a MACS mouse neutrophil isolation kit (Miltenyi Biotech #130-097-658, Germany) or a Sony MA900 sorter as described previously (3). Neutrophil purity and percentage of cells expressing OLFM4 was evaluated by flow cytometry as described elsewhere (3,54). For TEER assays, sorted neutrophils were added to CMT-93 (mouse colonic epithelial cells; ATCC) monolayer on 96-well E-plate (Agilent # 300601010), and changes in TEER were measured every 15 minutes for up to 72hrs as described previously (71) and in Supplementary methods.…”

“…Although tissue collection and sequencing of cells from the uninfected host was performed at a later data than the C. difficileinfected host, we used an age-matched WT C57Bl/6 male mouse from the same colony and sequenced the samples with same protocols and on the same instruments. All samples were collected at ~10 am (or 4 hours after the lights turn on, Zeitgeber time 4), and processed as previously described (3,30,31,70). Neutrophil sorting buffer was supplemented with 1U/µl of RNase inhibitor.…”

“…To investigate these findings in vivo, we examined the association between colonic neutrophil numbers and epithelial damage using a well-established mouse model of CDI (3,(30)(31)(32)(33) (Fig. 1b & Supp Fig.…”