Fig 2: Monocyte sub-populations are altered early in ARDS.(a) Representative plots and proportions of monocyte sub-populations based on CD14 and CD16 expression early and (b) late (c). Each data point = one individual patient/ healthy donor control (HC), b, c Data+mean, one-way ANOVA with Holm-Sidak post-test.

Fig 3: Irradiation abrogates myeloid cell precursor and mature myeloid cell expansion and impacts IFN?R1 and IFN?R2 expression. (A–C) Mice were injected with KR158B-luc cells and 21 days later, half of the mice were subjected to 5 Gy total body irradiation. Seven days later, bone marrow was obtained from both groups and frequencies of IFNyR1+ and IFNyR2+ cells were evaluated on lineage- HSPCs. Mice subjected to total body irradiation were found to possess more IFNyR1+ and IFNyR2+ lineage- cells via flow cytometry. (D–F) A cohort of non-tumor-bearing mice were injected with KR158B-luc cells while a separate cohort remained non-tumor-bearing. Twenty-eight days after implantation, both cohorts were sacrificed and lineage- HSPCs were FACS sorted and then either non-tumor-bearing or glioma-bearing HSPCs were transferred into C57BL/6 that were subjected to 9 Gy total body irradiation the day prior. Twenty-eight days after HSPC transfer, bone marrow was collected and stained for flow cytometry. No significant differences in the frequencies of lin-cKit+Sca-1-CD16/32hi GMPs and CD11b+Gr-1+ MDSCs between mice that received non-tumor-bearing HSPCs and those that received glioma-bearing HSPCs. All data represent the mean±SD. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by Mann-Whitney t-test (n=5 biological replicates). HSPCs, hematopoietic stem and progenitor cells.

Fig 4: Patients with ARDS are monocytopenic early in the disease with phenotypically distinct circulating monocytes.a,b, Lowest (a) and all (b) partial pressures of oxygen (PaO2) from clinical arterial blood samples from ARDS patients, 24 h preceding research blood sampling (green: normal range). c,d, Highest recorded FiO2 (c) and highest recorded arterial plasma lactate level within 24 h of research sampling (d) in ARDS patients (red samples: lactate =upper limit of normal; green: normal local reference (0.5–1.6 mmol l-1)). e, Blood leukocyte counts, monocyte proportions and monocyte counts from ARDS patients, collected within 48 h of diagnosis (early ARDS) and a healthy volunteer cohort (HC). f, Blood leukocyte count, monocyte proportions and monocyte counts from ARDS patients collected between 48 h and 7 d (late ARDS) and HC. g, Monocyte HLA-DR and CD11b expression in HC and ARDS patients. h, CD14++CD16- classical monocyte proteomic data from ARDS patients, relative to HC, for proteins associated with a human monocyte, in vitro hypoxic gene signature13. i, Classical (CD14++CD16-) monocyte proteome volcano plot from HC and ARDS patients. Significantly upregulated granule-associated proteins in ARDS patients versus HC (blue), a sample of known hypoxia-regulated proteins (orange). j, Classical monocytes proteinase 3, myeloperoxidase and azurocidin 1 copy numbers in HC and ARDS patients. k,l, Pearson’s correlation (k) and heatmap (l) of differentially expressed genes from HC and ARDS patient blood monocytes. Data in a–c are mean ± s.d. expressed as median (e,f) or shown as mean (g,j). In a,c–g and j each datapoint represents one patient/HC; in b, each datapoint represents one independent clinical sample. Statistical testing used was: unpaired, two-tailed Student’s t-test (e–f and j) and Mann–Whitney U-test (g).

Fig 5: Il1b is driver of emergency hematopoiesis.(A) Granulocyte, monocyte, and lymphocyte numbers in the blood of Il1a-/-, Il1b-/-, and WT mice 24 hours p.i. (GAS) and in uninfected controls (PBS), as determined using a hematology analyzer. ANOVA with Dunnett’s correction for multiple comparisons, n = 8 to 14 per genotype. **P < 0.01. (B) Neutrophil (CD45+ CD11b+ Ly6Cmed Ly6G+) and monocyte (CD45+ CD11b+ Ly6G- Ly6Chi) percentages (of living cells) in BM from Il1a-/-, Il1b-/-, and WT mice 24 hours p.i. (GAS) and from uninfected controls (PBS), as determined flow cytometry. One-way ANOVA, 10 to 16 per genotype. ****P < 0.0001. (C) Lin- progenitor cells (CD5- CD45R- CD11b- Gr-1- 7-4- Ter-119-) and early lymphoid precursors (LSK) (Lin- Sca-1+ c-Kithi) in BM from Il1a-/-, Il1b-/-, and WT mice 24 hours p.i. (GAS) and from uninfected mice (PBS), as determined by flow cytometry. ANOVA with Dunnett’s correction for multiple comparisons, n = 7 to 10 per genotype. (D) Common myeloid progenitors (CMPs) (Lin- Sca-1- c-Kithi CD16/32lo, CD34+) and granulocyte-macrophage progenitors (GMPs) (Lin- Sca-1- c-Kithi CD16/32hi, CD34lo) in BM from Il1a-/-, Il1b-/-, and WT mice 24 hours p.i. (GAS) and from uninfected controls (PBS), as determined by flow cytometry. ANOVA with Dunnett’s correction for multiple comparisons, n = 7 to 10 per genotype. **P < 0.01. (E and F) G-CSF protein (encoded by the Csf3 gene) (E) and Csf3 mRNA levels (F) at the site of infection from Il1a-/-, Il1b-/-, and WT mice 24 hours p.i. (GAS) and (for Csf3 mRNA) from uninfected controls (PBS), as determined by enzyme-linked immunosorbent assay (ELISA) and reverse transcription quantitative polymerase chain reaction (RT-qPCR), respectively. Kruskal-Wallis with Dunn’s multiple comparisons (E), n = 22 to 24 per genotype. ***P < 0.001; ****P < 0.0001.