Commensal microbiota stimulate systemic neutrophil migration through induction of serum amyloid A

Abstract

Neutrophils serve critical roles in inflammatory responses to infection and injury, and mechanisms governing their activity represent attractive targets for controlling inflammation. The commensal microbiota is known to regulate the activity of neutrophils and other leucocytes in the intestine, but the systemic impact of the microbiota on neutrophils remains unknown. Here we utilized in vivo imaging in gnotobiotic zebrafish to reveal diverse effects of microbiota colonization on systemic neutrophil development and function. The presence of a microbiota resulted in increased neutrophil number and myeloperoxidase expression, and altered neutrophil localization and migratory behaviours. These effects of the microbiota on neutrophil homeostasis were accompanied by an increased recruitment of neutrophils to injury. Genetic analysis identified the microbiota-induced acute phase protein serum amyloid A (Saa) as a host factor mediating microbial stimulation of tissue-specific neutrophil migratory behaviours. In vitro studies revealed that zebrafish cells respond to Saa exposure by activating NF-κB, and that Saa-dependent neutrophil migration requires NF-κB-dependent gene expression. These results implicate the commensal microbiota as an important environmental factor regulating diverse aspects of systemic neutrophil development and function, and reveal a critical role for a Saa-NF-κB signalling axis in mediating neutrophil migratory responses.

Fig. 1

Microbiota regulates neutrophil localization, number and stimulates inflammatory biomarkers. A. Live 6dpf Tg(mpx:GFP) zebrafish show GFP(+) neutrophil localization as a function of microbial status. Note the increased neutrophil localization in the kidney (white arrow) in CONVD animals. B. Flow cytometry analysis reveals that the percent frequency of GFP(+) neutrophils in dissociated 6dpf Tg(mpx:GFP) zebrafish is higher in CONVD compared with GF animals. C. qRT-PCR for mpx mRNA levels in sorted neutrophils. D. Quantification of mean total number of GFP(+) neutrophils associated with dissected intestines by segment. Data are representative of 8–10 guts per microbial condition from two biological replicates. Significant Student's t-test P-values are shown.

Fig. 2

Microbiota induces systemic neutrophil migration. Neutrophil tracking analysis of the CHT from 15-min movies of live 6dpf GF (A) and CONVD (B) Tg(mpx:GFP) zebrafish reveals increased migratory activity in CONVD. C. Quantification of neutrophil migration velocity and meandering index in 6dpf GF and CONVD zebrafish (calculated from 29 neutrophils per condition). Significant Student's t-test P-values are shown. See also Movies S1 and S2.

Fig. 3

Microbiota promotes neutrophil recruitment to tail wounds in GF and CONVD Tg(mpx:GFP) zebrafish. A. Brightfield and GFP fluorescence images of 6dpf GF and CONVD zebrafish tails 3 h post injury. B. Mean numbers of neutrophils recruited to wound site (posterior to the end of the notochord marked by white dashed line) at time points post injury as indicated. Data represents 8–10 fish per condition per time point. Significant Student's t-test P-values are shown: *P < 0.05 and **P < 0.005 vs GF.

Fig. 4

saa mediates systemic neutrophil migration in response to the microbiota. Comparisons of 6dpf GF and CONVD Tg(mpx:GFP) zebrafish injected with either standard control (Ctrl MO) or a morpholino targeting saa (saa MO). A. Images of whole live 6dpf Tg(mpx:GFP) zebrafish show GFP(+) neutrophil localization including increased concentration of neutrophils in the kidney (white arrow) and intestine (white arrow head) in CONVD Ctrl MO and saa MO zebrafish. B. Neutrophil units quantified by GFP densitometry from whole animal images similar to those shown in A. C. qRT-PCR for mpx mRNA in sorted neutrophils. qRT-PCR for ncf1 (D) and il1b (E) mRNA in whole 6dpf zebrafish. Quantification of neutrophil velocity (F) and meandering index (G) in the CHT, intestine (Int), and fin (calculated from 7–29 neutrophils per tissue per condition). Student's t-test P-values are indicated: a, P < 0.05 compared with GF condition in same genotype; b, P < 0.05 compared with Ctrl MO in same microbial condition. See also Movies S3–S6.

Fig. 5

SAA stimulation of a zebrafish cell line results in activation of the canonical NF-κB pathway and induces expression of NF-κB target genes. A. Western blot of zebrafish PAC-2 cells shows rapid phosphorylation of IκBα proteins after LPS (10 μg ml⁻¹) or SAA (4 μM) stimulation. B. Zebrafish PAC-2 cells transfected with ikbaa-luciferase gene reporter (pikbaa:Luc) show increased luciferase activity upon stimulation with LPS (10 μg ml⁻¹) or SAA (1, 4 μM). C and D. qRT-PCR using primers for mmp9 and ikbaa, predicted NF-κB target genes, demonstrate induction upon stimulation of PAC-2 cells with LPS (10 μg ml⁻¹) or SAA (1, 4 μM) normalized to 18S ribosomal RNA [rRNA]). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6

SAA promotes neutrophil migration and requires NF-κB and protein synthesis. A. Peritoneal isolated murine neutrophils were pretreated for 1 h with BAY 11-7082 (BAY, 25 μM) or cycloheximide (CHX, 50 μg ml⁻¹) and then plated into the top well of a Transwell system. The cells' migration in response to SAA (2.08 μM, 25 μg ml⁻¹) in the bottom well was enumerated after 2.5 h. Representative light images of neutrophils migrated into bottom wells. Magnification 200×. B. Quantitative measurements of migrated neutrophils. Data are expressed as mean ± SEM. *P < 0.05. Results are representative of two independent experiments.