Attenuated mRNA expression of inflammatory mediators in neonatal rat lung following lipopolysaccharide treatment

Figures & data

Figure 1 Overview of LPS/TLR-4 signaling pathway.

Notes: LPS is delivered to CD14 by LBP, which in turn transfers LPS to TLR-4. Upon LPS binding, TLR-4 initiates the MyD88-dependent and MyD88-independent pathways. The MyD88-dependent pathway ultimately upregulates the production of inflammatory mediators including cytokines and chemokines, and the MyD88-independent pathway ultimately increases the production of Type I interferons.
Abbreviations: LBP, LPS-binding protein; LPS, lipopolysaccharide; MCP, monocyte chemotactic protein; MIP, macrophage inflammatory protein; MyD88, myeloid differentiation marker 88; TLR, toll-like receptor; TNF-α, tumor necrosis factor-α; TRIF, TIR domain-containing adaptor inducing IFN-β.

Figure 2 Relative mRNA expression of TLRs in the lung of P1, P21, and P70 animals. P1, P21, and P70 animals were treated with saline (S) or 0.25 mg/kg lipopolysaccharide (L) for 2 hours. Relative mRNA levels of TLR-2 (A), TLR-4 (B), TLR-5 (C), and TLR-6 (D) in the lung of these animals were measured by real-time reverse transcription PCR.

Notes: Data were presented as means ± SE; n = 4. *Represents significant difference from P1 S at P < 0.05; ^#represents significant difference from P1 L; ^&represents significant difference from P21 S; ^¥represents significant difference from P21 L; ^§represents significant difference from P70 S (two-way ANOVA followed by Bonferroni post-test).
Abbreviations: mRNA, messenger RNA; P1, postnatal day 1; PCR, polymerase chain reaction; TLR, toll-like receptor.

Table 1 Fold changes in the relative mRNA levels of inflammatory genes in the lung of animals at P1, P21, and P70 following treatment with saline (S) or lipopolysaccharide (L)

Gene name	P1 L vs P1 S	P21 L vs P21 S	P70 L vs P70 S	P21 S vs P1 S	P70 S vs P1 S	P21 L vs P1 L	P70 L vs P1 L
TLR-2 ^a	2.00	6.18*	8.59*	1.87	3.91	5.80*	16.82*
TLR-4 ^a	−1.09	−1.20	−1.75*	1.27	3.93*	1.14	2.44*
TLR-5 ^a	1.18	1.37	1.81*	1.26	1.47	1.47	2.26*
TLR-6 ^a	1.01	1.80	2.56*	1.10	2.71*	1.97	6.88*
LBP ^a	1.16	−1.06	1.74*	5.19*	5.99*	4.21*	8.99*
MyD88 ^b	−1.06	1.31*	1.48*	1.09	1.08	1.52 *	1.70*
CD14 ^b	1.19	2.46*	2.75*	1.04	1.12	2.13*	2.58*
TNF-α ^b	19.06*	10.21*	10.75*	2.64	3.89	1.41	2.19*
IL-1β ^b	9.54*	4.28*	3.17*	4.38*	6.31*	1.97*	2.10*
IL-6 ^b	1.17	21.71*	11.15*	2.32	4.77	43.09*	45.56*
IL-10 ^b	3.76*	5.85*	2.35*	1.78	3.98*	2.77*	2.49*
IL-1ra ^b	1.73	2.30*	2.57*	2.31	2.82	3.07*	4.18*
MIP-1β ^b	2.45	8.98*	3.44*	1.99	4.49*	7.31*	6.32*
MCP1^b	−1.01	4.94*	2.78*	−2.63*	−1.79*	1.88*	1.57*
MIP-2^b	11.79*	31.89*	11.04*	−1.08	2.48	2.52*	2.33*

Notes: P1, P21, and P70 animals were treated with saline (s) or 0.25 mg/kg lipopolysaccharide (L) for 2 hours. Relative mRNA levels of inflammatory mediators in the lung of these animals were measured by real-time reverse transcription PCR (^a) or semi-quantitative reverse transcription PCR (^b). Data were presented as fold changes in relative mRNA levels of inflammatory genes between different treatment groups.

* Represents significant difference between the treatment groups (two-way ANOVA followed by Bonferroni post-test).

Abbreviations: IL, interleukin; MCP-1, monocyte chemotactic protein-1; MIP, macrophage inflammatory protein; mRNA, messenger RNA; P1, postnatal day 1; PCR, polymerase chain reaction; TLR, toll-like receptor; TNF-α, tumor necrosis factor-α.

Figure 3 Relative mRNA expression of CD14, MyD88, and LBP in the lung of P1, P21, and P70 animals. P1, P21, and P70 animals were treated with saline (S) or 0.25 mg/kg lipopolysaccharide (L) for 2 hours. Relative mRNA levels of CD14 (A and B) and MyD88 (A and C) in the lung of these animals were measured by semi-quantitative reverse transcription PCR and the relative mRNA level of LBP (D) was measured by real-time reverse transcription PCR.

Notes: Data were presented as means ± SE; n = 4. *Represents significant difference from P1 S at P < 0.05; ^#represents significant difference from P1 L; ^&represents significant difference from P21 S; ^¥represents significant difference from P21 L; ^§represents significant difference from P70 S (two-way ANOVA followed by Bonferroni post-test).
Abbreviations: LBP, lipopolysaccharide binding protein; MyD88, myeloid differentiation marker 88; P1, postnatal day 1; mRNA, messenger RNA; PCR, polymerase chain reaction.

Figure 4 Relative mRNA expression of proinflammatory cytokines in the lung of P1, P21, and P70 animals. P1, P21, and P70 animals were treated with saline (s) or 0.25 mg/kg lipopolysaccharide (L) for 2 hours. Relative mRNA levels of TNF-α (A and B), IL-1β (A and C), and IL-6 (A and D) in the lung of these animals were measured by semiquantitative reverse transcription PCR.

Notes: Data were presented as means ± SE; n = 4. *Represents significant difference from P1 S at P < 0.05; ^#represents significant difference from P1 L; ^&represents significant difference from P21 S; ^¥represents significant difference from P21 L; ^§represents significant difference from P70 S (two-way ANOVA followed by Bonferroni post-test).
Abbreviations: IL, interleukin; P1, postnatal day 1; PCR, polymerase chain reaction; TNF, tumor necrosis factor.

Figure 5 Relative mRNA expression of anti-inflammatory cytokines in the lung of P1, P21, and P70 animals. P1, P21, and P70 animals were treated with saline (S) or 0.25 mg/kg lipopolysaccharide (L) for 2 hours. Relative mRNA levels of IL-10 (A and B) and IL-1ra (A and C) in the lung of these animals were measured by semiquantitative reverse transcription PCR.

Notes: Data were presented as means ± SE; n = 4. *Represents significant difference from P1 S at P < 0.05; ^#represents significant difference from P1 L; ^&represents significant difference from P21 S; ^§represents significant difference from P70 S (two-way ANOVA followed by Bonferroni post-test).
Abbreviations: IL-10, interleukin-10; IL-1ra, interleukin-1 receptor antagonist; mRNA, messenger RNA; P1, postnatal day 1.

Table 2 Ratios of the relative mRNA levels of proinflammatory to anti-inflammatory cytokines in the lung of P1, P21, and P70 animals following treatment with saline (S) or lipopolysaccharide (L)

Ratio	P1 S	P21 S	P70 S	P1 L	P21 L	P70 L
TNF-α:IL-10	0.27 ± 0.04	0.43 ± 0.09	0.32 ± 0.07	1.46 ± 0.48	0.75 ± 0.11*	1.20 ± 0.06*
TNF-α:IL-1ra	0.12 ± 0.03	0.13 ± 0.01	0.15 ± 0.01	1.18 ± 0.28	0.62 ± 0.12	0.66 ± 0.05
IL-1β:IL-10	0.50 ± 0.06	1.30 ± 0.21	0.97 ± 0.23	1.35 ± 0.36	0.93 ± 0.13	1.05 ± 0.04
IL-1β:IL-1ra	0.21 ± 0.02	0.40 ± 0.03	0.46 ± 0.03	1.15 ± 0.28	0.79 ± 0.20	0.60 ± 0.09
IL-6:IL-10	0.25 ± 0.05	0.30 ± 0.02	0.33 ± 0.07	0.07 ± 0.03	1.14 ± 0.10*	1.28 ± 0.04*
IL-6:IL-1ra	0.10 ± 0.02	0.10 ± 0.01	0.16 ± 0.01	0.06 ± 0.02	0.99 ± 0.25*	0.71 ± 0.06*

Notes: P1, P21, and P70 animals were treated with saline (S) or 0.25 mg/kg lipopolysaccharide (L) for 2 hours. Relative mRNA levels of proinflammatory and anti-inflammatory cytokines in the lung of these animals were measured by semi-quantitative RT-PCR, and the ratios of relative mRNA levels of proinflammatory to anti-inflammatory cytokines were calculated. Data were presented as means ± SE.

* Represents significant difference from P1 L (two-way ANOVA followed by Bonferroni post-test).

Abbreviations: IL, interleukin; mRNA, messenger RNA; P1, postnatal day 1; PCR, polymerase chain reaction; TNF-α, tumor necrosis factor-α.

Figure 6 Relative mRNA expression of chemokines in the lung of P1, P21, and P70 animals. P1, P21, and P70 animals were treated with saline (S) or 0.25 mg/kg lipopolysaccharide (L) for 2 hours. Relative mRNA levels of MIP-1β (A and B), MIP-2 (A and C), and MCP-1 (A and D) in the lung of these animals were measured by semiquantitative reverse transcription PCR.

Notes: Data were presented as means ± SE; n = 4. *Represents significant difference from P1 S at P < 0.05; ^#represents significant difference from P1 L; ^&represents significant difference from P21 S; ^§represents significant difference from P70 S (two-way ANOVA followed by Bonferroni post-test).
Abbreviations: MCP-1, monocyte chemotactic protein-1; MIP, macrophage inflammatory protein; P1, postnatal day 1.

Figure 7 Enzymatic activity of myeloperoxidase (MPO) in the lung of P1, P21, and P70 animals. P1, P21, and P70 animals were treated with saline (S) or 0.25 mg/kg lipopolysaccharide (L) for 2 hours. MPO activity in the lung of these animals was then measured by enzymatic assay.

Notes: Data were presented as means ± SE; n = 4. ^#Represents significant difference from P1 L; ^¥represents significant difference from P21 L; ^§represents significant difference from P70 S (two-way ANOVA followed by Bonferroni post-test).
Abbreviation: P1, postnatal day 1.