Western Blot analysis of inflammasome components after stimulation with NTHi.

<p>A+B show caspase-1 (A) and NLRP3 (B) expression over a period of 6 h after stimulation with NTHi 10⁶ cfu/ml in murine macrophages. NLRP3 was detected in murine macrophages after stimulation with inactivated NTHi as well (C). Densitometric analysis of NLRP3 in murine macrophages after stimulation with NTHi 10⁶ cfu/ml for 24 h is shown in (D).</p

Immunohistochemical and immunocytochemical staining for caspase-1 in human lung tissue and human alveolar macrophages.

<p>Human lung tissue was stimulated with NTHi 10⁶ cfu/ml for 24 h. Tissue was fixated with HOPE-solution and caspase-1 detected via IHC. The images show the expression of caspase-1 in alveolar macrophages (A+B), bronchial epithelial cells (C), in the medium control (D) and in the capillary endothelium (arrow) (E). Human alveolar macrophages from BAL were stained for caspase-1. Increased expression can be observed after stimulation with NTHi (G) in contrast to the medium control (F).</p

<i>Nontypeable</i> Haemophilus Influenzae Infection Upregulates the NLRP3 Inflammasome and Leads to Caspase-1-Dependent Secretion of Interleukin-1β — A Possible Pathway of Exacerbations in COPD

<div><p>Rationale</p><p><i>Nontypeable</i> Haemophilus influenzae (NTHi) is the most common cause for bacterial exacerbations in chronic obstructive pulmonary disease (COPD). Recent investigations suggest the participation of the inflammasome in the pathomechanism of airway inflammation. The inflammasome is a cytosolic protein complex important for early inflammatory responses, by processing Interleukin-1β (IL-1β) to its active form.</p><p>Objectives</p><p>Since inflammasome activation has been described for a variety of inflammatory diseases, we investigated whether this pathway plays a role in NTHi infection of the airways.</p><p>Methods</p><p>A murine macrophage cell line (RAW 264.7), human alveolar macrophages and human lung tissue (HLT) were stimulated with viable or non-viable NTHi and/or nigericin, a potassium ionophore. Secreted cytokines were measured with ELISA and participating proteins detected via Western Blot or immunohistochemistry.</p><p>Measurements and Main Results</p><p>Western Blot analysis of cells and immunohistochemistry of lung tissue detected the inflammasome key components NLRP3 and caspase-1 after stimulation, leading to a significant induction of IL-1β expression (RAW: control at the lower detection limit vs. NTHi 505±111pg/ml, p<0.01). Inhibition of caspase-1 in human lung tissue led to a significant reduction of IL-1β and IL-18 levels (IL-1β: NTHi 24 h 17423±3198pg/ml vs. NTHi+Z-YVAD-FMK 6961±1751pg/ml, p<0.01).</p><p>Conclusion</p><p>Our data demonstrate the upregulation of the NRLP3-inflammasome during NTHi-induced inflammation in respiratory cells and tissues. Our findings concerning caspase-1 dependent IL-1β release suggest a role for the inflammasome in respiratory tract infections with NTHi which may be relevant for the pathogenesis of bacterial exacerbations in COPD.</p></div

Cytokine secretion in human lung tissue and murine macrophages after challenge with nigericin, KCl, glibenclamide and NAC.

<p>A. Human lung tissue was stimulated either with nigericin 10 µM alone or together with NTHi 10⁶ cfu/ml. Sole challenge with nigericin did not lead to elevated IL-1β levels, whereas the addition of nigericin to NTHi primed macrophages enhanced the cytokine secretion; (n.s. = not significant). B. RAW cells were pretreated for 30 minutes with KCl 30 mM or glibenclamide 250 µM and then stimulated with NTHi 10⁶cfu/ml. IL-1β concentrations decreased significantly in both settings. C. RAW cells were preincubated with NAC 20 mM for 60minutes prior to stimulation with NTHi 10⁵ cfu/ml. IL-1β levels were reduced significantly in comparison to the control.</p

Cytokine secretion in murine macrophages after stimulation with non-viable NTHi.

<p>Murine macrophages were stimulated with inactivated NTHi for 24 h. IL-1β concentrations were significantly reduced compared to stimulation with viable NTHi (A). TNF-α and CXCL-2 levels were better preserved after stimulation with non-viable NTHi in comparison to IL-1β (B+C).</p

Overview of inflammasome activation.

<p>Two different types of stimuli are able to activate the inflammasome, pathogen associated molecular patterns (PAMPs) on the one hand and damage associated molecular patterns (DAMPs) on the other. A 2-hit-theory has been postulated, stating that for inflammasome activation two distinct signals are required. Only thereafter proIL-1β is cleaved by caspase-1, so that mature IL-1β can be secreted by macrophages and promote the inflammatory response.</p

Immunohistochemical and immunocytochemical staining for NLRP3 in human lung tissue and human alveolar macrophages.

<p>Human lung tissue was stimulated with NTHi 10⁶ cfu/ml for 24 h. Tissue was fixated with HOPE-solution and NLRP3 detected via IHC. The images show the expression of NLRP3 in alveolar macrophages (A+B), bronchial epithelial cells (C), in the medium control (D) and in alveolar type II cells (ATII) (E). A specific ATII staining (human surfactant protein B) was performed in HLT (F, upper image). NLRP3-positive cells (F, lower image) were identified in consecutive slides from the same tissue section (arrows). Human alveolar macrophages from BAL were stained for NLRP3. Increased expression can be observed after stimulation with NTHi (H) in contrast to the medium control (G).</p

Cytokine secretion in macrophages and human lung tissue after stimulation with NTHi.

<p>Human lung tissue (HLT) and murine macrophages were stimulated for 24 h with NTHi 10⁶ cfu/ml. IL-1β and IL-18 concentrations were significantly increased in HLT (A+D). 6 h after stimulation with NTHi a caspase-1 inhibitor (Z-YVAD-FMK 100 µM) was added. After inhibition IL-1β and IL-18 concentrations in HLT and macrophages were significantly reduced (B,C and E).</p

IDO-mediated antimicrobial effect is lost under low oxygen concentrations.

<p>(A) Growth of <i>Staphylococcus aureus</i> in supernatants of unstimulated or IFN-γ stimulated (500 U/mL) human fibroblasts, incubated for 72 h under different oxygen concentrations (1%–20% O₂). Bacterial growth was determined 24 h after infection by optical density at 620 nm +/− SEM. (B) Proliferation of <i>Toxoplasma gondii</i> in HFF cells that have been pre-stimulated with 500 U/mL IFN-γ or not for 72 h under different oxygen concentrations (1%–20% O₂). Then the cells were infected with the parasites and the Toxoplasma proliferation was determined after 48 h by the incorporation of ³H-uracil. (C) Replication of herpes simplex virus type 1 (HSV1) in pre-stimulated (500 U/mL IFN-γ or not) HFF cells that have been incubated for 72 h under normoxia (20% O₂) or hypoxia (1% O₂). After pre-stimulation cells were infected with HSV1 and viral replication was detected after additional 72 h via real-time PCR. A significant inhibition of bacterial, parasitic and viral growth, respectively, as compared to the stimulated normoxia positive control is marked with an asterisk (*), n = 3.</p

IFN-γ signalling and cell survival under different oxygen conditions.

<p>(A) FACS analysis of unstimulated or IFN-γ stimulated (100 U/mL) HeLa cells that have been incubated for 72 h under normoxia (20% O₂) or hypoxia (1% O₂) in cell culture medium with supplemental L-tryptophan (100 µg/mL). Cells were stained for HLA-DR. (B-D) Cell viability assay. Indicated cell numbers of 86HG39 glioblastoma cells (B), HeLa cells (C) or human foreskin fibroblasts (HFF; D) were incubated under normoxia or hypoxia (24–72 h). Then alamarBlue was added to the cells and the reducing power of living cells in the samples was measured quantitatively via absorbance at OD₅₇₀/OD₆₀₀+/− SD. Data of one representative experiment, performed in triplicates. (E) FACS analysis of unstimulated or IFN-γ stimulated (100 U/mL) HeLa cells, incubated for 72 h under normoxia, hypoxia or anoxia in cell culture medium with supplemental L-tryptophan (100 µg/mL). Cells were stained for DAPI as indicator for cell survival. PFA-fixed cells served as positive control for cell death. (F) Kynurenine amount in supernatants of unstimulated or IFN-γ stimulated (500 U/mL) HeLa cells in the presence of different amounts of L-tryptophan (0, 6 or 50 µg/mL). Cells were incubated for 72 h in normoxia or hypoxia and subsequently reoxygenated for 48 h in normoxia. Then the kynurenine content in the cell culture supernatants was determined by optical density at 492 nm +/− SEM, using Ehrlichs reagent. A significant inhibition of kynurenine production as compared to the stimulated normoxia positive control is marked with asterisks, n = 3.</p