The regulatory mechanism of TNF-α and c-Jun NH2-terminal kinase (JNK) signaling pathways in PA VAP-induced lung injury.

<p><i>P</i>. <i>aeruginosa</i>, <i>Pseudomonas aeruginosa</i>; ICAM, intracellular adhesion molecule; VCAM, vascular cell adhesion molecule; IL, interleukin.</p

Mechanical ventilation (MV) after PA instillation induced mRNA expression of cytokines and NF-κB as well as AP-1 DNA-binding activity in the lungs.

<p>(A) MV after PA instillation significantly induced mRNA expression of ICAM and VCAM in the lungs in WT mice as compared with PA instillation or MV alone group. n = 5/group. (B). MV after PA instillation induced TNF-α, IL-1β and IL-6 production in BALF of WT mice as compared to those in PA instillation or MV alone group. The concentrations of TNF-α, IL-1β and IL-6 in the BALF were determined by enzyme-linked immunosorbent assay (ELISA). n = 5/group. (C) The DNA-binding activity of NF-κB and AP-1 in the lungs of WT mice receiving MV after normal saline or PA (10⁴ or 10⁶ CFU) instillation. Signal intensity was determined by densitometry and normalized to free probe band. *P<0.05, **P<0.01. n = 4/group. CFU, colony forming unit; NS, normal saline. *P<0.05, **P<0.01.</p

Effects of mechanical ventilation (MV) after instillation of supernatants from <i>ex vivo</i> PA stimulated AMs on pulmonary MPO activity and total protein concentration in BALF in WT and in JNK1^-/- mice.

<p>MV after supernatant instillation induced TNF-α levels (A) as well as total protein concentration (B) in BALF in WT mice but not in JNK1^-/- mice. n = 6/group. The concentrations of TNF-α in the BALF were determined by enzyme-linked immunosorbent assay (ELISA). (C) MV after supernatant instillation induced pulmonary MPO activity in WT mice but not in JNK1^-/- mice. Also, MV after TNF-α instillation induced lung MPO activity in WT mice but not in JNK1^-/- mice. Mice were instilled with TNF-α or supernatants from PA-stimulated AMs and received MV for 3 h at 1 h after supernatant instillation. n = 5-6/group. (D) Mechanical ventilation after instillation of supernatants from <i>ex vivo</i> PA stimulation of AMs induced a higher mortality in WT mice than in JNK1^-/- mice. PA, <i>P</i>. <i>aeruginosa</i>; Super, supernatants from <i>ex vivo</i> PA stimulation of AMs. *P<0.05, **P<0.01, ***P<0.001.</p

Mechanical ventilation (MV) after PA instillation induced mRNA expression of cytokines in the lungs in WT mice but not in JNK1^-/- mice.

<p>(A) mRNA expression of ICAM, VCAM, IL-6, and MIP-2 in the lungs in WT mice and JNK1^-/- mice after PA instillation and MV treatment. Signal intensity was determined by densitometry and normalized to GAPDH band. n = 4/group. (B) The levels of proinflammatory cytokines (TNF-α, IL-1β and IL-6) in BALF in WT and JNK1^-/- mice after PA instillation and MV treatment. The concentrations of TNF-α, IL-1β and IL-6 in the BALF were determined by enzyme-linked immunosorbent assay (ELISA). n = 5-6/group. *P<0.05, **P<0.01.</p

Effects of <i>P</i>. <i>aeruginosa</i> (PA) instillation on mechanical ventilation-induced lung injury.

<p>(A) WT mice were intranasally instilled with live PA (1 × 10⁴ or 1 × 10⁶ CFU) or normal saline at 2 days before receiving mechanical ventilation (MV) for 3 h and lung tissues were harvested and assayed. (B) Positive bacterial cultures of lung tissues shown as CFU counted on the LB plates. (C) PA instillation before ventilation increased MPO activity of the lung. (D) PA instillation before MV increased nitrite concentration in BALF. Nitrite in BALF was assayed by Griess reagent. PA, <i>P</i>. <i>aeruginosa</i>; MPO, myeloperoxidase; BALF, bronchoalveolar lavage fluid; NS, normal saline. *P<0.05, **P<0.01, ***P<0.001. n = 6/group.</p

Instillation of supernatants from <i>ex vivo</i> PA-stimulated AMs enhanced mechanical ventilation-induced lung injury.

<p>(A) AMs collected from BALF of WT mice were stimulated with live PA for 4 h. After stimulation, supernatants were collected for TNF-α analysis by ELISA or for nitrite production assay by using Griess reagent. n = 6/group. (B) Mechanical ventilation (MV) after instillation of supernatants from <i>ex vivo</i> PA-stimulated AMs increased TNF-α as well as IL-1β levels in BALF. The concentrations of TNF-α and IL-1β in the BALF were determined by enzyme-linked immunosorbent assay (ELISA). Mice were instilled with supernatants from PA-stimulated AMs and received MV for 3 h at 1 h after supernatant instillation. n = 4/group. (C) MV after instillation of TNF-α (100 ng) or supernatants from <i>ex vivo</i> PA-stimulated AMs induced pulmonary MPO activity. Mice were instilled with TNF-α or supernatants from PA-stimulated AMs and received MV for 3 h at 1 h after supernatant instillation. n = 6/group. (D) MV after instillation of supernatants from <i>ex vivo</i> PA-stimulated AMs or TNF-α (100 ng) induced total protein concentration in BALF. n = 5/group. PA, <i>P</i>. <i>aeruginosa</i>; Super, supernatants from <i>ex vivo</i> PA stimulation of AMs. *P<0.05, **P<0.01, ***P<0.001.</p

Animal models in examining PA VAP-induced lung injury.

<p>(A) Animals were instilled with 10 μl of normal saline as the control or with equal volume of PA (ATCC 27853, 10⁴ or 10⁶ CFU) via nostrils into the lungs. Two days after PA instillation, mice received MV with high tidal volume for 3 hr. (B) To examine the role of AMs on PA VAP-induced lung injury, AMs collected from BALF of WT mice were stimulated with live PA for 4 h. The anesthetized mice were instilled with 10 μl of supernatants and received MV for 3 hr at 1 hr after supernatant instillation. PA, <i>P</i>. <i>aeruginosa</i>; VAP, Ventilator-associated pneumonia; AMs, alveolar macrophages; BALF, bronchoalveolar lavage fluid; NS, normal saline.</p

<i>Pseudomonas aeruginosa</i> Ventilator-Associated Pneumonia Induces Lung Injury through TNF-α/c-Jun NH2-Terminal Kinase Pathways

<div><p>Ventilator-associated pneumonia (VAP) is a common nosocomial infection among intensive care unit (ICU) patients. <i>Pseudomonas aeruginosa</i> (PA) is the most common multidrug-resistant Gram-negative pathogen and VAP caused by PA carries a high rate of morbidity and mortality. This study examined the molecular mechanism of PA VAP-induced lung injury. C57BL/6 wild-type (WT) mice and JNK1 knockout (JNK1^-/-) mice received mechanical ventilation (MV) for 3 h at 2 days after receiving nasal instillation of PA. The WT and JNK1^-/- mice also received MV after the induction of lung injury by instillation of supernatants from PA-stimulated alveolar macrophages (AMs). AMs isolated from WT, IκB-kinase (IKK)β^ΔMye (IKKβ was selectively deleted in macrophages), and JNK1^-/- mice were <i>ex vivo</i> stimulated with live PA and supernatants were collected for cytokine assay. Intranasal instillation of 10⁶ PA enhanced MV-induced NF-κB DNA binding activity in the lungs and nitrite levels in BALF. MV after PA instillation significantly increased the expression of ICAM and VCAM in the lungs and TNF-α, IL-1β, and IL-6 levels in bronchoalveolar lavage fluid (BALF) of WT mice, but not in JNK1^-/- mice. MV after supernatant instillation induced more total protein concentration in BALF and neutrophil sequestration in the lungs in WT mice than JNK1^-/- mice and cytokine assay of supernatants indicated that TNF-α is a critical regulator of PA VAP-induced lung injury. E<i>x vivo</i> PA stimulation induced TNF-α production by AMs from WT as well as JNK1^-/- mice but not IKKβ^ΔMye mice. In summary, PA colonization plays an important role in PA VAP-induced lung injury through the induction of JNK1-mediated inflammation. These results suggest that the pathogenesis mechanism of PA VAP involves production of TNF-α through activation of IKK/NF-κB pathways in AMs and JNK signaling pathway in the lungs.</p></div

E<i>x vivo</i> PA stimulation of AMs from IκB-kinase (IKK)β^ΔMye (IKKβ was selectively deleted in macrophages) mice induces less TNF-α production in supernatants than those from JNK1^-/- mice as well as WT mice but not IKKβ^ΔMye mice.

<p>AMs isolated from mice were <i>ex vivo</i> stimulated with PA (10⁴ or 10⁶ CFU) for 4 hr and the supernatants were examined. (A) E<i>x vivo</i> PA stimulation of AMs from IKKβ^ΔMye mice induces less TNF-α production in supernatants than those from IKKβ^{ΔMye +/-} mice. The concentrations of TNF-α were determined by enzyme-linked immunosorbent assay (ELISA). n = 5/group. (B) E<i>x vivo</i> PA stimulation of AMs from JNK1^-/- as well as WT mice induce TNF-α production in supernatants. n = 5/group. PA, <i>P</i>. <i>aeruginosa</i>; Super, supernatants from <i>ex vivo</i> PA stimulation of AMs. *P<0.05, **P<0.01, ***P<0.001.</p

Fructooligosaccharides (FOS) or dead <i>L</i>. <i>salivarius</i> feeding decreased the intestinal bacterial overgrowth in STZ-DM mice.

<p>(A) Relative abundance of bacteria across difference groups, as indicated by 16S rRNA gene sequencing. Values represent the mean abundance of Genus found at >1% relative abundance in at least one sample. (B) The collected mucosa from the terminal ileum were weighed and homogenized. Total bacteria counts were significantly increased in STZ-DM mice and FOS or dead <i>L</i>. <i>salivarius</i> feeding decreased them. (C) <i>E</i>. <i>coli</i> and <i>K</i>. <i>pneumoniae</i> of intestinal lumen were significantly increased in STZ-DM mice in comparison with those in SPF WT mice. FOS or dead <i>L</i>. <i>salivarius</i> feeding decreased the growth of pathogenic bacteria such as <i>E</i>. <i>coli</i> and <i>K</i>. <i>pneumoniae</i> in STZ mice. (D) The growth of <i>lactobacilli/bifidas</i> in intestinal lumen was significantly decreased in STZ-DM mice compared with SPF mice. FOS feeding significantly increased them in STZ-DM mice. STZ, streptozotocin; DM, diabetes mellitus; FOS, fructooligosaccharides; dLac, dead <i>L</i>. <i>salivarius</i>. *P<0.05, **P<0.01. n = 5/group.</p