Acute alcohol intoxication inhibits the lineage- c-kit+ Sca-1+ cell response to Escherichia coli bacteremia

Abstract

Alcohol abuse predisposes the host to bacterial infections. In response to bacterial infection, the bone marrow hematopoietic activity shifts toward granulocyte production, which is critical for enhancing host defense. This study investigated the hematopoietic precursor cell response to bacteremia and how alcohol affects this response. Acute alcohol intoxication was induced in BALB/c mice 30 min before initiation of Escherichia coli bacteremia. Bacteremia caused a significant increase in the number of bone marrow lineage (lin(-))-c-kit(+)Sca-1(+) cells. Marrow lin(-)c-kit(+)Sca-1(+) cells isolated from bacteremic mice showed an increase in CFU-granulocyte/macrophage activity compared with controls. In addition to enhanced proliferation of lin(-)c-kit(+)Sca-1(+) cells as reflected by BrdU incorporation, phenotypic inversion of lin(-)c-kit(+)Sca-1(+)Sca-1(-) cells primarily accounted for the rapid increase in marrow lin(-)c-kit(+)Sca-1(+) cells following bacteremia. Bacteremia increased plasma concentration of TNF-alpha. Culture of marrow lin(-)c-kit(+)Sca-1(+)Sca-1(-) cells with murine rTNF-alpha for 24 h caused a dose-dependent increase in conversion of these cells to lin(-)c-kit(+)Sca-1(+) cells. Sca-1 mRNA expression by the cultured cells was also up-regulated following TNF-alpha stimulation. Acute alcohol intoxication inhibited the increase in the number of lin(-)c-kit(+)Sca-1(+) cells in the bone marrow after E. coli infection. Alcohol impeded the increase in BrdU incorporation into marrow lin(-)c-kit(+)Sca-1(+) cells in response to bacteremia. Alcohol also suppressed the plasma TNF-alpha response to bacteremia and inhibited TNF-alpha-induced phenotypic inversion of lin(-)c-kit(+)Sca-1(+)Sca-1(-) cells in vitro. These data show that alcohol inhibits the hematopoietic precursor cell response to bacteremia, which may serve as one mechanism underlying the impaired host defense in alcohol abusers with severe bacterial infections.

Figure 1

Representative dot plots of c-kit-APC vs. Sca-1-PE-Cy7 of lineage negative bone marrow cells. Saline: mice received i.p. saline; EtOH: mice received i.p. alcohol; Control: mice received i.v. saline; 1×10⁶ E. coli: mice received i.v. 1×10⁶ E. coli; 5×10⁷ E. coli: mice received i.v. 5×10⁷ E. coli.

Figure 2

Changes in lin-c-kit+Sca1+ and lin-c-kit+Sca-1- cells in the bone marrow. Saline: mice received i.p. saline plus i.v saline; EtOH: mice received i.p. alcohol plus i.v. saline; 1×10⁶ E. coli: mice received i.p. saline plus i.v. 1×10⁶ E. coli; EtOH/1×10⁶ E. coli: mice received i.p. alcohol plus i.v. 1×10⁶ E. coli; 5×10⁷ E. coli: mice received i.p. saline plus i.v. 5×10⁷ E. coli; EtOH/5×10⁷ E. coli: mice received i.p. alcohol plus i.v. 5×10⁷ E. coli. Data are mean ± SEM (n = 4∼7). Bars with different letters (a – d) at the same time point in each panel are statistically different (p < 0.05).

Figure 3

BrdU incorporation into bone marrow lin-ckit+Sca1+ cells. Saline: mice received i.p. saline plus i.v. saline; EtOH: mice received i.p. alcohol plus i.v. saline; E. coli: mice received i.p. saline plus i.v. 1×10⁶ E. coli; EtOH/E. coli: mice received i.p. alcohol plus i.v. 1×10⁶ E. coli. Data are mean ± SEM (n = 5). Bars with different letters (a – c) in each panel are statistically different (p < 0.05).

Figure 4

Plasma cytokine levels post i.v. challenge with 1×10⁶ E. coli. The baseline level of each cytokine in saline or alcohol treated group is the pooled values of the corresponding cytokine in animals treated with saline or alcohol without bacterial challenge at different time points. Data are mean ± SEM (n = 5). *: p < 0.05 compared to the Saline group at the same time point; †: p < 0.05 compared to the baseline value.

Figure 5

A: TNF-α-induced in vitro inversion of lin-c-kit+Sca1- cells to lin-c-kit+Sca-1+ cells. B and C: the effects of alcohol on TNF-α (1 ng/ml) induced in vitro inversion of lin-c-kit+Sca1- cells to lin-c-kit+Sca-1+ cells and Sca-1 mRNA expression by lin-c-kit+Sca-1- cells. D: the effect of acute alcohol intoxication on the alteration of marrow lin-c-kit+Sca-1+ cell population in response to i.v. challenge with TNF-α. Data are mean ± SEM (n = 5). Bars with different letters (a – d) in each panel are statistically different (p < 0.05).

Figure 6

A and C: CFU activity of bone marrow lin-ckit+Sca1+ cells in M3434 and M3534 media. Data are mean ± SEM (n = 10). Bars with different letters (a – b) in each panel are statistically different (p < 0.05). B and D: changes in lin-c-kit+Sca1+ and lin-c-kit+Sca-1- cells in the systemic circulation. Data are mean ± SEM (n = 4∼7). Bars with different letters (a – b) at the same time point in each panel are statistically different (p < 0.05).

Figure 7

A: blood PMN counts at 24 h post i.v. challenge. Data are mean ± SEM (n = 5). Bars with different letters (a – b) are statistically different (p < 0.05). B: E. coli CFU counts in cultures of the liver and spleen tissue samples at 24 h post i.v. challenge with 1×10⁶ E. coli. Data are mean ± SEM (n = 5). *: p < 0.05 compared to E. coli group. C: E. coli CFU counts in cultures of the blood, liver, and spleen samples from mice challenged with 5×10⁷ E. coli. Data are mean ± SEM (n = 5). *: p < 0.05 compared to E. coli group. D: changes of survival rate following i.v. challenge with 5×10⁷ E. coli. Data are mean ± SEM (n = 5). *: p < 0.05 compared to E. coli group.