Temporal Regulation of Neutrophil Toll-like Receptors 2 and 4 in Acute Inflammation

Kay E, Nourshargh S, Scotland RS. Queen Mary University of London, London, UK

Background: Toll-like receptors (TLRs) have a key role in innate immune responses, detecting microbial infection and tissue-derived stress signals via pathogen-associated and damage-associated molecular patterns, respectively. TLR2 and TLR4 have been identified as novel therapeutic targets in diverse inflammatory disorders, with several blocking treatments in development. Several innate immune cell subtypes are reported to express TLR mRNA but the relative distribution and regulation of TLR protein during inflammation has not been investigated in detail. The aim of the current study was to characterise expression profile of neutrophil (PMN) TLR2 and TLR4 during onset and resolution phases of an acute inflammatory response. In addition, since we have recently demonstrated that female tissue macrophages express more TLR2 and TLR4 than male cells1 we have also investigated TLR expression between the sexes.

Methods: Cell-surface TLR on murine PMNs was quantified by flow cytometry. Male and female C57BL/6 mice (8-11wks) were treated with zymosan (1mg, ip) to induce peritonitis or PBS. Animals were killed by Schedule I and leukocytes were isolated from blood (cardiac puncture), peritoneal cavity (10ml lavage), spleen, and femur bone marrow (BM) at 3h or 24h. Erythrocytes were lysed with ACK lysis buffer, and leukocytes were resuspended in ice-cold PBS with 1% goats serum. Leukocytes were counted using a haemocytometer and incubated (20min, 4°C) with Fc-blocking antibody (anti-CD16/CD32; eBioscience) prior to addition of PE-antiTLR2 [clone T2.5; eBioscience], or PE-antiTLR4 [clone MTS510; eBioscience], or respective isotype control Ab (1µg/ml/105cells). PMNs were identified as GR1hi/CD115-ve. Samples were run on Fortessa cytometer (BD Biosciences) with data analysed using FlowJo (v.7). TLR expression was calculated as relative fluorescence intensity (RFI) compared to isotype control. Data are expressed as mean ±SEM and analysed by 1-way ANOVA followed by Bonferroni’s post-test (GraphPad Prism).

Results: Under basal conditions, PMN TLR expression in males was similar in all compartments (n=6) [TLR2: 2.1± 0.14 (blood) 2.0± 0.14 (BM) 2.2± 0.18 (spleen) and TLR4: 2.2± 0.28 (blood) 2.6± 0.34 (BM) 2.6± 0.21 (spleen). Zymosan induced PMN mobilisation from BM and influx into the peritoneal cavity at 3h but did not alter leukocyte TLRs (n=7, P>0.05). At 24h, levels of both TLRs were elevated on PMNs (n=7, P<0.05) [TLR2: 2.4± 0.29 (blood), 2.3± 0.28 (BM), 3± 0.33 (spleen) and TLR4: 3.3± 0.39 (blood), 3.2± 0.47 (BM), 3.5± 0.43 (spleen)]. TLR levels on PMNs recruited into the cavity at 3h and 24h were similar (n=7, P>0.05) to that on circulating cells [TLR2: 1.7± 0.12 and 2.8± 0.42, TLR4: 2.2± 0.24 and 3.3± 0.61]. Expression and regulation of TLRs on female PMNs was identical to males (n=7, P>0.05).

Conclusions: This study demonstrates that surface expression of TLR2 and TLR4 is higher on PMNs during later phases of a resolving inflammation in both sexes. This suggests a potential role for PMNs and TLRs in the resolution phase of inflammation.

Acknowledgements: EK is funded by a British Heart Foundation Studentship.

References: 1Scotland RS, et al (2011). Blood. 118, 5918-27.