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Peripheral TREM1 responses to brain and intestinal immunogens amplify stroke severity

Abstract

Stroke is a multiphasic process in which initial cerebral ischemia is followed by secondary injury from immune responses to ischemic brain components. Here we demonstrate that peripheral CD11b+CD45+ myeloid cells magnify stroke injury via activation of triggering receptor expressed on myeloid cells 1 (TREM1), an amplifier of proinflammatory innate immune responses. TREM1 was induced within hours after stroke peripherally in CD11b+CD45+ cells trafficking to ischemic brain. TREM1 inhibition genetically or pharmacologically improved outcome via protective antioxidant and anti-inflammatory mechanisms. Positron electron tomography imaging using radiolabeled antibody recognizing TREM1 revealed elevated TREM1 expression in spleen and, unexpectedly, in intestine. In the lamina propria, noradrenergic-dependent increases in gut permeability induced TREM1 on inflammatory Ly6C+MHCII+ macrophages, further increasing epithelial permeability and facilitating bacterial translocation across the gut barrier. Thus, following stroke, peripheral TREM1 induction amplifies proinflammatory responses to both brain-derived and intestinal-derived immunogenic components. Critically, targeting this specific innate immune pathway reduces cerebral injury.

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Fig. 1: TREM1 is induced on peripheral myeloid cells that infiltrate the ischemic brain.
Fig. 2: TREM1 is elevated in peripheral infiltrating myeloid cells early after MCAo.
Fig. 3: Genetic ablation of TREM1 improves outcome after MCAo.
Fig. 4: The TREM1 decoy peptide LP17 reduces stroke injury.
Fig. 5: Visualization of TREM1 induction using PET imaging demonstrates activation of peripheral myeloid cells in spleen and intestine after MCAo.
Fig. 6: Increased gut permeability after MCAo induces TREM1 in lamina propria and blood Mo/MΦ subsets.
Fig. 7: Intestinal TREM1 activation amplifies gut barrier permeability and bacterial translocation to the periphery.

Data availability

The data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

This work was supported by National Institutes of Health (NIH) grant (nos. R01NS045727, R21NS087639, R01NS100180 and RO1AG053001 to K.I.A.), The Paul and Daisy Soros Fellowship for New Americans (to P.S.M.), the Gerald J. Lieberman Fellowship (to P.S.M.) and Stanford Medicine Dean’s Fellowship (to E.N.W.). The authors would like to thank W. Lu for LC–MS expertise, the Stanford Shared FACS Facility, the Stanford Human Immune Monitoring Center, and the Protein and Nucleic Acid Facility.

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Q.L., E.M.J., R.K.L, Q.W., E.N.W., P.S.M., M.S.S., S.S.M. and X.Y. designed and performed the experiments and analyzed the data. H.B.Y., S.T. and J.W. performed the experiments. M.S., S.S.Y and C.M. provided advice. J.D.R. and L.L. designed and performed metabolic measurements and analyzed the data. E.M.J. and M.L.J. designed and performed PET measurements and analyzed the data. Q.L., M.L.J. and K.I.A. conceived and supervised the project, designed the experiments, interpreted the data and wrote the manuscript.

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Correspondence to Katrin I. Andreasson.

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Peer review information: Laurie Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Integrated supplementary information

Supplementary Figure 1 Temporal dynamics of myeloid cells and TREM1 expression in ipsilateral (IL) hemisphere after MCAo.

Flow cytometry gating strategy of brain myeloid cells in the ischemic hemisphere 48 h after MCAo. Time courses were carried out over 7 days post MCAo in C56Bl/6J 2–3 mo male mice. Representative plots of CD11b+CD45+ myeloid cell populations at day 0 before MCAo, and at 2 and 6 days after MCAo. Time course of percents of macrophages, neutrophils, and microglia in ischemic hemisphere out to Day 7 post MCAo; n=6 biologically independent samples at days 0 and 7, and n=7 at days 2,4,and 6, mean +/- SEM;1 way ANOVA for each cell type: microglia P=0.015, Mo/MΦ P<0.0001, PMNs P<0.0001. TREM1 surface expression on CD11b+CD45hi Mo/MΦ and CD11b+CD45hiLy6G+ PMNs 2 days after MCAo compared to isotype control antibody. Representative plots of CD11b+CD45+TREM1+ cells at day 2 and day 6 after MCAo in ischemic ipsilateral (IL) and non-infarcted contralateral (CL) hemispheres. Percentages of TREM1+ macrophages, neutrophils, and microglia in IL and CL hemispheres and in sham IL and CL hemispheres at day 2 and day 6 (n= 6 biologically independent samples for sham, n=7 for MCAo, mean +/- SEM; two-way ANOVA for IL MCAo vs IL sham hemispheres: for macrophages, effect of MCAo P <0.0001, effect of time P <0.01, effect of interaction, P <0.05; Bonferroni post-hoc day 2 IL MCAo vs IL sham **** P <0.0001; post-hoc day 6 IL MCAo vs day 6 IL sham hemisphere ## P <0.01; for microglia, effect of MCAo ** P =0.006).

Supplementary Figure 2 TREM1 surface expression is induced early after MCAo in peripheral Mo/MΦ cells.

TREM1 surface expression was quantified in blood at 0h before sham or MCAo, and 1h, 4.5h, 48h, and 144h (6 days) after sham surgery or MCAo. Percents of TREM1+ Mo/MΦ and PMNs in blood are shown (n=3–12 biologically independent samples per time point, mean +/- SEM; two-way ANOVA for macrophages, effects of MCAo, time, and interaction P<0.0001; Bonferroni post hoc **** P <0.0001 at 4.5h for MCAo vs sham). TREM1 surface expression was quantified in spleen at 0h before sham or MCAo, and 1h, 4.5h, 48h, and 144h (6 days) after sham surgery or after MCAo. Percents TREM1+ Mo/MΦ and PMNs in spleen are shown (n=3–12 biologically independent samples per time point, represented as mean +/- SEM; two-way ANOVA for macrophages, effect of MCAo and time, P<0.0001; effect of interaction P<0.001, Bonferroni post hoc **** P <0.0001 at 4.5h for MCAo vs sham). TREM1 MFI in blood and IL hemisphere Mo/MΦ subsets 2 days (n=5–7 biologically independent samples per group, mean +/- SEM) and 6 days (n=4–7 biologically independent samples per group, mean +/- SEM) after MCAo (Student’s two tailed t-test, ** P <0.01).

Supplementary Figure 3 Genetic ablation of Trem1 improves outcome after MCAo.

Percent TREM1 expression in CD11b+CD45hiLy6Ghi PMNs, CD11b+CD45hi Mo/MΦ, and CD11b+CD45int microglia 2 days after MCAo from Trem1+/+, Trem1+/-, and Trem1-/- ischemic hemispheres (n=5–10 biologically independent samples per cell type per genotype, mean +/- SEM). Representative plots of CD11b+CD45hiLy6Ghi PMNs, CD11b+CD45hi Mo/MΦ, and CD11b+CD45int microglia that were sorted and isolated for transcriptomic analysis at 2 days after MCAo. Top three Gene Ontology pathways (FDR<0.05, absolute fold ≥2). KEGG lysosomal genes are induced in Trem1-/- vs Trem1+/+ PMNs (note log10 scale). Heat map of macrophage genes differentially regulated and FDR corrected (P<0.05) in Trem1+/+ vs Trem1-/- ischemic hemispheres at 2 days after MCAo (absolute fold ≥2). Histogram of TREM2 antibody vs isotype control. BV2 microglia were stimulated with LPS 10 ng/ml and surface expression of TREM1 determined at 2, 6, 10, and 20 h after stimulation. Raw macrophages and BV2 microglia were stimulated with LPS 10 ng/ml and qRT-PCR performed for TREM1 and TREM2 expression at 0, 4, and 20 h (n=3 biologically independent samples per time point per group, mean +/- SEM; 1-way ANOVA, P values for TREM1 in red and TREM2 in blue). Percent CD11b+CD45+ myeloid cells in Trem1+/+, Trem1+/-, and Trem1-/- IL and CL hemispheres 2 days after MCAo (n=8 biologically independent samples per genotype for Trem1+/+ and Trem1+/- mice and n=9 for Trem1-/- mice, mean +/- SEM; two-way ANOVA, effect of genotype P <0.0001, effect of hemisphere P <0.05; post-hoc * P <0.05 and *** P <0.001). Percent CD11b+CD45hiLy6Ghi PMNs, CD11b+CD45hi Mo/MΦ, and CD11b+CD45int microglia in Trem1+/+, Trem1+/-, and Trem1-/- IL and CL hemispheres 2 days after MCAo (n=8 biologically independent samples for Trem1+/+ and Trem1+/- mice and n=9 samples for Trem1-/- mice, mean +/- SEM; two-way ANOVA, effect of hemisphere P <0.0001 all three cell types, effect of genotype P <0.01 for Mo/MΦ only; post-hoc *** P <0.001).

Supplementary Figure 4 Administration of LP17 to Trem1-/- mice.

LP17 reduces mortality post-stroke (n=26 mice per group; Log-rank test * P =0.011). LP17 administered at 4.5h after MCAo does not affect survival (n=28–36 mice per group). Neuroscores of Trem1-/- mice that underwent MCAo and received LP17 or scrambled peptide at the time of reperfusion (n=8 mice per group; mean +/- SEM). Percent infarct volume in Trem1-/- mice treated with LP17 or scrambled peptide (n= 8 mice per group, mean +/- SEM). Representative histogram of TREM2 expression on CD11b+CD45+Ly6G+ PMNs 2 days after MCAo +/- LP17 or scrambled peptide treatment at time of reperfusion. Body weights of sham and MCAo mice from Fig. 4m-n. Number of left front and left hind paw slips in beam-tested mice (n=5 sham, n=8 scrambled, n=11 LP17).

Supplementary Figure 5 TREM1 signal is increased in peripheral myeloid tissues after MCAo.

64Cu-labeled anti-TREM1-mAb (that is, [64Cu]TREM1-mAb) was generated with high specific radioactivity (>0.400 MBq/μg), radiochemical purity (>99%), and labeling efficiency (70–95%), and formulated in phosphate-buffered saline [0.1 mol/L NaCl, 0.05 mol/L sodium phosphate (pH 7.4)] (see Methods). HEK293 cells transiently expressing murine Trem1 cDNA and control empty vector transfected cells were assayed for binding of [64Cu]TREM1-mAb at 1 hour (n=3–4 biologically independent samples per group, mean +/- SEM). Unlabeled TREM1 antibody was used to block binding. Quantification of PET signal in peripheral organs from MCAo and sham mice (36 h post-MCAo; n=9 biologically independent samples per group, mean +/- SEM). Quantification of TREM1 signal from ex vivo biodistribution studies of blood, heart, liver and lungs (n=8–15 biologically independent samples per group, mean +/- SEM). Quantification of spleen TREM1 signal from ex vivo biodistribution studies shows higher uptake of [64Cu]TREM1-mAb in MCAo mice compared to shams and compared to MCAo and sham mice injected with [64Cu]Isotype control (n=12, MCAo-[64Cu]TREM1; n=10, sham-[64Cu]TREM1; n=8, MCAo-[64Cu]ISO-Ctrl; n=3, Sham-[64Cu]ISO-Ctrl) mice, mean +/- SEM). Ex vivo biodistribution of brain hemispheres corroborates brain PET imaging findings (n=10 MCAo-[64Cu]TREM1, n=9 sham-[64Cu]TREM1 biologically independent samples, mean +/- SEM). All comparisons are two-tailed unpaired Student’s t-test **** P <0.001, ** P <0.01, * P <0.05.

Supplementary Figure 6 TREM1 is induced in the intestinal inflammatory Mo/MΦ subset after MCAo.

ß-adrenergic inhibition with propranolol (ppl) has no effect on neutrophil TREM1 expression after MCAo. TREM1 MFI in neutrophils in sham, MCAo, and MCAo treated with propranolol (n=5 sham, n=8 MCAo and n=9 MCAo+ppl mice, mean +/- SEM). Percent change in volume of IL hemisphere in sham and MCAo mice +/- ppl 4.5h after MCAo (n=5 sham, n=9 MCAo and n=9 MCAo+ppl, mean +/- SEM). Immune factor changes at 4.5h in small intestine lamina propria (n=3–6 biologically independent samples per group, mean +/- SEM.; ** P <0.01 Student’s two tailed t-test). Quantification of bacterial colonies that grew out of blood collected from Trem1+/+ and Trem1-/- mice at 4.5h after MCAo (n=3 sham Trem1+/+, n=6 MCAo Trem1+/+ and n=4 MCAo Trem1-/- mice, mean +/- SEM).

Supplementary Figure 7 Model of dual TREM1 amplification of immune responses after cerebral ischemia.

(Left side) Cerebral injury activates the sympathetic nervous system (SNS) within hours of MCAo, leading to early disruption of the gut barrier and translocation of bacterial PAMPs across the epithelial barrier. There, PAMPs induce and activate TREM1 signaling in lamina propria Mo/MΦ subsets, amplifying the innate immune response and further disrupting gut barrier integrity and facilitating translocation of bacteria to the periphery. (Right side): Cerebral infarction induces the release of sterile DAMPs that activate TREM1 on circulating peripheral and splenic myeloid cells. Thus, TREM1 is induced in myeloid cells in two spatially distinct processes after MCAo. Dual brain-derived and intestinal-derived TREM1 responses converge and amplify the post-stroke innate immune response, increasing cerebral injury.

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PET imaging of TREM1

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Liu, Q., Johnson, E.M., Lam, R.K. et al. Peripheral TREM1 responses to brain and intestinal immunogens amplify stroke severity. Nat Immunol 20, 1023–1034 (2019). https://doi.org/10.1038/s41590-019-0421-2

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