Long-term immune reprogramming of classical monocytes with altered ontogeny mediates enhanced lung injury in sepsis survivors

Abstract

Patients who survive sepsis are predisposed to new hospitalizations for respiratory failure, but the underlying mechanisms are unknown. Using a murine model in which prior sepsis predisposes to enhanced lung injury, we previously discovered that classical monocytes persist in the lungs after long-term recovery from sepsis and exhibit enhanced cytokine expression after secondary challenge with intra-nasal lipopolysaccharide. Here, we hypothesized that immune reprogramming of post-sepsis monocytes and altered ontogeny predispose to enhanced lung injury. Monocyte depletion and/or adoptive transfer was performed three weeks and three months after sepsis. Monocytes from post-sepsis mice were necessary and sufficient for enhanced LPS-induced lung injury and promoted neutrophil degranulation. Prior sepsis enhanced JAK-STAT signaling and AP-1 binding in monocytes and shifted monocytes toward the neutrophil-like monocyte lineage. In human sepsis and/or pneumonia survivors, monocytes were predictive of 90-day mortality and exhibit transcriptional and proteomic neutrophil-like signatures. We conclude that sepsis reprograms monocytes into a pro-inflammatory phenotype and skews bone marrow progenitors and monocytes toward the neutrophil-like lineage, predisposing to neutrophil degranulation and lung injury.

Figure 1.. Monocyte depletion improves lung injury and inflammation in post-CLP mice.

(A) Experimental design for monocyte depletion with anti-CCR2 antibody (αCCR2) or isotype prior to i.n. LPS. Lung permeability assessed by BAL albumin (B), change in permeability relative to isotype-treated for each condition (C), epithelial injury assessed by BAL RAGE (D), neutrophil recruitment (E), and BAL IL-6 (F) shown 72 hours after i.n. LPS. n = 4–8 per group, 3 cohorts; 2 of 18 mice in the iso-treated CLP group died by 72 hours, no other deaths were recorded. BAL protein levels expressed relative to the mean of isotype-treated unoperated control mice. Mean ± SEM, Sidak post-hoc p-value (B, D-F), Welch’s t-test (C) shown. * p < 0.05, ** p < 0.01, *** p < 0.001. CLP, Cecal Ligation and Puncture. LPS, Lipopolysaccharide. BAL, bronchoalveolar lavage. RAGE, receptor for advanced glycation end-products. PMN, polymorphonuclear.

Figure 2.. Adoptive transfer of post-CLP monocytes enhances the lung injury response to LPS.

Bone marrow Ly6C^hi monocytes from Ccr2^−/−, Ccr2^wt age-matched unoperated control, and Ccr2^wt 3-wk post-CLP mice were isolated and administered into Ccr2^−/− i.v. with concurrently with i.n. LPS (A). Alveolar permeability assessed by BAL albumin or total protein (B, C), neutrophil recruitment (D), and BAL IL-6 (E) shown 72 hours after i.n. LPS. BAL protein measurements expressed relative to the mean of unoperated control Ccr2^wt transfer for each cohort. n = 3–6 per group, 3 cohorts. Mean ± SEM, Welch’s t-test p-value shown. * p < 0.05, ** p < 0.01. CLP, Cecal Ligation and Puncture. LPS, Lipopolysaccharide. BAL, bronchoalveolar lavage. PMN, polymorphonuclear.

Figure 3.. Post-CLP monocytes promote activation and degranulation of neutrophils.

BAL S100A8/A9 following adoptive transfer (A) and antibody-mediated depletion of bone marrow Ly6C^hi monocytes (B). Monocyte and neutrophil S100A8/A9 production in supernatants following stimulation with LPS or monocyte conditioned media (CM), respectively (C). Neutrophil granule proteins were measured to assess degranulation induced by monocyte CM (D). n = 4 mice per group, 2 cohorts. Supernatant protein expressed relative to the mean of unoperated control monocyte CM. Mean ± SEM, Welch’s t-test p-value shown. * p < 0.05, ** p < 0.01, *** p <0.01. CLP, Cecal Ligation and Puncture. LPS, Lipopolysaccharide. BAL, bronchoalveolar lavage. MPO, Myeloperoxidase. NGAL/LCN2, neutrophil gelatinase-associated lipocalin. MMP9, matrix metallopeptidase-9.

Figure 4.. Monocyte mediated enhanced lung injury in post-CLP mice is durable.

3-mo post-CLP and age-matched control mice were administered i.n. LPS (A). Alveolar permeability assessed by BAL albumin (B), epithelial injury assessed by BAL RAGE (C), neutrophil recruitment (D), and BAL IL-6 (E) measured 72-hr after i.n. LPS. Bone marrow Ly6C^hi monocytes were isolated from Ccr2^wt age-matched unoperated control, and Ccr2^wt 3-mo post-CLP mice and administered into Ccr2^−/− mice i.v. concurrently with i.n. LPS, alveolar permeability assessed by BAL albumin (F). n = 5–10 per group, 2 cohorts (A-E), n = 2–6 per group, 3 cohorts (F). Mean ± SEM, Welch’s t-test p-value shown. * p < 0.05, ** p < 0.01, *** p < 0.001. CLP, Cecal Ligation and Puncture. LPS, Lipopolysaccharide. BAL, bronchoalveolar lavage. RAGE, receptor for advanced glycation end-products. PMN, polymorphonuclear. BM, bone marrow.

Figure 5.. Transcriptional and epigenetic pathways contributing to post-CLP monocyte priming.

Ly6C^hi monocytes were isolated from the bone marrow for transcriptomic and epigenomic profiling. Top 10 transcriptomic pathways enriched in 3-wk post-CLP monocytes relative to control (A). Selected transcriptomic pathways enriched in post-CLP monocytes with upregulated and downregulated genes per pathway (B). In a separate experiment, ATAC-seq was performed, unique peaks were identified, and pathway enrichment performed on promoter (C) and enhancer regions (D). Known motif enrichment performed relative to control monocytes genomic background (E). Quantification of peaks containing the AP-1 binding motif in post-CLP and control monocytes (F). n = 4–5 mice per group. CLP, Cecal Ligation and Puncture. TSS, transcription start site.

Figure 6.. Neutrophil-like monocytes are expanded in post-CLP mice.

Monocyte differentiation pathways (A). Differential expression in Ly6C^hi monocytes highlighting neutrophil- and DC-specific genes (B). Enrichment of GMP-derived and MDP-derived monocyte signatures in Ly6C^hi monocytes (C). Scatter properties of Ly6C^hi monocytes in post-CLP and unoperated control (D). Gfi1-tdTomato fluorescent intensity in Neutrophils and FSC^hi or FSC^lo monocytes (E). Proportions of FSC^hiGfi1-tdTomato Ly6C^hi monocytes in post-CLP and unoperated control mice (F). n= 4–5 per group, 2 cohorts (D), n=3–5 mice per group, 1 cohort (B, C, E, F). Mean, Mean ± SEM and Welch’s t-test (D-F) are shown. * p < 0.05, ** p < 0.01.. MDP, monocyte-dendritic cell progenitor. cMoP, common monocyte progenitor. GMP, granulocyte-monocyte progenitor. MP, monocyte progenitor. CLP, cecal ligation and puncture. FSC, forward scatter. SSC, side scatter.

Figure 7.. CLP reprograms progenitors toward GMP-lineage.

Monocyte committed BM progenitors (MP+cMoP enriched) were isolated from BM and tested for enrichment of MP (GMP-derived) and cMoP (MDP-derived) signatures (A). Quantification of myeloid progenitors by flow cytometry (B). Peripheral blood counts (C). n = 4–5 per group, 1 cohort (A-C). Mean, Wilcoxon rank-sum (A), Mean ± SEM and Welch’s t-test (B, C) are shown. * p < 0.05, ** p < 0.01, *** p <0.01. MDP, monocyte-dendritic cell progenitor. cMoP, common monocyte progenitor. GMP, granulocyte-monocyte progenitor. MP, monocyte progenitor. CLP, cecal ligation and puncture. WBC, white blood cell.

Figure 8.. Neutrophil-like monocyte lineage in patients with sepsis.

Unadjusted Cox proportional hazard regression (95% CI, dark gray) with relative hazard ratio for 90-day mortality for absolute monocyte count (AMC), histogram of AMC distribution shown below (A). Light gray boxes indicate values outside of the normal clinical range. Three-dimensional interaction surface showing the relationship of AMC and absolute neutrophil count (ANC) with 90-day mortality (B). Selected neutrophil-like and DC-like gene expression in CD14+ monocytes in patients with acute sepsis (n = 4 sepsis, 5 healthy) and community acquired pneumonia (n = 69 CAP, 41 healthy) (C, D). Conserved human-mouse (E) and mouse-specific (F) neutrophil-like signature proteins in classical monocytes from patients with sepsis. Proteins detected in more than 1 sample across conditions were included in the heatmap, missing values were imputed as described in the methods. Conserved human-mouse (G) or mouse-specific signatures (H) across monocyte substates (MS1–4) in scRNAseq performed in patients with acute bacterial infection. Mean, 25^th and 75^th percentile shown (C, D). Multivariate-ANOVA p-value shown (E, F). * p < 0.05, ** p < 0.01, *** p < 0.001. HC, healthy control. Sep, sepsis. CAP, community acquired pneumonia.