Epidemic curve of influenza-like illness in Japan.

<p>Number of reported cases of influenza-like illness per sentinel surveillance site per week in Japan, from week 36, 2004 through week 35, 2010.</p

Epidemic curve of influenza-associated encephalopathy and other encephalopathy/encephalitis in Japan.

<p>Number of cases of influenza-associated encephalopathy and acute encephalitis/encephalopathy other than influenza in Japan, from week 36, 2004 through week 35, 2010.</p

Age distribution of influenza-A associated encephalopathy by patient visits.

<p>Number of influenza A-associated encephalopathy cases per estimated 1,000,000 patient visits by age, 2006–2010.</p

Age distribution of patients with influenza-like illness who consulted a medical facility.

<p>Estimated number of patients with influenza-like illness who consulted with a medical facility by age, 2006–2010.</p

Age distribution of influenza-A associated encephalopathy per population.

<p>Number of influenza A-associated encephalopathy cases per 1 million population by age, 2004–2010.</p

Number of reported cases of influenza-associated encephalopathy (fatal cases) by virus type in Japan, 2004–2010.

<p>ILI: influenza like illness.</p><p>IAE: influenza-associated encephalopathy.</p>*<p>Reported as influenza A from week 36, 2004 through week 27, 2009.</p>†<p>Reported as influenza B from week 36, 2004 through week 35, 2010.</p>‡<p>Reported as influenza A (H1N1) 2009 or influenza A from week 28, 2009 through week 35, 2010.</p>§<p>Reported as influenza A (H3N2) in week 24, 2010.</p

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

<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-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