Involvement of Alveolar Macrophages and Neutrophils in Acute Lung Injury After Scorpion Envenomation: New Pharmacological Targets

Hadjer Saidi,1 Julie Bérubé,2 Fatima Laraba-Djebari ,1,3 and Djelila Hammoudi-Triki1

Abstract—

Androctonus australis hector (Aah) scorpion venom is well known to induce a systemic inflammatory response associated with cell infiltration in lung and edema formation. The present study investigate (i) in vivo the evolution of lung and systemic inflammation triggered by Aah venom and (ii) analyze in vitro the signaling cascade, upstream of inflam- matory cytokine expression after Aah venom-stimulated mouse alveolar macrophage (MH- S), the main resident immune cells in the lung. The inflammation induced by Aah venom was assessed in mice through inflammatory cell count, nitric oxide metabolite, and lactate dehydrogenase (LDH) activity in blood, concordantly with neutrophil sequestration in tissue and lung histology. In the in vitro study, MH-S cells are stimulated with Aah venom in the presence of signaling pathway inhibitors, NG25 an inhibitor of transforming growth factor β- activated kinase (TAK1), PD184352 MAP kinase (MKK)1/2 inhibitor, BI605906 an inhibitor of IKκ-β (inhibitor of nuclear factor kappa B), and BIRB0796 an inhibitor of p38 MAPK. Obtained results showed that leukocyte transmigration is important in some area of the lung and is closely associated with systemic increase of nitric oxide and LDH. The in vitro study showed that Aah venom induce significantly an increase of the expression of TNF-α, IL-1β, and MIP-2 in MH-S cells. The pretreatment with inhibitors showed that cytokine increase involves TAK1, IKκ-β, and ERK1/2 pathways, similarly to Toll-like receptor activation. These findings highlight the contribution of alveolar macrophage and their secretory products to tissue damage and made of TAK1 and ERK1/2, an interesting target in scorpion envenomation.

KEY WORDS: Androctonus australis hector venom; alveolar macrophage; acute lung injury; TAK1; ERK1/2.

INTRODUCTION

Androctonus australis hector (Aah) venom is a het- erogeneous mixture of biomolecules known for their abil- ity to provoke an intense inflammatory reaction, through leukocyte recruitment; increased levels of proinflammatory cytokines such as IL-6, IL-1β, and TNF-α; and an eleva- tion of intercellular adhesion molecules 1 (ICAM-1) ex- pression and reactive oxygen species (ROS) in the lung [1– 4]. The contribution of proinflammatory mediators in scor- pion venom pathogenesis has been demonstrated by many clinical and experimental studies [5–9]. ROS was released from activated monocyte/macrophage, induced lung inju- ry, and initiated cascades of proinflammatory reactions multiplying pulmonary and systemic stress [10].
The pulmonary environment is a source of signals with important potential for macrophage activation. Alve- olar macrophages (AMs) represent the first line of innate immune defense in the respiratory bronchioles and air- spaces. Their location at the interface of host and environ- ment emphasizes their pivotal role in the defense of the lung through their ability to scavenge inhaled particles; phagocytosis of pathogen components and subsequent pro- cessing leads to the presentation of antigens [11–13]. The AMs are necessary for the lung function, but they may also act to damage the organ when inflammatory responses are too severe through the production of immunological medi- ators such as TNF-α, IL-1ß, and IL-6, which can increase capillary leakage and recruit inflammatory leukocytes such as neutrophils following the release of MIP2 [14–16].
Studies performed in vitro with different scorpion venoms reported an immunomodulatory activity of macro- phages and hematopoietic cells. Aah venom seems to directly stimulate murine hematopoietic cells proliferation, differentiation toward granulocyte cells and induced a sig- nificant TNF-α and nitrite levels in culture supernatants [17]. Tityus serrulatus venom (TsV), its toxin 1 (Ts1) elicited liberation of inflammatory mediators such as TNF-α, IFN-γ, IL-6, IL-1β, PGE2, LTB4, and ROS fol- lowing murine macrophage activation in vitro [9, 18, 19]. Interestingly, the stimulation of murine macrophage cell line with TsV and its neurotoxins targeting different ion channels showed that TsV, Ts1, and Ts6 induced the release of NO, IL-6, and TNF-α; Ts2 stimulated increased IL-10 production, indicating that Ts1 and Ts2 bind on Na+ channels, presenting opposite effects regarding NO, TNF- α, IL-6, and IL-10. Additionally, Ts1 and Ts6 showed similar effects despite the fact that they act on Na+ and K+ ion channels, respectively [20]. Furthermore, the same authors showed the interaction of TsV with Toll-like re- ceptors 2 (TLR2) and TLR4 induced in part. Myeloid differentiation factor (MyD88)-dependent activation of NF-κB and AP-1, in another part via TLR4, induce MyD88-independent pathway involving ERK1/2 and p38 activation [19]. All these pathways play critical roles at multiple levels of the immune system including the inflam- matory response and cell cycle progression [21–23].
Downstream signaling events of Toll-like receptors (TLR) engagement have been shown to mediate the acti- vation of TGF-beta activated kinase (TAK1), essential fac- tor for the activation of MAPK including extracellular signal-regulated kinases (ERK)1/2, the only substrates of MEK, p38, and NF-κB known by their involvement in inflammatory cytokine expression and secretion [24–26].
In the present study, an association of in vivo and in vitro approaches was undertaken to investigate the mechanisms through which Aah venom mediated lung injury and bring out new pharmacological targets. We followed the neutrophil mobilization and their accumula- tion in lung parenchyma related to nitric oxide and LDH liberation in sera. In the in vitro study, we explored the potential effect of Aah in AMs which represent the pre- dominant innate cellular defense of the lung and we inves- tigated the role of TAK-1, p38, MEK, and IKκ-β on IL-1β, TNF-α, and MIP2 expression in alveolar macrophage (MH-S) upon Aah venom stimulation.

MATERIALS AND METHODS

Biological Materials

Venom

Aah venom was provided by the Laboratory of Cel- lular and Molecular Biology of the Biological Sciences Faculty at USTHB in lyophilized form.

Animals

NMRI mice (22 ± 2 g), provided by the Pasteur Insti- tute of Algeria, were used for all experiments. The animals were kept under controlled environment and received food and water ad libitum. The experiments were achieved in accord with the guidelines for the care of laboratory ani- mals published by the European Union.

Alveolar Macrophages

Mus musculus alveolar macrophages cell line MH-S (ATCC® CRL-2019™) was purchased from ATCC (Rockville, MD, USA).

Non-biological Materials

SB203580 was obtained from InvivoGen (San Diego, CA). PD184352 was purchased from US Biological (Swampscott, MA). BIRB0796 was kindly provided by Professor Sir Philip Cohen (Medical Research Council Protein Phosphorylation Unit, University of Dundee, UK). All chemical products used in these experiments were acquired from Leica Biosystems (USA), Merck (Germa- ny), Panreac (Spain), Sigma (USA), and Fisher.

Methods

In Vivo Study

The animals were divided into two groups. The first group, used as control, was injected subcutaneously with 200 μL of physiological saline solution (0.9% NaCl). The second group received a subcutaneous (s.c.) injection of sublethal dose (0.5 mg/kg body weight) of Aah venom; this group is divided into subgroups which were sacrificed at different time intervals 90, 180, and 360 min following the Aah venom injection.

Inflammatory Cell Count

Blood smears were prepared from the animals and stained with May–Grunwald–Giemsa. The slides were ex- amined under a common light microscopy at × 1000 mag- nification. The percentage of cell subpopulations was cal- culated based on the count of 100 cells.

Myeloperoxydase Activity

The importance of neutrophil accumulation in tissue was measured by assaying myeloperoxidase (MPO) activ- ity as previously described [27]. The lungs removed at different times were homogenized in physiological saline solution (0.9% NaCl) then centrifuged at 6000 rpm for 20 min. The first supernatant (S1) was conserved at − 20 °C and the second supernatant (S2) was recovered after three freeze-thaw cycles of the pellet followed by a ho- mogenization and a centrifugation at 6000 rpm for 40 min. Twenty microliters of S2 were added to 300 μL of chromogene substrate (0.68 mM O-dianisidine, 3 mM po- tassium bromide prepared in Tris–HCl 50 mM; pH 6.6 and H2O2 8.8 mM). Enzyme activity was assessed by measur- ing the changes in absorbance every 60 s over a period of 4 min at 460 nm. Results were expressed as changes in absorbance per 1 mg of proteins. Protein concentration was determined according to the method of Bradford [28], using BSA as standard.

Measurement of Nitric Oxide Production

The concentration of nitrite (NO −), a soluble oxidation product of NO, was measured in sera collected after blood centrifugation at 1000g for 10 min using Griess reagent (0.1% N-1-napthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% phosphoric acid) [29]. Fifty microliters of deproteinized sera were mixed with an equal volume of the Griess reagent and optic density was measured at 540 nm. Sodium nitrite was used as a standard to calculate NO − concentrations.

Lactate Dehydrogenase Activity

The activity of lactate dehydrogenase (LDH: EC 1.1.1.27) was determined in sera collected as cited above, by using Spinreact commercial kit (Spain). The enzyme values were expressed in international units (IU/L) and represent the mean of four replicate determinations (means
± SEM).

Histological Analysis
Lungs were carefully harvested from sacrified ani- mals and immediately immersed into a formalin fixative so- lution (4%, pH 7.4), dehydrated in ethanol, cleared in xylene, and embedded in paraffin. Histological sections (1 μm thick) were cut and stained with hematoxylin- eosin (H & E) for microscopic examination (ZEISS Axiolab microscope, HIROCAM MA88-500).

In vitro Study

Alveolar Macrophage Culture and Stimulation

MH-S were seeded in 24-well microtiter plates at a concentration of 2 105 cells/well and cultured in RPMI- 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FCS), 100 U/mL penicillin/streptomy- cin, 5 mM HEPES, and 2 mM glutamine. After 24-h incubation at 37 °C in a humidified atmosphere of 5% CO2, these cells were exposed to Aah venom at concentra- tion of 25 μg/mL for 1 h in the same incubation conditions cited above; supernatants are removed and cells are rinsed with cold PBS then collected to mRNA extraction.

RNA Extraction and cDNA Synthesis

Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer’s protocol. The RNA was quantified, and 1 μg was treated with DNase I (Invitrogen) and reverse-transcribed using Superscript II Reverse Tran- scriptase (Invitrogen), according to the manufacturer’s protocols.

Real-Time PCR

Semiquantitative real-time PCR was performed in 96- well plate format using SYBR green-based detection on a Step-One-Plus machine (ABI) with each 10-μl reaction containing ∼ 50 ng of cDNA, 0.3 μm sense and antisense primers with 1× QuantiTect SYBR Green supermix (Qiagen), (Table 1). The plate was sealed and cycled under the following conditions: 95 °C for 10 min, 50 cycles of 95 °C for 10 s, and 60 °C for 45 s; mRNA levels of β-actin were used for normalization, and fold induction was deter- mined from Ct values using the Pfaffl method [30].

Statistical Analysis

The obtained data were expressed as mean ± standard error of the mean (S.E.M.) and analyzed by student test; the differences were considered significant if probability values (P) were less than 0.05 (p < 0.05).

RESULTS

In Vivo Study

Inflammatory Cell Mobilization to Blood

The time course of inflammatory cell count in blood of envenomed mice showed that the venom induced a significant elevation in neutrophil percentage at 180 and 360 min post injection with 53 ± 5.84 and 45.5 ± 3.27% respectively. Monocyte numeration exhibited an elevation at 180 min without statistical significance when compared to the control group (Fig. 1).

Effect of Aah Venom on MPO Activity

Neutrophil cells are usually the first cell type to reach the site of injury and predominate in an immediate inflam- matory reaction. The myeloperoxidase (MPO) activity was estimated in pulmonary tissue to evaluate the accumulation of neutrophils. The obtained results showed that Aah ven- om induced a significant elevation of neutrophil infiltration into lung at 180 min (3-fold) and 360 min (1.8-fold) in comparison to control lungs (Fig. 2).

Effect of Aah Venom on Nitric Oxide Level

Nitrites are metabolic products of NO and often used as markers to indicate NO formation. Nitrite concentration was measured in sera 90, 180, and 360 min following Aah venom injection (Fig. 3). Aah venom displayed a monophasic increase of nitrites. Indeed an increase at 90 min (15.83 ± 2.97) followed by a very significant ele- vation measured at 180 min (19.8 ± 2.85 versus 5± 1.19 μM in control sera) was observed; this level decreased at 360 min but remains significantly higher than in the control group (18.58 ± 3.91 μM).

Effect of Aah Venom on LDH Activity

Enzymatic activities are usually examined in sera as markers to evaluate tissue injuries [1]. In this study, mea- surement of lactate dehydrogenase activity showed that Aah venom administration induced 2.5-fold (1696.5 ± 191.36 IU/L) and 1.9-fold (1268.08 ± 212.16 IU/L) elevations in sera of envenomed mice at 180 and 360 min respectively (Fig. 4).

Lung Histology

The pulmonary parenchyma from the control group revealed normal structure of the tissue (Fig. 5a, f). In contrast, the lung tissues from treated animals with Aah venom revealed significant alterations, with congestion of blood vessels, intense hemorrhage, and interstitial edema, thickening of alveolar wall following infiltration of inflam- matory cells into interstitium and alveolar spaces at 180 min post envenomation (Fig. 5b, g, g1). Cell recruit- ment in some area of the lung revealed their orientation in response to chemokines secreted by lung resident cells (Fig. 5b, d). These histological changes are also observed on venom-treated group at 360 min but were less pro- nounced (Fig. 5c, e, h).

In vitro Study

Aah Venom Downstream Signaling Cascade on Mouse Alveolar Macrophage Stimulation of MH-S by Aah venom induced signif- icant elevation on mRNA expression of proinflammatory cytokines TNF-α and IL-1β (Fig. 6a, b) and the potent neutrophil chemoattractant MIP-2 (Fig. 6c).
Previous studies have shown that expression and secretion of cytokines following cell stimulation required different signaling cascade; among them are MAP kinases and NF-κB [31–33]. Obtained results showed that the pretreatment of MH-S by some MAPK signaling inhibitors prevented mRNA expression of studied cytokines.
The NG25 and PD 184352 inhibitors of TAK1 (MAP3K7) and MKK1/2 respectively were the most ef- fective inhibitors with 100% of cytokine production, sug- gesting the involvement of TAK1 and ERK1/2 (the only known substrates of their activators MKK1/2), in signaling pathways (Fig. 6a–c).
The addition of IKκ-β inhibitor (BI 605906) to cells before cell stimulation with Aah venom showed partial prevention of cytokine up expression (Fig. 6a–c). The only inhibitor that has no effect on the three cytokine mRNA expression is the p38 inhibitor, BIRB 0796, indicating that the expression of these cytokines following MH-S stimu- lation by Aah venom is independent of p38 (Fig. 6a–c).

DISCUSSION

Although the precise nature of the immune system’s role in scorpion envenomation has not been fully elucidat- ed, we know that acute lung inflammation is characterized by complex interactions among cytokines, chemokines, adhesion molecules, leukocytes, such as IL-6, TNF-α, IL-1β, and nitric oxide.
In the present work, the evolution of inflamma- tion induced by Aah venom in vivo showed that cell influx in lung is tightly linked to lung edema forma- tion and correlated positively with systemic over pro- duction of NO and LDH elevation suggesting the involvement of inflammatory cell activation in lung tissue damages and systemic inflammation. This hy- pothesis is supported by previous studies which have demonstrated that upregulated release of nitrogen and oxygen species by phagocyte cells such as macrophage and neutrophils, along with proinflammatory cyto- kines, induces the peroxidative degradation of mem- brane phospholipids [34]. These alterations contribute to lung tissue injury manifested by disruption of the alveolar/epithelial barrier and the development of in- terstitial edema [35].
Neutrophil sequestration induced by Aah venom and its major toxin Aah I in lung parenchyma has been explained by the involvement of different systems, cholinergic and adrenergic [3], kinin-kallikrein [36], and mastocytes [37] showing the complexity of lung injury induced by this scorpion venom. The present study focused on alveolar macrophages considered to be the principal defense cells of the respiratory system and responsible for the acute phases of the inflamma- tory response [38]. Among chemokines and cytokines secreted by these cells, MIP 2 is known as a potent chemotactic factor for neutrophils [39, 40], TNF-α, and IL-1β proinflammatory cytokines, early elevated in sera of Androctonus envenomed mice [1, 3] and related to the severity of scorpionic envenomation [6]. Our experiments in vitro showed that MH-S stimulation by Aah venom induced a significant elevation of MIP-2, TNF-α, and IL-1β expressions. These data joint previous studies which displayed enhancement of these potent proinflammatory cytokines following macrophages activation by TsV [18, 19]. Furthermore, Zoccal and collaborators [19] have demonstrated that TLR2, TLR4, and CD14 receptors sense TsV and its major component, toxin 1 (Ts1), to mediate cytokine and lipid mediator production.
In this study, the use of different protein kinase inhib- itors that target element downstream of TLR signaling, we showed that TAK1, IKκ-β, and MEK regulate TNF-α, IL- 1β, and MIP-2 expression on MH-S cells in response to Aah venom. Similarly to the obtained results, the involve- ment of MEK in proinflammatory mediator mRNA, like TNF-α, IL-1β, MCP-1 Cox2, IL-6, IL-8, has been showed following stimulation of macrophages [41–43], and non- immune cells like airway epithelial cells [33, 44], exhibiting the importance of this pathway in cell signaling, independently of the JNK and p38 pathways.
Based on the obtained results and literature data concerning MEK activation, we suggest this signaling cascade TAK1 → IKκ-β → MEK downstream Aah ven- om interaction with TLRs. In fact, TLR stimulation phosphorylates the TAK1 which is essential for the activation of MAPK and NF-κB as TAK1-deficient cells cannot activate these pathways in response to TLR ligands [45]. TAK1 activate IκB leading to the activation of a transcription factor NF-κB [46] and following TPL2 (MAP3K8) activation trigger the acti- vation of MEK1 and MEK2 [32, 41, 47]. In macro- phages, TPL2 under basal conditions is stoichiometri- cally associated with NF-κB1 p105, an IκB family member. Like other IκB members, p105 is also phosphorylated by IκB kinase complex upon stimula- tion by TLRs and this process results in the partial degradation of p105, which then releases TPL2. While the p105-bound TPL2 is inactive in terms of MEK1/2 activation, the free TPL2 is active and phosphorylates MEK1/2, which then activates ERK1/2. However, the free TPL2 is also unstable and is targeted for degrada- tion [42, 48–51].Upstream TPL2, IKκ-β is able to phosphorylate IκB and marked it for degradation in the cytoplasm. The released of NF-κB dimer can then be activated by RelA (also known as p65) phosphory- lation and can translocate into the nucleus, where NF- κB triggers the transcription of target genes [52, 53]. The genetic deficiency of RelA inhibits the LPS- induced pulmonary expression of the chemokines KC and MIP-2, resulting in decreased neutrophil migration [54]. In the present study, we have not assessed directly whether NF-κB is needed for cytokine mRNA expres- sion in MH-S cells exposed to Aah venom. Beside IKκ- β, it is well known that TAK-1 signaling can also result in the activation of p38 and c-Jun NH2-terminal kinases (JNK) MAPKs.p38 pathways seems to be not involved in upregulation of studied cytokines Since macrophages pretreated with BIRB0796, a p38 inhibitor showed no differences with Aah venom-stimulated cells.
On top of signaling cascade proposed in our study, we find the TAK1; this MAP3K functions as an upstream signaling mediator during TLR engagement in different immune cell types including macrophages [55, 56]. In support of the notion that TLR initiation of innate immune responses is finely tuned to the activating stimulus, an increasing complexity of hierarchic regulation has been revealed. This complexity begins with the specific TLR isoform engaged with at least 12 membrane-bound family members identified. These receptors then recruit the cyto- solic adapter proteins MyD88 and TRIF, [57] to propagate their signals to intracellular effector molecules. Most TLRs use MyD88 with the exception of TLR3, which exclusive- ly recruits TRIF [58]. TLR4 is the only member of the TLR family that exploits both MyD88 and TRIF to induce the downstream targets of the signaling cascade [59, 60]. MyD88-dependent and MyD88-independent pathways have been activated below TLR4 stimulation by Tityus serrulatus venom (TsV) on macrophage, inducing MyD88-dependent increase of NF-κB and MyD88- independent activation of both p38 and ERK1/2. Signaling events upstream p38 and ERK1/2 remain unclear [19]. In the present study, the TAK1 activation could be initiated by the binding of MyD88 and/or TRIF since previous works showed phosphorylated TAK1 results of both adaptors signaling [61, 62]. The preceding data combined illustrat- ing further the importance of protein kinases downstream TLRs in the context of acute inflammation caused by scorpion venom.

CONCLUSION

In summary, the present investigation revealed the direct effect of Aah venom on alveolar macrophage which seemed, through secreted molecules, to be important cell in triggering lung and systemic inflammation involving TAK1 and ERK1/2 signaling pathways. The understanding of deregulated signaling pathways is still need to be elucidated.

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