Intestinal Serum amyloid A suppresses systemic neutrophil activation and bactericidal activity in response to microbiota colonization

Abstract

The intestinal microbiota influences the development and function of myeloid lineages such as neutrophils, but the underlying molecular mechanisms are unresolved. Using gnotobiotic zebrafish, we identified the immune effector Serum amyloid A (Saa) as one of the most highly induced transcripts in digestive tissues following microbiota colonization. Saa is a conserved secreted protein produced in the intestine and liver with described effects on neutrophils in vitro, however its in vivo functions remain poorly defined. We engineered saa mutant zebrafish to test requirements for Saa on innate immunity in vivo. Zebrafish mutant for saa displayed impaired neutrophil responses to wounding but augmented clearance of pathogenic bacteria. At baseline, saa mutants exhibited moderate neutrophilia and altered neutrophil tissue distribution. Molecular and functional analyses of isolated neutrophils revealed that Saa suppresses expression of pro-inflammatory markers and bactericidal activity. Saa's effects on neutrophils depended on microbiota colonization, suggesting this protein mediates the microbiota's effects on host innate immunity. To test tissue-specific roles of Saa on neutrophil function, we over-expressed saa in the intestine or liver and found that sufficient to partially complement neutrophil phenotypes observed in saa mutants. These results indicate Saa produced by the intestine in response to microbiota serves as a systemic signal to neutrophils to restrict aberrant activation, decreasing inflammatory tone and bacterial killing potential while simultaneously enhancing their ability to migrate to wounds.

Fig 1. Saa mediates neutrophil behavior in vivo.

(A-B) Imaging and quantification of lyz:EGFP⁺ neutrophils recruited to tail wound margin over 6 hours following caudal fin amputation (dashed red line indicates wound margin; scale bar = 250 μm) (n ≥ 24 larvae / genotype at 6 hour time point). (C) Measurement of lyz:DsRed⁺ neutrophil speed from time-lapse imaging in caudal fin tissue over 6 hour period following amputation (n = 4 larvae / genotype, 87–112 cells tracked / genotype). (D-E) Representative images of 6 dpf Tg(lyz:DsRed) WT and saa^-/- larvae (scale bar = 500 μm). (F) Enumeration of intestine-associated lyz:EGFP⁺ cells in 6 dpf larvae (n = 32–40 larvae / genotype). (G-J) Quantitative analysis of lyz:EGFP⁺ neutrophil behavior from time-lapse imaging of distinct anatomical compartments (intestine and trunk, ROIs in panel D) in 6 dpf larval zebrafish (6 larvae / genotype, ≥ 23 cells analyzed / genotype / tissue). Data analyzed by t-test. For panel B, statistical comparisons were performed within each time point. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig 2. Saa regulates neutrophil abundance and maturation.

(A) Flow cytometry analysis of lyz:EGFP⁺ neutrophil abundance from whole 6 dpf WT and saa mutant zebrafish larvae (results are combined from 3 independent experiments, ≥ 4 replicates / genotype / experiment, 60–90 larvae / replicate). (B-C) Morphological analysis of lyz:DsRed⁺ neutrophil cytospins stained with Wright-Giemsa from adult WT and saa mutant zebrafish kidneys (5–6 adult zebrafish kidneys / genotype / experiment, 2 independent experiments, n ≥ 199 cells analyzed / genotype / experiment) (scale bar = 10 μm). (D-F) qRT-PCR of leukocyte-associated transcripts lysozyme C (lyz), l-plastin (lcp), and colony-stimulating factor 3a (csf3a) from 6 dpf whole zebrafish larvae (n = 4 replicates / genotype / timepoint, 25–30 larvae / replicate). Data in panel A analyzed by t-test. Data in panel C analyzed by chi-squared test. Data in panels D-F analyzed by one-way ANOVA with Tukey’s multiple comparisons test. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig 3. Saa suppresses neutrophil transcriptional activation and bactericidal activity.

(A) qRT-PCR of pro-inflammatory mRNAs from sorted neutrophils from 6 dpf WT and saa mutant zebrafish larvae (5,000–12,000 lyz:EGFP⁺ cells / replicate, 3–6 replicates / genotype / experiment, 60–90 larvae / replicate). (B) Microscopic analysis revealed neutrophils isolated from adult zebrafish kidneys extend protrusions in response to bacterial signals ex vivo (white arrows; scale bar = 20 μm). (C) il1b expression in un-stimulated and E. coli exposed lyz:EGFP⁺ neutrophils from WT and saa mutant zebrafish following 4 hours ex vivo culture (3–5 replicates / genotype / condition). (D) CFU quantification of bacterial concentration following 4 hour co-culture of isolated lyz:EGFP⁺ neutrophils from WT and saa mutant zebrafish with E. coli (MOI 2). (E) Quantification of intracellular ROS levels by CellROX fluorescence from neutrophils cultured ex vivo with and without E. coli (lyz:EGFP⁺ cells isolated from 6 zebrafish adult kidneys / genotype, quantification of ≥ 177 cells / condition). In panels C and E, a one-way ANOVA with Tukey’s multiple comparisons test was used. Data in panels A and D were analyzed with a t-test. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig 4. Intestinally-derived Saa regulates systemic neutrophil activity.

(A) qRT-PCR of saa from whole 6 dpf larvae of the indicated genotypes (n = 4 replicates / genotype, 25–30 larvae / replicate) (B) Enumeration of intestine-associated lyz:DsRed⁺ neutrophils along the anterior to posterior axis (segment 1 to segment 3) in 6 dpf larvae (n ≥ 25 larvae / genotype). (C) lyz:DsRed⁺ neutrophil recruitment to caudal fin wound 6 hours following amputation in 6 dpf zebrafish larvae (n ≥ 25 larvae / genotype at 6 hour time point). (D) CFU quantification of bacterial concentration following 4 hour co-culture of lyz:DsRed⁺ adult zebrafish neutrophils with P. aeruginosa (P.a., MOI 0.2) (8 replicates / genotype). (E) Representative stereoscope images of IEC specific mCherry expression in 6 dpf Tg(-0.349cldn15la:mCherry)^rdu65 larvae compared to non-transgenic (NTG) controls. White dashed line indicates the intestine (scale bar = 500 μm). (F) Representative confocal micrograph of immunostained transverse section of Tg(-0.349cldn15la:mCherry) 6 dpf larvae labeled with the absorptive cell brush border-specific antibody 4E8 (scale bar = 20 μm). (G,H) qRT-PCR of cldn15la, fabp2, and fabp10a (G) from sorted Tg(-0.349cldn15la:mCherry)⁺ IECs and saa (H) from cldn15la:mCherry⁺ and negative cells isolated from 6 dpf larvae of indicated genotypes (13,000 –0.349cldn15la:mCherry⁺ or mCherry negative cells / replicate, 4 replicates / genotype, 30 larvae / replicate). (I) qRT-PCR of saa from 6 dpf larval dissected digestive tissue of the indicated genotypes (n = 4 replicates / genotype, 25–30 larvae / replicate) (J) Enumeration of intestine-associated lyz:DsRed⁺ neutrophils in 6 dpf larvae (n = 30 larvae / genotype). (K) lyz:DsRed⁺ neutrophil recruitment to caudal fin wound 6 hours following amputation in 6 dpf zebrafish larvae (n ≥ 18 larvae / genotype at 6 hour time point). (L) CFU quantification of bacterial concentration following 4 hour co-culture of lyz:DsRed⁺ adult zebrafish neutrophils with P. aeruginosa (P.a., MOI 0.2) (3–6 replicates / genotype). (M) il1b qRT-PCR from lyz:DsRed⁺ neutrophils co-cultured with and without P.a. ex vivo for 4 hours (n ≥ 2 replicates / condition). (N) CFU quantification of in vivo P.a. bacterial burden following systemic infection of larval zebrafish at 5 days post infection (dpi) (data from 3 independent experiments, n ≥ 30 larvae / genotype). Data in panels A-D and H-N were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. A Mann-Whitney test was applied to panel G. For panels C and K, statistical comparisons were performed amongst samples within the same time point. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig 5. Microbiota-induced Saa conditions neutrophils in vivo.

(A) qRT-PCR of saa from gnotobiotic zebrafish larvae following microbiota colonization (CV) at 3 dpf versus germ free (GF) (3 replicates / condition / timepoint, n ≥ 20 larvae / replicate). (B) qRT-PCR of neutrophils isolated from 6 dpf gnotobiotic WT and saa mutant zebrafish larvae (3 replicates / genotype / condition, n ≥ 27 larvae / replicate). (C) lyz:EGFP⁺ neutrophil recruitment to caudal fin wound margin 6 hours after amputation in 6 dpf gnotobiotic WT and Tg(cldn15la:saa) sibling zebrafish larvae (n ≥ 18 larvae / genotype / condition at the 6 hour timepoint). (D) Working model depicting signals from the microbiota evoking production of intestinal and hepatic Saa leading to shared and distinct effects on systemic neutrophil function. Statistical comparisons of data in panel in A were performed within each time point and analyzed by t-test. Data in panel B analyzed by two-way ANOVA with p values reported in the table. Data in panel C analyzed by one-way ANOVA with Tukey’s multiple comparisons test. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.