Combined immunomodulator and antimicrobial therapy eliminates polymicrobial sepsis and modulates cytokine production in combined injured mice

Figures & data

Figure 1. Probit plot of radiation dose response of female B6D2F₁/J mice exposed to ⁶⁰Co γ-radiation. Groups of mice were given 8.0 (40 mice), 8.5 (40 mice), 9.0 (32 mice), 9.5 (28 mice), 10.0 (28 mice), 10.5 (32 mice), 11.0 (40 mice), 11.5 (40 mice), or 12.0 Gy (40 mice) at 0.4 Gy/min mid-line tissue from bilaterally positioned Co-60 gamma-radiation sources in the AFRRI cobalt high-dose-rate irradiation facility. Survival at 30 days was evaluated by probit analysis.

Figure 2. Incidence of Gram-negative and Gram-positive bacteria detected in ventricular heart blood, liver, and/or spleen of euthanized, moribund B6D2F₁/J mice vs. time (days) post-irradiation (9.5 Gy or 9.75 Gy Co-60 gamma-radiation).

Table I. Bacterial translocation in combined injured (CI) and radiation injured (RI) mice given 9.75 Gy ⁶⁰Co gamma-photon radiation.

Number of mice with number of isolates	Treatment
	CI	RI
Mice sampled	46	44
Mice, septic	45	41
Single isolate	20	26
Multiple isolates	25	15
2 isolates per mouse	16	13
1 GN^a + 1 GP^b	15	10
2 GN	1	0
2 GP	0	3
3 isolates per mouse	9	2
1 GN + 2 GP	8	2
2 GN + 1 GP	1	0

^aGN, Gram-negative; ^bGP, Gram-positive.

Figure 3. Incidence of isolates of bacterial species from ventricular heart blood, liver, and/or spleen of lethally irradiated (RI, 9.75 Gy Co-60 gamma-radiation) mice compared to CI (RI + skin wound) mice vs. time (days) post-irradiation. Numbers (e.g., 2/0) above the bars indicate total number of infected mice from which the indicated bacterial species were isolated/number of mice with no detectable bacterial growth on each day.

Figure 4. Survival of lethally irradiated (RI, 9.75 Gy ⁶⁰Co gamma-radiation) mice (n = 20) administered oral antimicrobial therapy with LVX (90 mg/kg q.d. po) and AMX (300 mg/kg q.d. po) for 14 days from day 8 through day 21 after irradiation. *p < 0.01 (RI + water vs. RI + LVX), #p < 0.05 (RI + AMX vs. RI + LVX), @p < 0.05 (RI + water vs. RI + LVX AMX).

Figure 5. Survival of CI (lethally irradiated, 9.75 Gy, then wounded) B6D2F₁/J mice (n = 20) administered an immunomodulator (STDCM-MPL, 100 μg, ip, 1 h after irradiation), and antimicrobial agents (LVX, 90 mg/kg q.d. po and AMX, 325 mg/kg q.d. po). Antimicrobial agents were administered for 21 days from day 1 through day 21 after irradiation. *p < 0.01 (CI + STDCM-MPL + LVX + AMX vs. CI + SQL + LVX + AMX).

Figure 6. (A) Induction of cytokines in serum (IL-1α, IL-1β, IL-6, IL-10, G-CSF, and GM-CSF) by STDCM-MPL and LVX in irradiated and CI mice. Mice were given 9.75 Gy Co-60 gamma-radiation (or sham irradiated) and wounded within 1 h of irradiation. Mice received treatments of STDCM-MPL (100 μg, ip, 1 h after irradiation and wounding) and/or LVX (90 mg/kg q.d., po, days 1–7). Blood samples were collected (n = 5 mice per group per time point) at indicated time points (day 0 [4–5 h], 1, 3, 7 d after irradiation) and serum samples were analyzed by multiplex Luminex assay for cytokines (pg/ml ± SEM). Numbers above bars denote significant difference (p < 0.05) compared to respective controls: 1 signifies a statistically significant difference between groups R and R + W; 2, between R + W + water and R + W + LVX; 3, between R + SQL and R + STDCM-MPL; 4, between R + W + SQL and R + W + STDCM-MPL; 5, between R + STDCM-MPL and R + W + STDCM-MPL; 6, between Sham R and Sham R + W; and 7, between R + LVX and R + W + LVX. The apostrophe (’) indicates significant reduction of cytokine or chemokine production compared to the respective control. (B) Induction of cytokines in serum (IFN-γ, KC, MCP-1, MIP-1α, MIP-1β, and TNF-α) by STDCM-MPL and LVX in irradiated and CI mice. Mice were given 9.75 Gy Co-60 gamma-radiation (or sham irradiated) and wounded within 1 h of irradiation. Mice received treatments of STDCM-MPL (100 μg, ip, 1 h after irradiation and wounding) and/or LVX (90 mg/kg q.d., po, days 1–7). Blood samples were collected (n = 5 mice per group per time point) at indicated time points (day 0 [4–5 h], 1, 3, 7 d after irradiation) and serum samples were analyzed by multiplex Luminex assay for cytokines (pg/ml ± SEM). Numbers above bars denote significant difference (p < 0.05) compared to respective controls: 1 signifies a statistically significant difference between groups R and R + W; 2, between R + W + water and R + W + LVX; 3, between R + SQL and R + STDCM-MPL; 4, between R + W + SQL and R + W + STDCM-MPL; 5, between R + STDCM MPL and R + W + STDCM-MPL; 6, between Sham R and Sham R + W; and 7, between R + LVX and R + W + LVX. The apostrophe (’) indicates significant reduction of cytokine or chemokine production compared to the respective control.

Figure 7. (A) Induction of cytokines in spleen lysates (IL-1α, IL-1β, IL-6, IL-10, G-CSF, and GM-CSF) by STDCM-MPL and LVX in irradiated and CI mice. Mice were given 9.75 Gy Co-60 gamma-radiation (or sham irradiated) and wounded within 1 h of irradiation. Mice received treatments of STDCM-MPL (100 μg, ip, 1 h after irradiation and wounding) and/or LVX (90 mg/kg q.d., po, days 1–7). Spleen lysates were collected (n = 5 mice per group per time point) at indicated time points (day 0 [4–5 h], 1, 3, 7 d after irradiation) and spleen lysate samples were analyzed by multiplex Luminex assay for cytokines (pg/ml ± SEM). Numbers above bars denote significant difference (p < 0.05) compared to respective controls: 1 signifies a statistically significant difference between groups R and R + W; 2, between R + W + water and R + W + LVX; 3, between R + SQL and R + STDCM-MPL; 4, between R + W + SQL and R + W + STDCM-MPL; 5, between R + STDCM-MPL and R + W + STDCM-MPL; 6, between Sham R and Sham R + W. The apostrophe (’) indicates significant reduction of cytokine or chemokine production compared to the respective control. (B) Induction of cytokines in spleen lysates (IFN-γ, KC, MCP-1, MIP-1α, MIP-1β, and TNF-α) by STDCM-MPL and LVX in irradiated and CI mice. Mice were given 9.75 Gy Co-60 gamma-radiation (or sham irradiated) and wounded within 1 h of irradiation. Mice received treatments of STDCM-MPL (100 μg, ip, 1 h after irradiation and wounding) and/or LVX (90 mg/kg q.d., po, days 1–7). Spleen lysates were collected (n = 5 mice per group per time point) at indicated time points (day 0 (4–5 h), 1, 3, 7 d after irradiation) and spleen lysate samples were analyzed by multiplex Luminex assay for cytokines (pg/ml ± SEM). Numbers above bars denote significant difference (p < 0.05) compared to respective controls: 1 signifies a statistically significant difference between groups R and R + W; 2, between R + W + water and R + W + LVX; 3, between R + SQL and R + STDCM-MPL; 4, between R + W + SQL and R + W + STDCM-MPL; 5, between R + STDCM-MPL and R + W + STDCM-MPL; 6, between Sham R and Sham R + W. The apostrophe (’) indicates significant reduction of cytokine or chemokine production compared to the respective control.

Table II. Bacterial isolation from ventricular heart blood, liver, and/or spleen from euthanized moribund or recently deceased (< 2 h) irradiated (RI) or combined injured (CI) B6D2F₁/J mice.

Microbial species	Number of mice
	RI						CI
	None (18)^a	Vehicle 1 (SQL^b) ip. (14)	Vehicle 2 (H2O) po (12)	STDCM- MPL^c (10)	SQL LVX^d + AMX^e (13)	STDCM-MPL LVX + AMX (9)	None (17)	Vehicle 1 (SQL) ip (16)	Vehicle 2 (H2O) po (13)	STDCM- MPL (15)	SQL LVX + AMX (17)	STDCM-MPL LVX + AMX (17)
Gram-negative
Escherichia coli	11	11	8	8	0	1^f	16	13	13	13	0	0
Enterobacter cloacae	1	0	0	2	0	0	2	3	0	3	0	0
Gram-positive
Enterococcus faecalis	6	3	3	2	0	2^f	8	3	4	0	0	0
E. gallinarum	0	0	0	0	0	0	0	2	6	0	0	0
S. xylosus	5	1	6	1	0	0	2	0	5	0	0	0
S. cohnii	0	1	0	0	0	0	0	0	0	0	0	0
Lactobacillus sp.	0	0	0	0	0	0	2	0	0	0	0	0
Yeast
Candida parapsilosis	0	0	0	0	0	0	0	0	0	0	0	2
No microbial growth	2	1	0	0	12	6	1	0	0	0	17	15

Mice were treated with STDCM-MPL, 100 μg i.p., 1 h after irradiation and oral antimicrobial therapy with levofloxacin (LVX), 90 mg/kg q.d. po, and amoxicillin (AMX), 325 mg/kg q.d. po, for 14 days from Day 8 through Day 21 after RI or from Day 1 through Day 21 after CI. ^a (#), total number of mice evaluated in each treatment group; ^bSQL, 2% squalene-0.2% Tween 80-water; ^cSTDCM-MPL, synthetic trehalose dicorynomycolate and monophosphoryl lipid A adjuvant; ^dLVX, levofloxacin; ^eAMX, amoxicillin; ^fE. coli was isolated from one RI mouse early on d 8, the first day of antimicrobial therapy and E. faecalis was isolated from two RI mice on d 25 and 29, several days after the end of therapy, that were treated with the combination STDCM-MPL, LVX, and AMX from two CI mice on d 25 and 29, several days after the end of therapy, that were treated with the combination STDCM-MPL, LVX, and AMX.