Interleukin-36γ and IL-36 receptor signaling mediate impaired host immunity and lung injury in cytotoxic <i>Pseudomonas aeruginosa</i> pulmonary infection: Role of prostaglandin E2

<div><p><i>Pseudomonas aeruginosa</i> is a Gram-negative pathogen that can lead to severe infection associated with lung injury and high mortality. The interleukin (IL)-36 cytokines (IL-36α, IL-36β and IL-36γ) are newly described IL-1 like family cytokines that promote inflammatory response via binding to the IL-36 receptor (IL-36R). Here we investigated the functional role of IL-36 cytokines in the modulating of innate immune response against <i>P</i>. <i>aeruginosa</i> pulmonary infection. The intratracheal administration of flagellated cytotoxic <i>P</i>. <i>aeruginosa</i> (ATCC 19660) upregulated IL-36α and IL-36γ, but not IL-36β, in the lungs. IL-36α and IL-36γ were expressed in pulmonary macrophages (PMs) and alveolar epithelial cells in response to <i>P</i>. <i>aeruginosa in vitro</i>. Mortality after bacterial challenge in IL-36 receptor deficient (IL-36R^-/-) mice and IL-36γ deficient (IL-36γ^-/-) mice, but not IL-36α deficient mice, was significantly lower than that of wild type mice. Decreased mortality in IL-36R^-/- mice and IL-36γ^-/- mice was associated with reduction in bacterial burden in the alveolar space, bacterial dissemination, production of inflammatory cytokines and lung injury, without changes in lung leukocyte influx. Interestingly, IL-36γ enhanced the production of prostaglandin E2 (PGE2) during <i>P</i>. <i>aeruginosa</i> infection <i>in vivo</i> and <i>in vitro</i>. Treatment of PMs with recombinant IL-36γ resulted in impaired bacterial killing via PGE2 and its receptor; EP2. <i>P</i>. <i>aeruginosa</i> infected EP2 deficient mice or WT mice treated with a COX-2-specific inhibitor showed decreased bacterial burden and dissemination, but no change in lung injury. Finally, we observed an increase in IL-36γ, but not IL-36α, in the airspace and plasma of patients with <i>P</i>. <i>aeruginosa</i>-induced acute respiratory distress syndrome. Thus, IL-36γ and its receptor signal not only impaired bacterial clearance in a possible PGE2 dependent fashion but also mediated lung injury during <i>P</i>. <i>aeruginosa</i> infection.</p></div

Impact of PGE2-EP2 signal in <i>P</i>. <i>aeruginosa</i> infection <i>in vivo</i>.

<p>(A-C) WT and PGE2 receptor 2 deficient (EP2^-/-) mice were intratracheally infected with 2.0×10⁵ CFU <i>P</i>. <i>aeruginosa</i>. After 24 h infection, (A) bacterial counts in BAL (upper panel) and homogenized spleen samples (lower panel), (B) the production of TNF-α and IL-6 in BAL and (C) the albumin concentration in BAL were examined. Each group consisted of 4–5 mice. (D, E) WT mice were administrated COX-2 inhibitor (NS398; 10μM) or vehicle intraperitoneally 1 h prior to 2.0 × 10⁵ CFU <i>P</i>. <i>aeruginosa</i> challenge. (D) Bacterial counts in BAL (left panel) and homogenized spleen samples (right panel), (E) the albumin concentration in BAL were examined. Each group consisted of 4–5 mice. Data are shown as mean (A, D) or means ± SD (B, C and E). * <i>p</i><0.05, # <i>p</i><0.01, ¶ <i>p</i><0.0001, significant compared with untreated mice or as indicated.</p

IL-36γ impairs PM bacterial killing during <i>P</i>. <i>aeruginosa</i> and dependence on PGE2-EP2 signal.

<p>Primary PMs were harvested from WT, IL-36R^-/- or EP2^-/- mice, and treated with or without recombinant IL-36γ (100ng/ml) for 18h. (A) Vehicle or rIL-36γ treated PMs (1 × 10⁶ cells/well) isolated from WT and IL-36R-/- mice were incubated with for 2 h with FITC-labelling heat killed <i>P</i>. <i>aeruginosa</i> at a MOI 100. After 2h incubation, cells were collected and analyzed the phagocytic response as FITC positive cells by flow cytometry. Data showed the percentage of phagocytic PMs. (B) Vehicle or rIL-36γ treated PMs (1 × 10⁶ cells/well) from WT and IL-36R^-/- mice, (C) Vehicle or rIL-36γ treated PMs (1 × 10⁶ cells/well) isolated from WT and EP2^-/- mice were incubated with live <i>P</i>. <i>aeruginosa</i> at a MOI 100. PMs were harvested at 30 min to quantify CFU as the initial time point, or incubated further for an additional 90 min. CFU/10⁶ PMs were obtained each samples by subsequent dilution method. Data (means ± SEM) are representative of two independent experiments. * <i>p</i><0.05, § <i>p</i><0.001, ¶ <i>p</i><0.0001, compared with untreated mice or as indicated.</p

IL-36 receptor and IL-36γ deficient mice attenuated lung injury during <i>P</i>. <i>aeruginosa</i> infection.

<p>(A, C) Lung histopathological analysis in <i>P</i>. <i>aeruginosa</i> infected WT, IL-36R^-/- and IL-36γ-/- mice was performed at 10 h (A) and 24 h (C) post infection. H&E-stained lung tissue at magnification of 400X. (B) The quantification of lung injury in lung sections with infected-WT, IL-36R^-/- and IL-36γ^-/- mice at 10 h were evaluated as previously described (n = 3 per each group). Lung injury scoring system parameters include neutrophils in the alveolar space (i), neutrophils in the interstitial space (ii), hyaline membranes (iii), proteinaceous debris filling the airspaces (iv) and alveolar septal thickening (v). At least 20 random regions were scored 0–2 independently and the final lung injury score was calculated as below; score = [(20 × i) + (14 × ii) + (7 × iii) + (7 × iv) + (2 × v)] / (number of fields × 100). (D) Lung permeability was quantified by albumin concentration in BAL fluid from 6 h and 24 h post infection (n = 6–8 per each group). All data are shown as means ± SD. * <i>p</i><0.05, compared with WT mice.</p

IL-36 receptor and IL-36γ deficient mice were resistant to acute <i>P</i>. <i>aeruginosa</i> infection.

<p>WT, IL-36 receptor deficient (IL-36R^-/-) and IL-36γ deficient (IL-36γ^-/-) mice were intratracheally infected with 2.0 × 10⁵ CFU <i>P</i>. <i>aeruginosa</i>. (A) Survival rate were assessed every 12 h following <i>P</i>. <i>aeruginosa</i> infection. Each group consisted of 6–10 mice. Survival curves were analyzed using the log-rank (Mantel–Cox) test. (B) Bacterial burden in BAL (left upper) and bacterial dissemination in spleen (left panel) were assessed by subsequent dilution method (n = 6–8 per each group). 50 CFU/mL is the limit of detection. Horizon bar indicate mean values. (C) The number of total cell (left panel), neutrophils (middle panel) and monocytes and macrophages (right panel) in BAL were counted at 0 h, 6 h and 24 h after infection. Each group consisted of 4–8 mice. (D) The production of TNF-α, IL-6, IL-10 and IL-17 were measured by ELISA (n = 4–8 each group). (C, D) Data are shown as mean (B) or means ± SD. # <i>p</i><0.01, § <i>p</i><0.001, ¶ <i>p</i><0.0001, n.s.:not significant, n.a.: not available, compared with infected in WT mice or as indicated.</p

<i>P</i>. <i>aeruginosa</i> induced IL-36α and IL-36γ expression in primary pulmonary macrophages and alveolar epithelial cells.

<p>Primary pulmonary macrophages (PMs) and alveolar epithelial cells (AECs) isolated from WT mice were treated with LPS (1μg/ml), live <i>P</i>. <i>aeruginosa</i> or heat-killed <i>P</i>. <i>aeruginosa</i> at a multiplicity of infection (MOI) 10. (A, B) After 4 h and 24 h incubation, expression of IL-36α (left panel) and IL-36γ (right panel) mRNA in PMs (A) and AECs (B) were analyzed by real-time PCR. (C, D) After 24 h incubation, PMs and AECs were treated with or without ATP (5mM) during 20 min of incubation, and then culture medium (CM) were harvested. The protein levels of IL-36α (left panel) and IL-36γ (right panel) in CM of treated PMs (C) and AECs (D) were measured by ELISA. Data (means ± SEM) are representative of two independent experiments. N.D.; not detected. * <i>p</i><0.05, # <i>p</i><0.01, § <i>p</i><0.001, ¶ <i>p</i><0.0001, N.S.; not significant, compared with medium only or as indicated. LPS; lipopolysaccharide, PA; <i>Pseudomonas aeruginosa</i>, HK PA; heat killed <i>Pseudomonas aeruginosa</i>, ATP; adenosine triphosphate.</p

IL-36γ induced COX-2 mRNA expression and the production of PGE2 in PMs during <i>P</i>. <i>aeruginosa</i> infection.

<p>(A-C) Primary PMs isolated from WT and IL-36R^-/- mice were treated with recombinant IL-36α (100ng/ml) and IL-36γ (100ng/ml) for 24 h. (A) The expression of prostaglandin-endoperoxide synthase 2/cyclooxygenase 2 (Ptgs2/COX-2) mRNA in PMs was analyzed by real-time PCR. (B) The protein production of prostaglandin e 2 (PGE2) in CM by PMs was examined by ELISA. (C) PMs isolated from WT, IL-36R^-/- and IL-36γ^-/- mice were treated with live <i>P</i>. <i>aeruginosa</i> at a MOI 10 for 24 h. (C) PGE2 production in CM were examined. Data (means ± SEM) are representative of two independent experiments. * <i>p</i><0.05, # <i>p</i><0.01, significant compared with medium only or as indicated. (D-F) WT, IL-36R^-/- and IL-36γ^-/- mice were intratracheally infected with 2.0 × 10⁵ CFU <i>P</i>. <i>aeruginosa</i>. (D) PTGS2 mRNA and (F) the production of PGE2 in BAL in infected-WT, IL-36R^-/- and IL-36γ^-/- mice were examined (n = 4 per each group). (F) The expression of COX-2 in the lungs was determined by Western blotting. Band intensity represents relative density compared with β-actin, and fold changes compared with the lungs of naïve WT mice were presented. # <i>p</i><0.01, ¶ <i>p</i><0.0001, significant compared with untreated mice or as indicated.</p

Expression of IL-36 cytokines in lung of WT mice infected with <i>P</i>. <i>aeruginosa</i>.

<p>Wild-type (WT) mice were infected intratracheally with 2.0 × 10⁵ colony forming unit (CFU) <i>P</i>. <i>aeruginosa</i>. (A) Transcript products of IL-36α (left panel) and IL-36γ (right panel) in the lungs of untreated, 6 h and 24 h after <i>P</i>. <i>aeruginosa</i> infection. mRNA was analyzed by real-time PCR. (B, C) Protein expression of IL-36α (left panel) and IL-36γ (right panel) in (B) bronchial alveolar lavage (BAL) and (C) lung homogenized tissue were quantified by ELISA. All data are shown as means ± SD of 4–5 mice/group. * <i>p</i><0.05, # <i>p</i><0.01, § <i>p</i><0.001, significant compared with untreated mice. N.D.; not detected.</p

Distribution of antimicrobial resistance genes among ESBL-producing <i>Enterobacter</i> spp.

*<p>All <i>qnrA</i> were <i>qnrA1</i>.</p

Dendrogram and PFGE of <i>Xba</i>I-digested genomic DNAs from ESBL-producing <i>Enterobacter</i> species.

<p>EC: <i>Enterobacter cloacae</i>, EA: <i>Enterobacter aerogenes</i>. Strain No. and PFGE type correspond to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037967#pone-0037967-t002" target="_blank">Table 2</a>.</p