Inhibition of Caspase-1 with Tetracycline Ameliorates Acute Lung Injury

Abstract

Rationale: Acute respiratory distress syndrome (ARDS) is a heterogeneous syndrome with a mortality of up to 40%. Precision medicine approaches targeting patients on the basis of their molecular phenotypes of ARDS might help to identify effective pharmacotherapies. The inflammasome-caspase-1 pathway contributes to the development of ARDS via IL-1β and IL-18 production. Recent studies indicate that tetracycline can be used to treat inflammatory diseases mediated by IL-1β and IL-18, although the molecular mechanism by which tetracycline inhibits inflammasome-caspase-1 signaling remains unknown. Objectives: To identify patients with ARDS characterized by IL-1β and IL-18 expression and investigate the ability of tetracycline to inhibit inflammasome-caspase-1 signaling in ARDS. Methods: IL-1β and IL-18 concentrations were quantified in BAL fluid from patients with ARDS. Tetracycline's effects on lung injury and inflammation were assessed in two mouse models of direct (pulmonary) acute lung injury, and its effects on IL-1β and IL-18 production were assessed by alveolar leukocytes from patients with direct ARDS ex vivo. Murine macrophages were used to further characterize the effect of tetracycline on the inflammasome-caspase-1 pathway. Measurements and Main Results: BAL fluid concentrations of IL-1β and IL-18 are significantly higher in patients with direct ARDS than those with indirect (nonpulmonary) ARDS. In experimental acute lung injury, tetracycline significantly diminished lung injury and pulmonary inflammation by selectively inhibiting caspase-1-dependent IL-1β and IL-18 production, leading to improved survival. Tetracycline also reduced the production of IL-1β and IL-18 by alveolar leukocytes from patients with direct ARDS. Conclusions: Tetracycline may be effective in the treatment of direct ARDS in patients with elevated caspase-1 activity. Clinical Trial registered with www.clinicaltrials.gov (NCT04079426).

Keywords: antibacterial agents; immunomodulation; inflammasomes; influenza; precision medicine.

Figures

Figure 1.

Concentrations of IL-1β and IL-18 in BAL fluid (BALF) from patients with direct and indirect acute respiratory distress syndrome (ARDS). BALF of patients with direct (n = 23 viral and n = 25 bacterial origin) or indirect (n = 13) ARDS from UKB and MHH was obtained and analyzed via bead-based multiplex assay for (A) IL-1β and (B) IL-18. Mean, Mann-Whitney Test. MHH = Hannover Medical School; UKB = University Hospital Bonn.

Figure 2.

Tetracycline (TET) reduces lung injury and mortality and inhibits the production of IL-1β and IL-18 in mice with LPS-induced acute lung injury. C57BL/6J mice were challenged intratracheally with LPS (8.75 μg/g body weight) on Day 0 and treated daily with TET (75 μg/g body weight) or PBS intraperitoneally for 10 days. (A) Schematic procedure of the experiments. (B) Mice were evaluated for changes in survival (n ≥ 11 per group). The accumulation of (C) total protein, (D) albumin, and (E) neutrophils (n ≥ 5 per group) in BAL fluid (BALF) was determined by bicinchoninic acid assay (BCA), ELISA, and flow cytometry at the indicated time points. Lungs were removed at 96 hours and stained with hematoxylin and eosin. (F) Representative histologic sections are shown (magnification, 20×), and lung injury score was determined by examining 15 sections/lung/animal (n = 4 per group, magnification ×100). BALF was further analyzed for (G) IL-1β and (H) IL-18 (n ≥ 5 per group) using Luminex Assay and ELISA. Median with interquartile range of three or more independent experiments; Mann-Whitney test and log-rank test, respectively.

Figure 3.

Tetracycline (TET) reduces lung injury and inhibits IL-1β and IL-18 production in mice with influenza virus–induced acute lung injury. (A) C57BL/6J mice were challenged intratracheally with IAV (2,500 pfu/mouse) on Day 0 and treated daily with TET (75 μg/g body weight) or PBS intraperitoneally for 2 days. (B) Total protein, (C) albumin, (D) neutrophils, (E) IL-1β, and (F) IL-18 (n = 10 per group) were quantified in BAL fluid as described in Figure 2. Median with interquartile range of three independent experiments; Mann-Whitney test. IAV = influenza A virus.

Figure 4.

Tetracycline (TET) inhibits IL-1β production in vitro via selective inhibition of caspase-1 (Casp-1). Murine bone marrow–derived macrophages (BMDM) or immortalized murine BMDM overexpressing a fluorescently tagged ASC (apoptosis-associated speck-like protein containing a CARD domain)-mCerulean fusion protein were stimulated with either LPS (30 ng/ml) alone or in combination with nigericin (10 mM) or Poly (dA:dT) (0.5 μg/ml) and then treated with TET. (A and B) IL-1β, (C) TNF-α, and (D) LDH concentrations were measured in supernatants with ELISA or LDH assay. ASC speck formation was measured by fluorescence microscopy. (E) Six different images were taken of each condition at a magnification of ×40. (F) Immunoblots of lysates (Lys.) and supernatants (Sup.) of wild-type BMDM. Representative blots from four independent experiments. (G) Active recombinant Casp-1 was incubated with its substrate and TET. Reduction of recombinant Casp-1 activity was measured in relation to control without TET. Median with interquartile range of three or more independent experiments. *P < 0.05; Mann-Whitney test.

Figure 5.

Protection against LPS- and influenza virus–induced acute respiratory distress syndrome by tetracycline (TET) is mediated via inhibition of caspase-1 (Casp-1). Wild-type (WT) C57BL/6J and Caspase-1–knockout (KO) mice were challenged with LPS or IAV as indicated and treated with TET as described in Figures 2 and 3. Lungs from WT mice were homogenized, and pro–Caspase-1 (p45) and a subunit of activated Caspase-1 (p20) were measured via immunoblot. (A and B) Representative blot of three independent experiments and quantification of signal intensity is shown. (C) Mice were evaluated daily for loss of body weight (n = 5 per group). BAL fluid of Caspase-1ko/ko mice was obtained 8 hours after LPS instillation and monitored for (D) albumin, (E) neutrophils, and (F) IL-1β via bicinchoninic acid assay, flow cytometry, and ELISA, respectively (n ≥ 3 per group). Median with interquartile range. *P < 0.05; Mann-Whitney Test. Bonferroni correction for repeated statistical tests (body weight change) and adjustment of significance level to *P < 0.005. IAV = influenza A virus; ns = not significant.

Figure 6.

IL-1β expression by alveolar leukocytes from patients with direct acute respiratory distress syndrome (ARDS) is inhibited by tetracycline (TET). (A) BAL fluid from patients with direct ARDS was collected as described in Figure 1 and analyzed for neutrophils and macrophages via flow cytometry. Leukocytes were cultured and coincubated with increasing doses of (B and C) TET and (D) COL-3 (1, 3, 10, and 30 μg/ml) for 16 hours, and indicated cytokine production was quantified by ELISA. *P < 0.05 (vs. TET 0 μg/ml). Median with interquartile range of six patients; Mann-Whitney test.

Figure 7.

Proposed mechanism of acute respiratory distress syndrome (ARDS) treatment by tetracycline. Pathogen-associated molecular patterns (PAMPs) released during bacterial pneumonia activate pattern recognition receptors, stimulating assembly of an inflammasome complex with ASC (apoptosis-associated speck-like protein containing a CARD domain) oligomerization and caspase-1 activation. Pro–IL-1β/pro–IL-18 are cleaved by caspase-1 and secreted as active cytokines. These cause pulmonary inflammation and lung injury resulting in ARDS. Tetracycline selectively inhibits caspase-1 activation and subsequent IL-1β and IL-18 production. This reduces pulmonary inflammation, morbidity, and mortality.