Inhaled hydrogen sulfide protects against lipopolysaccharide-induced acute lung injury in mice

All animal experiments were performed in accordance with the guidelines of the local animal care commission (Ethics Committee University of Freiburg, Freiburg, Germany, permission No. G-07/25) and in conformance with the journals’ requirements for human and animal trials. C57BL/6 N mice weighing 22.4 g (± 0.3 g) were randomly assigned into four experimental groups: (1) intranasal (i.n.) application of endotoxin-free phosphate buffered saline (70 μl) + spontaneous breathing of room air (PBS + air), (2) PBS treatment + spontaneous breathing of air supplemented with 80 parts per million (ppm) hydrogen sulfide (H₂S, Air Liquide, Kornwestheim, Germany) (PBS + H₂S), (3) i.n. application of lipopolysaccharide (70 μl dissolved in PBS, LPS; 0,25 ng E.coli 055:B5; Sigma-Aldrich Chemie GmbH, Munich, Germany) + spontaneous breathing of air (LPS + air), (4) LPS treatment + spontaneous inhalation of 80 ppm H₂S (LPS + H₂S). Spontaneous breathing of the respective gas mixture started 1 h prior to PBS or LPS instillation. Instillation of PBS or LPS was conducted under short isoflurane anesthesia. Afterwards, mice were again placed in a sealed plexiglass chamber and subjected to either breathe room air or H₂S for another 6 h. H₂S concentration was continuously monitored using a portable gas monitor (MX6 iBrid, Industrial Scientific Corporation, Oakdale, PA).

At the end of each experiment all mice were sacrificed by an intraperitoneal, overdosed injection of ketamine (180 mg/kg) and acepromazine (1.8 mg/kg) and additional bleeding. Bronchoalveolar lavage fluid, lung tissue samples, and blood samples were gained and analyzed as described recently [11].

BAL aliquots were analyzed using interleukin-1β (IL-1β) and macrophage inflammatory protein-2 (MIP-2) ELISA kits (R&D Systems GmbH, Wiesbaden, Germany) according to the manufacturers’ instructions. Serum samples were tested using ELISA kits for myeloperoxidase (MPO) glycoprotein (HK210 ELISA, Hycult biotech GmbH, Beutelsbach, Germany) and IL-1β according to the manufacturers’ instructions.

The left lung was prepared, conserved, and cut into 12 μm thick cryosections for hematoxylin and eosin (H + E) staining as previously described [11]. From each lung, four representative photos were taken (magnification x200). Five high power fields (HPF) were randomly assigned to each photo. Subsequently, alveolar wall thickness and cellular infiltration were analyzed by Axiovision software (AxioVS40LE, Zeiss, Jena, Germany). For each HPF, the degree of lung damage was determined using a modified ALI score as described earlier [11]: In brief, (a) thickness of the alveolar walls, (b) infiltration or aggregation of inflammatory cells, and (c) hemorrhage were rated in a blinded fashion. Each item was graded according to the following point scale: 0: minimal damage, 1: mild damage, 2: moderate damage, 3: severe damage, 4: maximal damage. The degree of ALI was assessed by the sum of scores for each HPF, ranging from 0 to 12. The average of the sum of each field score per lung was compared among groups.

In the presented model of pulmonary sepsis H + E staining of lung cryosections showed that as compared to control conditions (PBS + air, PBS + H₂S, Figure 1A + B, respectively), LPS treatment clearly stimulated the formation of lung edema and the influx of immune-competent cells (LPS + air, Figure 1C). In sharp contrast, exposure to 80 ppm H₂S markedly reduced LPS-induced lung damage (LPS + H₂S, Figure 1D), reflected by decreased edema formation and cellular infiltration into the lung tissue. These findings were confirmed by quantitative analysis: H₂S inhalation prevented alveolar wall thickening (Figure 2A) and cellular infiltration (Figure 2B), that was otherwise observed in LPS-treated animals kept in room air. In addition, a reduction of lung damage to control levels by H₂S inhalation was also detected by rating an overall ALI score (Figure 2C), strongly suggesting a lung-protective role for H₂S inhalation in LPS-induced ALI.

These results add important information to the role of exogenous H₂S in sepsis. Conflicting data exist in models of cecal ligation and puncture demonstrating aggravation [12‐20] as well as substantial reduction of the resulting lung injury in response to application of H₂S donors [21, 22]. It is likely that the route of administration, dosage, timing, and the purity of H₂S donors may be accountable for the inconsistent data. This study is the first to show that inhalation of H₂S clearly prevents lung damage due to local LPS-induced injury, underlining the therapeutic potential of this gas under septic conditions. Our findings are further supported by a recent publication by Tokuda et al. [23]. Here, in contrast to our study, LPS was applied systemically. The study found inhalation of 80 ppm H₂S substantially increased survival. Although the authors did not analyze histopathological changes in the lung, the data suggest an organ-protective effect of H₂S inhalation. Moreover, we have shown recently that inhalation of H₂S in low dosage (80 ppm) ameliorated lung pathology in ventilator-induced lung injury [7]. In a related model, Francis et al. demonstrated, that inhalation of 60 ppm was sufficient to exert a series of lung protective effects [24]. These data suggest that application of lower concentrations as we used can achieve lung protection. Although we did not detect any toxic side effects of H₂S inhalation in our model (data not shown), future studies in this direction are needed to minimize H₂S exposure.

We next investigated whether the observed H₂S-mediated lung-protection was attributed to inhibition of LPS-induced inflammation. The development of lung damage in pulmonary sepsis is known to be critically dependent on the initiation of an inflammatory response, mainly characterized by neutrophil transmigration and activation and pro-inflammatory cytokine release [21, 25]. Neutrophil sequestration into the bronchoalveolar space was markedly increased by LPS application as compared to the control (Figure 3A). H₂S administration in LPS treated mice substantially reduced neutrophil numbers to control levels. Our data are in line with recent publications, where pulmonary neutrophil activity was found to be largely decreased in mice or rats, that had been protected from LPS-induced systemic inflammation both by inhaled H₂S [23] as well as by application of the slowly releasing H₂S donors S-diclofenac and GYY4137 [25, 26]. The findings of these trials clearly support the notion that exogenous H₂S can inhibit pro-inflammatory processes. In combination with neutrophil transmigration, the release of pro-inflammatory cytokines, e.g., IL-1β and MIP-2, is known to aggravate lung injury [21, 25]. In our study, quantitative analysis of IL-1β in the BAL revealed that it was nearly absent in both control groups (PBS + air and PBS + H₂S), whereas LPS instillation alone led to a vast increase of IL-1β readings (Figure 3B). In sharp contrast, H₂S inhalation reduced IL-1β to control levels. Likewise, LPS treatment increased MIP-2 protein that was partially prevented by H₂S (Figure 3C). The attenuation of neutrophil transmigration and pro-inflammatory cytokine release by administration of H₂S has also been shown in other models of ALI, e.g., ventilator-induced lung injury [7], oleic acid-induced lung injury [9, 10], caerulein-induced acute pancreatitis [27], or myocardial ischemia/reperfusion injury [28], strongly supporting our findings that gaseous H₂S substantially inhibits pulmonary inflammation and thereby limits LPS-induced lung damage. Future studies using different ALI models might discover the regulatory role of H₂S in each single aspect of human ALI / ARDS in order to define H₂S` therapeutic potential.

We finally questioned whether intranasal application of LPS would also induce systemic inflammation and whether H₂S would exert any systemic effects. As a marker for neutrophil activity we determined the concentration of the MPO glycoprotein in serum [23, 26]. In the present study, LPS treatment clearly increased serum MPO release. H₂S inhalation tended to reduce MPO levels, irrespective of the mode of additional PBS- or LPS-treatment (Figure 4A). Similar results were obtained by analyzing serum IL-1β levels (Figure 4B). Our findings compliment the results of two previous studies, where LPS-induced systemic inflammation (e.g., plasma nitrite/nitrate levels, pro-inflammatory cytokine release) was also clearly prevented by H₂S inhalation [23, 29]. However, on the basis of our results, we cannot clearly decipher, whether i.n. LPS directly induced a systemic inflammation or whether the inflammatory response observed resulted from lung injury. Therefore two scenarios concerning the role of H₂S appear possible: (1) H₂S inhalation may directly inhibit lung and systemic inflammation, or (2) H₂S inhalation may primarily inhibit lung inflammatory responses, secondary preventing a systemic inflammation. Either way, our data suggest that H₂S inhalation has the potential to inhibit both local as well as systemic inflammatory responses to septic insults.