An Early Myelosuppression in the Acute Mouse Sepsis Is Partly Outcome-Dependent

Abstract

Adult hematopoietic stem and progenitor cells (HSPCs) respond to bacterial infections by expansion to myeloid cells. Sepsis impairs this process by suppressing differentiation of stem cells subsequently contributing to an ineffective immune response. Whether the magnitude of HSPCs impairment in sepsis is severity-dependent remains unknown. This study investigated dynamics of the HSPC immune-inflammatory response in the bone marrow, splenic, and blood compartments in moribund and surviving septic mice. The 12-week-old outbred CD-1 female mice (n=65) were subjected to a cecal ligation and puncture (CLP) sepsis, treated with antibiotics and fluid resuscitation, and stratified into predicted-to-die (P-DIE) and predicted-to-survive (P-SUR) cohorts for analysis. CLP strongly reduced the common myeloid and multipotent progenitors, short- and long-term hematopoietic stem cell (HSC) counts in the bone marrow; lineage^-ckit⁺Sca-1⁺ and short-term HSC suppression was greater in P-DIE versus P-SUR mice. A profound depletion of the common myeloid progenitors occurred in the blood (by 75%) and spleen (by 77%) of P-DIE. In P-SUR, most common circulating HSPCs subpopulations recovered to baseline by 72 h post-CLP. Analysis of activated caspase-1/-3/-7 revealed an increased apoptotic (by 30%) but not pyroptotic signaling in the bone marrow HSCs of P-DIE mice. The bone marrow from P-DIE mice revealed spikes of IL-6 (by 5-fold), CXCL1/KC (15-fold), CCL3/MIP-1α (1.7-fold), and CCL2/MCP-1 (2.8-fold) versus P-SUR and control (TNF, IFN-γ, IL-1β, -5, -10 remained unaltered). Summarizing, our findings demonstrate that an early sepsis-induced impairment of myelopoiesis is strongly outcome-dependent but varies among compartments. It is suggestive that the HSCPC loss is at least partly due to an increased apoptosis but not pyroptosis.

Keywords: caspases; cecal ligation and puncture; hematopoietic stem and progenitor cells; immunity; infection; outcome prediction.

Figure 1

Outcome-related and longitudinal changes in the total cell count of the bone marrow progenitor cells in CD-1 mice subjected to CLP. (A) Comparative data for mice assigned as predicted to survive (P-SUR, green) and predicted to die (P-DIE, red) are shown for the white blood count (WBC), common myeloid progenitors (CMP), LSK cells, long-term hematopoietic stem cells (LT-HSC), short-term HSCs (ST-HSC), and multipotent progenitors (MPP). Subgroups were compared using Student t-test with Welch correction whenever required. Hash signs show differences between P-DIE and P-SUR groups; asterisks indicate differences between given subgroups of mice and control (healthy) mice, (B) Changes in the cell counts of given populations in the predicted to survive (P-SUR) mice only; dotted connecting lines indicate separate sets of P-SUR sacrificed at a given timepoint. Baseline (BL) n=10, 24 h n=9, 48 h n=12, 72 h n=9, 9 days n=5 Groups were compared using 1-way ANOVA with Tukey’s multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ^#p<0.05; ^##p<0.01.

Figure 2

Outcome-related and longitudinal changes in the total cell count of peripheral blood progenitor cells in CD-1 mice subjected to CLP. (A) Comparative data for mice assigned as predicted to survive (P-SUR) and predicted to die (P-DIE) are shown for the white blood count (WBC), common myeloid progenitors (CMP), LSK cells, long-term hematopoietic stem cells (LT-HSC), short-term HSCs (ST-HSC), and multipotent progenitors (MPP). Subgroups were compared using Student t-test with Welch correction whenever required. Hash show differences between P-DIE and P-SUR groups; asterisks indicate differences between given subgroups of mice and control (healthy) mice, (B) Changes in the cell counts of given populations in P-SUR mice only; dotted connecting lines indicate separate sets of P-SUR sacrificed at a given timepoint. Baseline n=10, 24 h n=10, 48 h n=10, 72 h n=12, 9 days n=5. Groups were compared using 1-way ANOVA test with Tukey’s multiple comparison test. *p<0.05, **p<0.01, ***p<0.001. ^#p<0.05.

Figure 3

Outcome-related and longitudinal changes in the total cell count of the spleen progenitor cells in CD-1 mice subjected to CLP. (A) Comparative data for mice assigned as predicted to survive (P-SUR) and predicted to die (P-DIE) are shown for the common myeloid progenitors (CMP), LSK cells, long-term hematopoietic stem cells (LT-HSC), short-term HSCs (ST-HSC), and multipotent progenitors (MPP). Subgroups were compared using Student t-test with Welch correction whenever required. Hash show differences between P-DIE and P-SUR groups; asterisks indicate differences between given subgroups of mice and control (healthy) mice, (B) Changes in the cell counts of given populations in P-SUR animals only; dotted connecting lines indicate separate sets of P-SUR mice sacrificed at a given timepoint; baseline (BL) n=10, 24 h n=8, 48 h n=8, 72 h n=9, 9 days n=5. Groups were compared using one-way ANOVA test with Tukey’s multiple comparison test. *p<0.05, **p<0.01, ***p<0.001. ^#p<0.05.

Figure 4

Activation of inflammatory and apoptotic caspases in the bone marrow hematopoietic stem and progenitor cells. (A) Lineage-negative mononuclear cells were analyzed for the expression of ckit and Sca-1 and the LSK subpopulation was analyzed for: (B) active caspases-3/-7 and (C) active caspase-1. Percentage of LSK cells with active caspases were compared between predicted to die (P-DIE) and predicted to survive (P-SUR) mice using 1-way ANOVA test with Tukey’s multiple comparison test. **p<0.01.

Figure 5

Outcome-related changes in the cytokine’s milieu of the bone marrow twenty-four hours after CLP. Total-protein normalized concentrations of (A) IL-6. (B) TNF. (C) IL-1β. (D) IFN-γ. (E) IL-5. (F) IL-10. (G) CXCL1/KC. (H) CCL3/MIP-1α. (I) CCL2/MCP-1 in the bone marrow supernatants of predicted-to survive (P-SUR) and predicted-to die (P-DIE) mice are shown. Concentration of cytokines between groups were compared using Student t-test with Welch correction whenever required. **p < 0.01, ****p < 0.0001 between control and septic mice. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 between P-DIE and P-SUR groups.

Figure 6

Expression of mRNA for selected cytokines by the bone marrow hematopoietic progenitors in sepsis. The LSK cells were analyzed twenty-four hours after CLP for the expression of mRNA transcripts for: (A) IL-6. (B) IL-1β. (C) TNF by flow cytometry. Groups were compared using Student t-test. **p<0.01.