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. 2020 Jul 23;9:e58855. doi: 10.7554/eLife.58855

Chronic ethanol consumption compromises neutrophil function in acute pulmonary Aspergillus fumigatus infection

Nathalia Luisa Sousa de Oliveira Malacco 1,✉, Jessica Amanda Marques Souza 1, Flavia Rayssa Braga Martins 1, Milene Alvarenga Rachid 2, Janaina Aparecida Simplicio 3, Carlos Renato Tirapelli 3, Adriano de Paula Sabino 4, Celso Martins Queiroz-Junior 5, Grazielle Ribeiro Goes 6, Leda Quercia Vieira 7, Danielle Glória Souza 8, Vanessa Pinho 5, Mauro Martins Teixeira 7, Frederico Marianetti Soriani 1,✉
Editors: Jos WM van der Meer9, Frank L van de Veerdonk10
PMCID: PMC7398701  PMID: 32701055

Abstract

Chronic ethanol consumption is a leading cause of mortality worldwide, with higher risks to develop pulmonary infections, including Aspergillus infections. Mechanisms underlying increased susceptibility to infections are poorly understood. Chronic ethanol consumption induced increased mortality rates, higher Aspergillus fumigatus burden and reduced neutrophil recruitment into the airways. Intravital microscopy showed decrease in leukocyte adhesion and rolling after ethanol consumption. Moreover, downregulated neutrophil activation and increased levels of serum CXCL1 in ethanol-fed mice induced internalization of CXCR2 receptor in circulating neutrophils. Bone marrow-derived neutrophils from ethanol-fed mice showed lower fungal clearance and defective reactive oxygen species production. Taken together, results showed that ethanol affects activation, recruitment, phagocytosis and killing functions of neutrophils, causing susceptibility to pulmonary A. fumigatus infection. This study establishes a new paradigm in innate immune response in chronic ethanol consumers.

Research organism: Mouse, Other

eLife digest

Alcoholism is a chronic disease that has many damaging effects on the body. Over long periods, excessive alcohol intake weakens the immune system, putting consumers at increased risk of getting lung infections such as pneumonia.

Some forms of pneumonia can be caused by the fungus Aspergillus fumigatus. This microbe does not tend to be a problem for healthy individuals, but it can be fatal for those with impaired immune systems. Here, Malacco et al. wanted to find out why excessive alcohol consumers are more prone to pneumonia.

To test this, the researchers used two groups of mice that were either fed plain water or water containing ethanol. After 12 weeks, both groups were infected with Aspergillus fumigatus. The results showed that alcohol-fed mice were more susceptible to the infection caused by strong inflammation of the lungs.

Normally, the immune system confronts a lung infection by activating a group of defense cells called neutrophils, which travel through the blood system to the infection site. Once in the right spot, neutrophils get to work by releasing toxins that kill the fungus. Malacco et al. discovered that after chronic alcohol consumption, neutrophils were less reactive to inflammatory signals and less likely to reach the lungs. They were also less effective in dealing with the infection. Neutrophil released fewer toxins and were thus less able to kill the microbial cells.

These findings demonstrate for the first time how alcohol can affect immune cells during infection and pave the way for new possibilities to prevent fatal lung infections in excessive alcohol consumers. A next step would be to identify how alcohol acts on other processes in the body and to find a way to modulate or even revert the changes it causes.

Introduction

Ethanol abuse is a leading cause of mortality worldwide (World Health Organisation, 2014). Chronic alcohol consumption has been correlated, as a comorbidity, to a wide range of health conditions, including alcoholic liver diseases, cirrhosis and cancers (Szabo and Saha, 2015; Pritchard and Nagy, 2005; Szabo, 1999; Bautista, 1999; Luján et al., 2010). Moreover, alcohol abusers are prone to develop severe lung inflammatory and infectious diseases, including acute respiratory distress syndrome (ARDS) (Liang et al., 2012), pneumonia caused by Streptococcus pneumoniae (Trevejo-Nunez et al., 2015; Tsuchimoto et al., 2015; Boé et al., 2001), Klebsiella pneumoniae (Yeligar et al., 2014; Ohama et al., 2015), Respiratory Syncytial Virus (RSV) infection (Meyerholz et al., 2008) and aspergillosis (Blum et al., 1978; Gustot et al., 2014). The mechanisms associated with this increased risk of disease and death are poorly understood, however studies have suggested that certain aspects of immune function may be altered by chronic ethanol intake (D'Souza El-Guindy et al., 2007; Lippai et al., 2013; Yen et al., 2017; Zhang et al., 2015; Gurung et al., 2009).

Aspergillus fumigatus is a ubiquitous and saprophytic fungus whose conidia are inhaled by humans on a daily basis (Latgé, 1999). Immunocompromised individuals are considered the risk group to develop the pulmonary invasive aspergillosis (IA) (Latgé, 2001) and mortality rates reach up to 95% (Brown et al., 2012). In normal conditions, inhaled conidia are cleared through mucociliary actions. However, if conidia pass through the initial barrier, alveolar macrophage (AM) phagocytosis takes place, resulting in a cascade of cytokine and chemokine release to recruit neutrophils to prevent fungal development (Dagenais and Keller, 2009; Caffrey-Carr et al., 2017). In all these circumstances, an altered leukocyte function may be a major risk factor for IA. Despite all advance in diagnosis and treatment, aspergillosis’ morbidity and mortality remain very high. Mildly immunocompromised conditions such as diabetes mellitus, low-dose corticosteroid therapy and alcoholism has been considered as predisposing factors (Blum et al., 1978; Baddley, 2011; Kousha et al., 2011).

Neutrophils have been shown to be essential to control fungal and bacterial burden in the site of infection and avoid the spread of these microbes and consequently, survival of the host (Gazendam et al., 2016; Romani, 2000; Robertson et al., 2008; Ng et al., 2019). During a pathogen-triggered inflammatory response, neutrophils are the earliest immune effector cells recruited to a site of infection (Kolaczkowska and Kubes, 2013; Sun et al., 2014; Rios-Santos et al., 2007). Neutrophil migration starts with the tethering and rolling of these cells on endothelial cells, a process mediated by selectins and their carbohydrate ligands on neutrophils and endothelial venules (Soethout et al., 2002). These interactions, together with chemokines signals such as CXCL1 and CXCL2 lead neutrophils to a crawling state through the endothelial vase (Kolaczkowska and Kubes, 2013; Phillipson and Kubes, 2011). G protein-coupled receptor on rolling neutrophils binds to the chemokines and changes β2 integrins conformation on the leukocyte surface, allowing a high affinity interaction with endothelial cells (Zemans et al., 2009).

Here, we describe that chronic ethanol consumption facilitates pulmonary infection by both A. fumigatus in mice. Mechanistically, chronic ethanol consumption impairs the normal neutrophil migration to the site of infection via release of high levels of circulating chemokine CXCL1 after infection, followed by downregulation of its receptor 2 (CXCR2). Additionally, ethanol consumption is responsible for an impaired neutrophil function characterized by less phagocytosis, killing, and oxidative burst leading to elevated lung pathology in mice and accentuated mortality rates after infection.

Results

Chronic ethanol consumption did not induce systemic inflammation

To assess the relevance of ethanol chronic consumption, we characterized several parameters during chronic ethanol consumption on host before the infection (Figure 1A). First, we checked weight change, food and liquid consumption during ethanol treatment. We found that there was no weight change in mice during the treatment with ethanol (Figure 1B). Moreover, food and liquid consumption were diminished in ethanol-treated mice compared with control group (Figure 1C and D). Second, blood ethanol levels in mice were about 200 mg of ethanol per deciliter of whole blood while control group ethanol level were not detectable, measured by gas chromatography after 12 weeks of ethanol treatment, (Figure 1E). In order to verify if ethanol consumption affected hematopoiesis we analyzed bone marrow precursors and blood cell counts. Mononuclear and neutrophil counts in peripheral blood did not show differences between groups (Figure 1F). Chronic ethanol consumption did not affect granulocyte progenitors in bone marrow (Supplementary file 1). In addition, no changes were observed in ALT levels in mice serum after the ethanol treatment compared to the control mice group (data not shown). Finally, serum levels of inflammatory mediators, such as TNF-α, IL-1β and CXCL1, have not been changed in ethanol-fed mice (Figure 1G–I).

Figure 1. Outcome of chronic ethanol treatment in mice.

Figure 1.

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(A) Experimental design: C57BL/6 mice received ethanol 5% (v/v) in the first week, followed by 10% (v/v) in the second week (to help mice acclimate with this intervention) and were treated during 10 weeks with ethanol 20% (v/v) in their drinking water. Control group received water. (B–D) During treatment, weight change, food and liquid consumption were measured. Data are presented as Mean ± SD (15 mice per group) *Significantly different (p<0.05) in t test. After ethanol treatment, blood was collected to evaluate (E) blood ethanol levels, (F) differential cell count and (G) TNF-α, (H) IL-1β and (I) CXCL1 in serum. Data are presented as Mean ± SD (4 to 9 mice per group) and analyzed with ANOVA test. Please, also see Supplementary file 1 and Figure 1—source data 1.

Figure 1—source data 1. Values for the outcome of ethanol treatment in mice.

Chronic ethanol consumption increased lethality and impaired pulmonary fungal clearance after A. fumigatus infection

After ethanol treatment, mice were intranasally infected with A. fumigatus conidia (Figure 2A). Ethanol-treated mice showed an increased lethality compared to control mice group (Figure 2B). In addition, weight loss was significantly higher in ethanol treated mice from days 3 to 7 after infection (Figure 2C). These clinical signs were accompanied by higher pulmonary fungal burden in ethanol-fed mice, demonstrating an impaired fungal clearance. Moreover, we were also able to identify hyphae into the airways of infected ethanol-treated mice, showed by red arrows at 24 hr after infection compared to control group (Figure 2D–F). Altogether, these results demonstrate that chronic ethanol consumption increased susceptibility to pulmonary A. fumigatus infection along with an impaired ability to clear the pathogen.

Figure 2. Chronic ethanol consumption leads to susceptibility associated with increased fungal load after A. fumigatus infection.

Figure 2.

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(A) Experimental design: C57BL/6 mice were treated for 12 weeks with ethanol. The next day after the end of the treatment, mice were intranasally infected with 3 × 108 conidia of A. fumigatus. (B) Comparative lethality curves with P = <0.0001 in Log-rank (Mantel-Cox) test. (C) Comparative weight change curves of ethanol-fed group and control group were performed (18 mice per group). Right lungs were collected 24 and 48 hr after the infection. Homogenate from right lungs were plated and CFUs were quantified. Left lungs were fixed with formaldehyde 4% and embedded in paraffin. Sections were stained with GMS and the percentage of germlings was counted (6–16 mice per group) P value indicated in the figure in t test. (D) Representative slides of GMS staining. The insets in 24 hr images represent magnification to show germlings (red arrows) into lung tissue. (E) Fungal load and (F) fungal germination in lungs p=0.0389 in t test. Bars represent 100 µm.

Ethanol consumption altered cytokine release in airways in mice after A. fumigatus infection

Next, we assessed cytokine and chemokine levels by ELISA after A. fumigatus infection to determine whether levels of inflammatory mediators in BALF supernatants were altered after ethanol consumption. We found no significant differences in the levels of neutrophil chemotactic mediator CXCL1 after A. fumigatus infection in BALF of ethanol-treated mice compared to control group (Figure 3A). Interestingly, another CXCR2 agonist and neutrophil chemotactic mediator, CXCL2 were increased at 24 hr of infection in BAL fluid of ethanol-treated mice compared to control mice (Figure 3B). Alcohol consumption declined IL-17 levels after 24 hr of infection (Figure 3C). We also observed no differences in TNF levels between control and ethanol-fed mice group (Figure 3D). Moreover, ethanol consumption was able to down modulate IL-1β and IL-10 levels after fungal infection (Figure 3E and F). These results suggest that chronic ethanol consumption dysregulate cytokine and chemokine release post A. fumigatus infection.

Figure 3. Ethanol consumption altered cytokine release in airways in mice after A. fumigatus infection.

Figure 3.

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BALF supernatants were harvested at 24 and 48 hr after infection and used for ELISA assay. (A) CXCL1, (B) CXCL2, (C) IL-17, (D) TNF, (E) IL-1β and (F) IL-10 levels in BALF. Experiments were assayed in triplicate. Data are presented as Mean ± SD (3 to 9 mice per group). *p<0.0247 in ANOVA test. Please, also see Figure 3—source data 1.

Figure 3—source data 1. Values for inflammatory mediators in BALF after A. fumigatus infection.

Ethanol consumption impaired neutrophil and lymphocyte recruitment into the airways during A. fumigatus infection

To clarify whether the diminished fungal clearance is due to an impaired inflammatory response, we next observed cell influx into the site of A. fumigatus infection. Although both groups exhibited a large recruitment of total cells into the airways, ethanol-treated mice had a significant reduction of recruited cells, compared to control group 24 hr after infection. However, at 48 hr after infection, there was a similar cell migration into the site of infection in both groups (Figure 4A). We demonstrate that almost all cells migrated to the airways represent neutrophils, one of the most important cells involved in killing and control of A. fumigatus infection (Erwig and Gow, 2016). Neutrophils and lymphocytes into airways were significantly decreased in ethanol-treated mice at 24 hr post infection (Figure 4B and C). In contrast, there were no differences in macrophages and eosinophils recruited to the site of infection (Figure 4D and E). Regarding to inflammatory cells recruited to lung tissue, we also observed a diminished neutrophils migration into the lung parenchyma in ethanol-treated mice, by MPO measurement, at 24 hr after infection (Figure 4F). Similar to alveoli, eosinophils and macrophages in pulmonary tissue exhibited no differences after A. fumigatus infection (Figure 4G and H). In addition, both ethanol-treated and control groups exhibited similar number of leukocytes in blood (Figure 4I). Besides, we characterized the populations of lymphocyte migrating to the airways of infected mice. We assessed CD3+CD4+IL17+ cells and results demonstrate that chronic ethanol consumption strongly reduced lymphocytes and IL-17 production in the site of infection after 24 hr of infection (Figure 4J–L). These results indicate that chronic ethanol consumption mostly affected specifically neutrophils recruitment to airways after A. fumigatus infection.

Figure 4. Chronic ethanol consumption affects cellular influx after A. fumigatus infection.

Figure 4.

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After ethanol treatment, mice were infected with A. fumigatus. BALF and lungs were harvested at 24 and 48 hr after infection for inflammatory cell infiltrates determination. (A) Total cells, (B) Neutrophil, (C) Macrophage, (D) Lymphocyte, (E) Eosinophil counts in BALF. (F) MPO, (G) NAG and (H) EPO assays in lungs. (I) Blood leukocyte count/mL. BALF cells were labeled with specific antibodies for flow cytometry. (J) Gate strategy for CD3+CD4+ T cells. (K) CD3+CD4+ cells and (L) CD3+CD4+IL-17+ cells in BALF. Experiments were assayed in triplicate. Data are presented as mean ± SD (4 to 7 mice per group). *Significantly different (p<0.01) in ANOVA test. Please, also see Figure 4—source data 1.

Figure 4—source data 1. Values for indirect measurement of cell infiltration in lung tissue and CD3+CD4+IL-17 cells in BALF after A. fumigatus infection.

Chronic ethanol consumption increased lung pathology after A. fumigatus infection

We assessed histopathology to determine the effect of ethanol consumption in pulmonary tissue after infection. Tissue sections of infected mice revealed a massive leukocyte recruitment into the lungs at 24 hr after infection, in which the inflammatory infiltrate covers a large part of the pulmonary parenchyma structure, including alveoli and perivascular regions that decreases after 48 hr of infection in the control group. The cellular infiltrate tissue remains more prominent after 48 hr of infection only in ethanol-fed mice (Figure 5A). Histopathology score results showed that both ethanol-treated and control groups had similar levels of inflammatory infiltrate, interstitial and alveolar edema and hemorrhage scores at 24 hr of infection. However, after 48 hr of infection, ethanol consumption showed a remaining cellular infiltrate, higher edema and hemorrhage, which increased the total pathology scores (Figure 5B–D).

Figure 5. Lung histopathology is affected by ethanol treatment in mice.

Figure 5.

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Lungs were harvested at 24 and 48 hr after infection fixed with formaldehyde 4% and embedded in paraffin. (A) Sections were stained with Hematoxylin and Eosin. Samples were graded on a 0 to 5-point scale score for (B) inflammatory infiltrate, (C) interstitial and alveolar edema, (D) hemorrhage and (E) total histopathologic score. Experiments were assayed in triplicate. Data are presented as mean ± SD (with 2 to 6 mice per group). *p<0.0142 in ANOVA test. Bars represent 100 µm.

Chronic ethanol consumption reduced leukocyte rolling, adhesion, and chemotaxis in mouse neutrophils

In order to investigate the transmigration process of neutrophils upon chemotaxis in vivo, we first performed an intravital microscopy to visualize migratory cells (Figure 6—source data 1) (Figure 6-rich media videos). Intracrostal administration of LPS was not able to induce a strong increase in rolling and adherence of cells to post capillary venules in ethanol-fed group after 2 hr of stimulation, compared to stimulated control group (Figure 6A–D). In addition, to confirm the impaired migratory ability of neutrophils was caused by ethanol consumption in mice, we performed ex vivo chemotaxis assay towards CXCL2, with mouse bone marrow-derived neutrophils (Figure 6E). As we expected because of previously results showed in Figure 4B, neutrophils from ethanol-treated mice had an impaired migration towards the chemokine stimuli compared to neutrophils from control non-treated mice (Figure 6F). Taken together, these results demonstrate that chronic ethanol ingestion can affect neutrophil rolling, adhesion and recruitment in different tissues.

Figure 6. Chronic ethanol consumption reduced leukocyte rolling and adhesion in vivo and neutrophils chemotaxis ex vivo.

(A) Experimental design: after the chronic ethanol treatment, mice received an intracrostal injection of LPS. After 2 hr, mice cremaster from mouse was exposed to examine the microcirculation by intravital microscopy. Post capillaries venules were recorded. (B) Representative images from the recorded videos (please see the rich media). (C) Number of rolling cells and (D) Number of adherent cells were counted in the videos. (E) Ex vivo neutrophil chemotaxis. Experimental design: after ethanol treatment, BM-derived neutrophils were separated by density gradient and a chemotaxis assay in a Boyden chamber towards CXCL2 was performed. (F) Number of migrated neutrophils in 60 min. Data are presented as Mean ± SD (3 to 5 mice per group) *Significantly different (p<0.0028) in ANOVA test. Please, also see Figure 6—source data 1 and Figure 6-rich media videos.

Figure 6—source data 1. Values for bone marrow neutrophil chemotaxis.

Figure 6.

Figure 6—video 1. Leukocyte rolling and adhesion in vivo-control mice + vehicle.
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After chronic ethanol treatment, mice received an intracrostal injection of LPS or vehicle (saline). After 2 hr, mice cremaster from mouse was exposed to examine the microcirculation by intravital microscopy. Post capillaries venules were recorded. Representative recorded videos from control mice + vehicle; (Figure 6—video 2) ethanol-treated mice + vehicle; (Figure 6—video 3) LPS-stimulated control mice and (Figure 6—video 4) LPS-stimulated ethanol treated mice.
Figure 6—video 2. Leukocyte rolling and adhesionin vivo-ethanol-treated mice + vehicle.
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Figure 6—video 3. Leukocyte rolling and adhesionin vivo-control mice + LPS.
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Figure 6—video 4. Leukocyte rolling and adhesionin vivo- ethanol-treated mice + LPS.
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Chronic ethanol consumption impairs neutrophil’s activation and recruitment by modulation of CD11b, CD62L and down regulation of CXCR2 in these cells during A. fumigatus infection

Neutrophils chemotaxis and recruitment is a complex process that requires leukocyte-endothelial interactions as well as inflammatory mediators’ release (Kolaczkowska and Kubes, 2013). Moreover, the main regulator of neutrophil migration in acute inflammation is CXCR2. To further investigate whether the impaired neutrophil migration is due a deficient neutrophil activation status during infection, we accessed the expression of the markers CD11b, CD62L and CXCR2 in circulating neutrophils after A. fumigatus infection using flow cytometry. While CD11b is mobilized from specific granules to the cell surface, enzymatic shedding in activated polymorphonuclear (PMN) rapidly down regulates CD62L. Both markers have their constitutive expression in resting PMNs. We found that neutrophils activation status was compromised, with CD62L up regulation and CD11b down regulation, in the peripheral blood neutrophils of infected ethanol-treated mice compared to infected non-treated mice (Figure 7A–C). In sepsis, the reduction of neutrophils migration is related to the down regulation of CXCR2 protein expression on circulating neutrophils surface (Alves-Filho et al., 2009). Indeed, CXCL1 serum levels were strongly augmented in ethanol-treated mice compared to the control group at 24 hr post infection (Figure 7D). We next analyzed the role of chronic ethanol intake in regulating CXCR2 expression in circulating neutrophils. At 24 hr after A. fumigatus infection, ethanol-treated mice exhibited significantly fewer circulating neutrophils expressing CXCR2 in the surface compared to the non-treated group (Figure 7E and F).

Figure 7. Chronic ethanol consumption impaired neutrophils activation and downregulated CXCR2 in a sepsis-like manner after A. fumigatus infection.

Figure 7.

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After ethanol treatment and A. fumigatus, blood was harvested at 24 hr post infection. Neutrophils were labeled with specific antibodies for flow cytometry. (A) Gate strategy to analyze neutrophils. Neutrophils were gated by size and cellular complexity and then gated again as Ly6G+CD11b+ cells. (B) CD62L (*p=0.001) and (C) CD11b expression in circulating neutrophils (*p=0.0479). (D) Serum levels of CXCL1 were measured by ELISA assay after 24 hr of infection (*p = 0.0001). (E) Ly6G+CXCR2+ cells (*p=0.0489) and (F) MFI of CXCR2 expression in blood (*p=0.007). Experiments were done at least twice. Data are presented as mean ± SD (3 to 8 mice per group). Analysis were made by ANOVA test. Dashed line represents basal levels of non-infected groups. Please, also see Figure 7—source data 1.

Figure 7—source data 1. Values of flow cytometry data.

Ethanol consumption decreases neutrophil functions of phagocytosis, killing and oxidative burst after A. fumigatus challenge

For the proper killing of A. fumigatus, fungal phagocytosis and ROS production by neutrophils are key events (Philippe et al., 2003). In this sense, we accessed phagocytosis and killing of A. fumigatus conidia by bone marrow-derived neutrophils from ethanol-treated and non-treated mice. We found that phagocytosis was significantly reduced in ethanol-treated mice either in vivo, evaluated by BALF recruited cells, or ex vivo, in bone marrow neutrophils, compared to control group (Figure 8A and B). Fungal killing was also reduced in ethanol intake mouse neutrophils (Figure 8C). Moreover, to evaluate the effect of chronic ethanol consumption in the promotion of respiratory burst of bone marrow-derived neutrophils we performed chemiluminescence experiments using luminol, which served as a probe for superoxide (O2●−)49. We observed that neutrophils from ethanol-fed mice produced lower levels of ROS triggered by A. fumigatus conidia compared to neutrophils from non-treated mice (Figure 8D). These results suggest that phagocytosis, killing and ROS production functions in neutrophils were affected by chronic ethanol consumption.

Figure 8. Ethanol consumption decreased neutrophils phagocytosis, killing and ROS production after A. fumigatus stimuli.

Figure 8.

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(A) Conidial phagocytosis was determined in BALF (*p=0.007). (B–D) After ethanol treatment, BM-derived neutrophils were separated by density gradient and incubated with A. fumigatus conidia. (B) Ex vivo phagocytosis was assessed by cytospin preparations from BALFs (*p=0.0013). (C) Killing assay was evaluated by cell lysis with water, the diluted samples were plated in fungal medium and colony-forming units (CFU) were determined after overnight incubation (*p=0.0238). (D) Luminometry assay was performed to evaluate neutrophil-mediated ROS production and area under curve analysis. (*p=0.057). Data are presented as mean ± SD (3 to 6 mice per group). Analysis were made by t student test.

Discussion

For several centuries, chronic ethanol consumption has been associated to increased susceptibility to infections as well as increased morbidity and mortality after injury (Lieber, 2005; Molina et al., 2010). Numerous studies have shown the effects of acute or chronic exposure to ethanol in inflammatory infections such as in models of K. pneumoniae infection and gut bacteria-associated sepsis (Ohama et al., 2015), intravenous Escherichia coli challenge (Bagby et al., 1998), S. pneumoniae (Trevejo-Nunez et al., 2015; Boé et al., 2001) and a few studies reporting this effect in Aspergillus infection (Blum et al., 1978).

In the present study, we demonstrate how chronic ethanol consumption alters immune and inflammatory pulmonary response after A. fumigatus infection. This involves several immunological phenomena, including defective leukocytes rolling and adhesion, impaired neutrophil migration by down regulation of CXCR2, failed neutrophil activation, impaired neutrophil effector functions (phagocytosis and killing), and reduction of polarized-Th17 innate response. Those features are responsible for an increased susceptibility of mice to A. fumigatus infection.

Chronic ethanol consumption did not alter the basal levels of inflammatory cytokines and chemokines before infection. Ethanol ingestion had an important role in modulating immune response after infection, which is in accordance with previous findings (D'Souza El-Guindy et al., 2007; Bhatty et al., 2011). Ethanol consumption also alters IL-6 and TNF expression of LPS-stimulated Kupffer cells (Maraslioglu et al., 2014). In the present work, we found reduction in IL-1β and IL-17 production after A. fumigatus infection in ethanol-fed mice, suggesting that the immunomodulatory effects caused by chronic ethanol consumption may contribute to the impaired inflammatory response against A. fumigatus.

It has been demonstrated that chronic ethanol consumption causes cellular abnormality in mice lung resident cells. Alveolar macrophages from ethanol-fed mice had deregulation in NADPH oxidase system, impairing phagocytosis and killing against K. pneumoniae (Yeligar et al., 2012). In fact, an impaired NADPH oxidase activity is a well-known risk factor to develop invasive aspergillosis and other life threatening diseases, as seen in Chronic Granulomatous Disease (CGD) patients (Cohen et al., 1981; King et al., 2016; Segal and Romani, 2009). Another study showed both lymphocytes and neutrophils response impaired by ethanol consumption in a model of cutaneous infection by Staphylococcus aureus, in which chronic ethanol-fed mice showed great skin lesions and bacteremia associated with reduced IL-17 and IL-1β production, suggesting that Th17-mediated neutrophilic response was impaired (Parlet et al., 2015).

Neutrophil recruitment to the site of infection is essential for the control of invading extracellular pathogens (Kolaczkowska and Kubes, 2013; Sun et al., 2014; Rios-Santos et al., 2007). Neutrophils are major cell type recruited for A. fumigatus conidia and hyphae killing and neutropenic patients are more susceptible to systemic fungal infections (Dagenais and Keller, 2009; Gazendam et al., 2016). In our data, neutrophil recruitment to infection site, in ethanol-fed mice, was impaired even though we did not observe decrease in both CXCL1 and CXCL2 BALF levels, indicating that ethanol consumption is responsible for compromising neutrophils activation and migration. Moreover, our data showed reduced leukocyte rolling and adhesion after LPS stimuli in ethanol-treated mice outside the airways. CXCL1 and CXCL2 chemokines bind to CXCR2 displaying an essential role in neutrophils activation and ensuing adhesion to endothelium (Kolaczkowska and Kubes, 2013; Phillipson and Kubes, 2011). In fact, it was demonstrated in vitro that chronic ethanol exposure impacted tight junction structures in epithelial cells, leading them vulnerable to endotoxemia (Wood et al., 2013). However, in our study vascular permeability was similar in both ethanol-fed mice and control mice groups (data not shown).

It has been well established that the decrease of CXCR2 expression impairs neutrophil migration (Russo et al., 2009), especially in sepsis (Rios-Santos et al., 2007; Alves-Filho et al., 2009), in which a great amount of inflammatory cytokines and chemokines is released to the blood, causing a complex systemic inflammation (Pierrakos and Vincent, 2010). In a model of severe sepsis, it was found that the migration failure and consequent mortality of individual was associated with the diminished expression of neutrophils CXCR2, which was due to great release of systemic CXCL1 (39). As seen in sepsis, our findings showed decrease CXCR2 expression and great amount of systemic CXCL1 levels in serum of ethanol-fed mice after A. fumigatus infection. To our knowledge, this is the first report establishing a relation between chronic ethanol intake and downregulation of CXCR2 receptor in mouse neutrophils. It is also important to mention that the cooperation between the GPCR receptor CXCR2 and P-selectin ligand, the P-selectin glycoprotein ligand-1 (PSGL-1) is essential to a successful neutrophil migration. The signaling events that ensure the adhesion cascade include the conversion of integrin αLβ2 from a low-affinity conformation to an extended high-affinity conformation that causes arrest and, consequently, perivascular crawling (Yago et al., 2018). Further studies are needed to address the role of chronic ethanol consumption in expression of P-selectin and PSGL-1. Our data indicate that chronic ethanol consumption drives host to a sepsis-like phenotype and this mechanism is responsible for impaired neutrophil migration. The mechanisms whereby CXCR2 is downregulated need further investigation.

To conclude, the findings presented here indicate a new paradigm in how chronic ethanol consumption strongly impairs neutrophils host pulmonary defense against A. fumigatus infection. This infection in mice causes a great inflammatory response, with release of cytokines and chemokines that act in favor to recruit neutrophils into the alveoli and these neutrophils are able to clear the fungus. In contrast, in a condition of chronic ethanol consumption, despite the correct induction of inflammatory response, neutrophils exhibit failure in activation, through the down regulation of CD11b and up regulation of CD62L in blood neutrophils and by accentuated release of CXCR2 ligands in blood flow. This leads to CXCR2 down regulation, which culminated in impaired neutrophils recruitment, increased fungal load and exacerbated lung pathology in mice. We also associate the lower neutrophils levels into the airways with lower innate polarized-Th17 immune response and reduced phagocytosis and killing of A. fumigatus conidia. In fact, we observed growing conidia and substantial fungal burden in lung from ethanol-fed mice, contributing to the highest susceptibility of ethanol-treated mice to A. fumigatus infection (Figure 9).

Figure 9. Mechanism related to chronic ethanol dysfunction in neutrophils after A. fumigatus infection.

Figure 9.

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In normal conditions, infection with A. fumigatus in mice causes a huge inflammatory response, characterized by neutrophil chemokines release. The recruited neutrophils clear the infection by phagocytosis and ROS-mediated killing. In contrast, in a condition of chronic ethanol consumption, despite the correct induction of inflammatory response, there is an increase of CXCR2 ligands in blood flow, causing CXCR2 downregulation. This leads to lower neutrophils recruitment, culminating in substantial fungal burden into the lungs from ethanol-fed mice.

Materials and methods

Key resources table.

Reagent type
(species) or
resource		 Designation Source or
reference		 Identifiers Additional
information
Strain, strain background (Mus musculus) C57BL/6J Multidisciplinary center for Biological Investigation on Laboratory Animal Science (CEMIB) -Unicamp C57BL/6JUnib
Genetic reagent (Aspergillus fumigatus) Strain A1163 A. fumigatus CEA17 isolate (CEA10 derivative) and converted to pyrG+ via A. niger pyrG gene ectopic insertion
Fedorova et al., 2008; Malacco et al., 2019.
Cell line (M. musculus) Primary bone marrow neutrophils This paper C57BL/6JUnib Freshly isolated from C57BL/6J (M. musculus)
Peptide, recombinant protein Recombinant murine MIP-2 (CXCL2) PeproTech Cat# 250–15 Chemotaxis (20 ng/ml)
Antibody Purified NA/LE CD16/CD32 Clone 2.4G2 - FC Block (Rat monoclonal) BD Biosciences Cat# 553140 FACS (1:100)
Antibody anti-CD3-FITC (Rat monoclonal) BD Biosciences Cat# 555274 FACS (1:100)
Antibody anti-CD4-APC (Rat monoclonal) BD Biosciences Cat# 553051 FACS (1:200)
Antibody anti-IL-17a-PE (Rat monoclonal) BD Biosciences Cat# 559502 FACS (1:100)
Antibody anti-Ly6G-BV421 (Rat monoclonal) BD Biosciences Cat# 562737 FACS (1:50)
Antibody anti-CXCR2-PE (Rat monoclonal) R and D Systems Cat# FAB2164P FACS (1:10)
Antibody anti-CD62L-APC (Rat monoclonal) BD Biosciences Cat# 553152 FACS (1:100)
Antibody anti-CD11b-FITC (Rat monoclonal) BD Biosciences Cat# 553310 FACS (1:100)
Commercial assay or kit Mouse TNF-a ELISA kit R and D Systems Cat# DY410
Commercial assay or kit Mouse IL-1b ELISA kit R and D Systems Cat# DY401
Commercial assay or kit Mouse CXCL1 ELISA kit R and D Systems Cat# DY453
Commercial assay or kit Mouse CXCL2 ELISA kit R and D Systems Cat# DY452
Commercial assay or kit Mouse IL-17 ELISA kit R and D Systems Cat# DY421
Commercial assay or kit Mouse IL-10 ELISA kit R and D Systems Cat# DY417
Software, algorithm Prism GraphPad
Software, algorithm FlowJo BD
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Ethics statement and mouse model of chronic ethanol consumption

All animal experiments received prior approval from the Animal Ethics Committee (CEUA) of Universidade Federal de Minas Gerais (UFMG), Brazil (Protocol number: 4/2015). Male C57BL/6J mice were randomly allocated into experimental groups. Mice were maintained in specific pathogen-free conditions. The chronic ethanol consumption model used was previously described (Ceron et al., 2018; Simplicio et al., 2017). Briefly, mice received ethanol 5% (v/v) in the first week, followed by 10% (v/v) in the second week (to help mice acclimate with this intervention) and were treated during 10 weeks with 20% (v/v) of ethanol in their drinking water. Control group received water. This protocol replicates blood ethanol levels after chronic ethanol consumption in human subjects, as described in studies with C57BL/6J or BALB/c mice (Ceron et al., 2018; Simplicio et al., 2017; Urso et al., 1981). Weight change, food and liquid consumption were measured weekly during ethanol treatment.

Cytokine and chemokine measurement

Cytokine and chemokine levels (TNF-α, IL-1β, CXCL1, CXCL2, IL-17, IL-10) were quantified in BAL, serum or plasma fluid using DuoSet ELISA kits (R and D Systems), in accordance to the manufacturer’s instructions.

Determination of blood ethanol levels

The determination of blood ethanol levels was made as previously described (Gonzaga et al., 2015). Briefly, mice were anesthetized and 100 μL of blood was collected and transferred into headspace vials. Blood ethanol levels were measured by gas chromatograph using a gas chromatographer as previously described (Ceron et al., 2018).

Mice infection

To determine the impact of chronic alcohol intake in fungal pulmonary infection, male C57BL/6J mice were treated for 12 weeks. After the last day of treatment, mice were infected intranasally with Aspergillus fumigatus A1163 strain (Fedorova et al., 2008). The fungus was grown in complete media for 48 hr at 37°C (Malacco et al., 2019). Fungal conidia were harvested by washing the media with sterile phosphate-buffered saline (PBS). After filtering, conidia were centrifuged at 1400 x g resuspended and counted in Neubauer chamber. Mice were infected with 3 × 108 conidia/animal, prepared in PBS.

BAL and tissue extraction

After 24 or 48 hr of infection, mice were anesthetized with ketamine (100 mg/kg) and xilazine (6 mg/Kg) and blood smear and serum or plasma were collected. After that, mice were euthanized and bronchoalveolar lavage fluid (BALF) was harvested as previously described (Malacco et al., 2019). BALF total cell counts were determined by counting leukocytes in Neubauer chamber. Differential cell count and in vivo phagocytosis count were obtained from cytospin preparations (Shandon III). BALF supernatants were used for cytokines, chemokines and total protein measurements. Protein amounts were quantified in BALF samples using the Bradford assay (Bradford, 1976).

Lung pathology analysis

At the indicated time points, lungs were collected. The right lobes were removed and frozen for subsequent analysis of myeloperoxidase (MPO) (Huang et al., 2016), N-acetyglucosaminidase (NAG) (Reiner et al., 1981), eosinophil peroxidase (EPO) (Strath et al., 1985) or measurement of fungal burden. The left lobes were fixed in formalin 4% (v/v) for histopathological analysis. Formalin-fixed tissue was dehydrated gradually in ethanol, embedded in paraffin, and 4 µm sections were stained with Hematoxylin and Eosin (H and E) or Grocott’s methenamine silver (GMS). The total histopathology score considered inflammatory infiltrate, interstitial and alveolar edema and hemorrhage (Hubbs et al., 1997). The percentage of germination of A. fumigatus conidia was counted in 200 to 300 fungal conidia in GMS-stained slides at x 100 magnification microscope.

Flow cytometry

Leukocytes obtained from BAL or blood samples were subjected to hypotonic lysis to remove residual erythrocytes, as described previously (Russo et al., 2009). Briefly, cells were treated with Fc block (R and D Systems), labeled with relevant antibodies, namely: CD3 - fluorescein isothiocyanate (FITC), CD4 - APC, IL-17 - phycoerythrin (PE), Ly6G - brilliant violet 421 (BV421), CXCR2 - PE, CD62L – APC and CD11b – FITC or isotype control. At least 30,000 events were acquired in a FACScan cytometer, and data were analyzed using FlowJo (Tree Star, Ashland, OR, USA) software. The relevant populations were gated, using accepted criteria for granularity, and sized and evaluated for staining of relevant surface and intracellular markers.

Intravital microscopy

The mouse cremaster preparation was used to study the behavior of leukocytes in the microcirculation and adjacent connective tissue, as previously described (Pinho et al., 2007). Briefly, 2 hr prior the surgery, mouse cremaster muscle were injected with LPS (250 ng/mL) diluted in saline. Then an incision was made in the scrotal skin to expose the cremaster muscle, which was then carefully removed from the associated fascia. A lengthwise incision was made on the ventral surface of the cremaster muscle using a cautery. The testicle and the epididymis were separated from the underlying muscle and were moved into the abdominal cavity. The muscle was then spread out over an optically clear viewing pedestal and was secured along the edges with a 4–0 suture. The exposed tissue was superfused with warm PBS. An intravital microscope (Olympus BX50F4) with x 20 magnification objective lens and x 10 times magnification eyepiece was used to examine the cremasteric microcirculation. A video camera (5100 HS; Panasonic) was used to project the images onto a monitor, and the images were recorded for playback analysis. The numbers of rolling and adherent leukocytes were determined offline during the video playback analyses. Leukocytes were considered adherent to the venular endothelium if they remained stationary for at least 30 s. Rolling leukocytes were defined as white cells moving at a velocity slower than that of the erythrocytes within a given vessel.

BM-derived neutrophils, phagocytosis and killing of fungal conidia

After isolation of mouse bone marrow (BM) from femur and tibia in RPMI 1640 medium, neutrophils were separated by density gradient centrifugation using Histopaque 1077 (density, 1.077 g/ml) in a 15 ml conical tube. Then, erythrocytes were lysed using ACK lysing buffer and the neutrophils were counted. Neutrophils purity was over than 80% (Swamydas and Lionakis, 2013). Phagocytosis and killing assay were performed by incubation of BM-derived neutrophils from mice treated and non-treated with ethanol with A. fumigatus conidia for 4 hr (phagocytosis) or 6 hr (killing) at 37° with 5% of CO2 in the ratio of 5:1. Phagocytosis was evaluated in cytospin preparations. To determine A. fumigatus killing, cells were lysed with distilled water and the diluted samples were plated in fungal medium and colony-forming units (CFU) were determined after overnight incubation at 37°C, and the percentage of killing was calculated as a percentage of the viability after incubation without neutrophils.

ROS detection

Luminometry assays were performed to evaluate the production of ROS by BM-derived neutrophils (Goes et al., 2016). Neutrophils (1 × 106 cells/well) were resuspended in complete RPMI medium without phenol red. Then the cells were plated in 96 well opaque plates (NUNC, Rochester, NY, USA) with 0.05 mM luminol (5-Amino-2,3-dihydro-1,4-phthalazinedione; Sigma-Aldrich) and A. fumigatus conidia in the proportion of 10 conidia to one neutrophil. Measurements were taken for 60 min with 2 min interval. Production of ROS was assayed by the light intensity generated by the reaction between ROS and luminol and expressed as fluorescent relative units.

Ex vivo chemotaxis assay

A modified Boyden chamber assay to examine the neutrophil chemoattractant response to CXCL-2 (kindly provided by Dr. José Carlos Alves-Filho, Universidade de São Paulo, Ribeirão Preto, Brazil) was performed using a 48-well microchamber (Neuro Probe) as previously described (Pinho et al., 2007). Murine bone marrow neutrophils were isolated as above described and resuspended in RPMI. Recombinant mouse CXCL-2 (20 ng/mL) diluted in running buffer (for wells containing neutrophils) or appropriate buffer control was added to the lower chambers of the apparatus. A 5-µm-pore polycarbonate membrane (Neuro Probe) was placed between the upper and lower chambers, and 5 × 104 cells in a volume of 50 µL were added to the top chambers of the apparatus. Cells were allowed to migrate into the membrane for 1 hr per treatment at 37°C with 5% CO2. Following incubation, the chamber was disassembled and the membrane was scraped and washed three times in PBS to remove nonadherent cells before being fixed in methanol and stained using the Diff-Quik system (Dade Behring). Each well-associated membrane area was scored using light microscopy to count the intact cells present in five random fields.

Statistical analysis

All experiments were made at least twice (biological replication) by independent experiments. The sample size estimation was done with G*Power 3.1 Software (Jacob Cohen's A power primer, 1992 in Psychological Bulletin Journal). Statistical analysis was performed with Graph Pad Prism six software (Graph Pad Prism Software, Inc, Sandiego, CA). Data are presented as the mean ± SD and were analyzed using One-way analysis of variance (ANOVA) followed by Tukey post-test to compare different groups. Student’s t test was used to compare two groups. Survival analysis was made by Log Rank test. Statistical significance was set as < 0.05. For more information, please see the transparent reporting form.

Acknowledgements

We would like to thank Universidade Federal de Minas Gerais for the opportunity to develop this work. We are thankful to Ilma Marçal and Rosemeire Oliveira for technical support.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Nathalia Luisa Sousa de Oliveira Malacco, Email: nathalialuisa2@gmail.com.

Frederico Marianetti Soriani, Email: fredsori@gmail.com.

Jos WM van der Meer, Radboud University Medical Center, Netherlands.

Frank L van de Veerdonk, Radboud University Medical Center, Netherlands.

Funding Information

This paper was supported by the following grants:

  • Fundação de Amparo à Pesquisa do Estado de Minas Gerais APQ-01756-10 to Jessica Amanda Marques Souza, Leda Quercia Vieira, Danielle Glória Souza, Vanessa Pinho, Mauro Martins Teixeira, Frederico Marianetti Soriani.

  • Coordenação de Aperfeiçoamento de Pessoal de Nível Superior 001 to Nathalia Luisa Sousa de Oliveira Malacco, Jessica Amanda Marques Souza.

  • Conselho Nacional de Desenvolvimento Científico e Tecnológico 474528-2012- 0 and 483184-2011-0 to Frederico Marianetti Soriani.

  • Instituto Nacional de Ciência e Tecnologia em Dengue e Interações Microrganismo-Hospedeiro to Nathalia Luisa Sousa de Oliveira Malacco, Jessica Amanda Marques Souza, Flavia Rayssa Braga Martins, Leda Quercia Vieira, Danielle Glória Souza, Vanessa Pinho, Mauro Martins Teixeira, Frederico Marianetti Soriani.

  • Universidade Federal de Minas Gerais 001 to Nathalia Luisa Sousa de Oliveira Malacco, Jessica Amanda Marques Souza, Flavia Rayssa Braga Martins, Milene Alvarenga Rachid, Celso Martins Queiroz-Junior, Grazielle Ribeiro Goes, Leda Quercia Vieira, Danielle Glória Souza, Vanessa Pinho, Mauro Martins Teixeira, Frederico Marianetti Soriani.

  • Fundação de Amparo à Pesquisa do Estado de Minas Gerais APQ-02198-14 and APQ-03950-17 to Jessica Amanda Marques Souza, Leda Quercia Vieira, Danielle Glória Souza, Vanessa Pinho, Mauro Martins Teixeira, Frederico Marianetti Soriani.

Additional information

Competing interests

No competing interests declared.

Author contributions

Investigation, Methodology, Writing - original draft.

Methodology.

Methodology.

Resources, Methodology.

Methodology.

Resources, Methodology.

Formal analysis.

Methodology.

Methodology.

Resources.

Resources.

Resources, Methodology.

Resources, Writing - review and editing.

Conceptualization, Resources, Supervision, Funding acquisition, Project administration, Writing - review and editing.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations of the CONCEA (Conselho Nacional de Controle de Experimentação Animal) from Brazil. All animal experiments received prior approval from the Animal Ethics Committee (CEUA) of Universidade Federal de Minas Gerais (UFMG), Brazil (Protocol number: 4/2015).

Additional files

Supplementary file 1. Percentage of neutrophil precursors in bone marrow after ethanol treatment.
elife-58855-supp1.docx (13.2KB, docx)

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

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Decision letter

Editor: Frank L van de Veerdonk1
Reviewed by: Frank L van de Veerdonk2, Agostinho Carvalho3, Mark Gresnigt

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Thank you for submitting your article "Chronic ethanol consumption compromises neutrophil function in acute pulmonary Aspergillus and Streptococcus infections" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Frank L van de Veerdonk as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Jos van der Meer as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Agostinho Carvalho (Reviewer #2); Mark Gresnigt (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.

Summary:

Malacco and colleagues addressed the role of chronic alcohol consumption in the immune response of neutrophils to infection with the fungal pathogen Aspergillus fumigatus. The results were to an extent validated in models of bacterial infection. The authors reveal an increased susceptibility of ethanol-treated mice to fungal infection as the result of an impaired neutrophil recruitment and activation at the site of infection. Neutrophils from treated mice also displayed a general defect of the antifungal effector functions, including phagocytosis, and killing, which contributed to the susceptibility phenotype. In general, the manuscript presents interesting and provocative findings, but there are several issues to be clarified

Essential revisions:

1) The rationale to study aspergillosis in relation to chronic alcohol consumption is poorly justified in the paper. Indeed the authors cite the literature that ARDS is more common in chronic alcohol abuse and list a number of pathogens, but not Aspergillus. The rationale to study aspergillosis specifically remains unclear.

2) The use of an infection model with Streptococcal is used to validate the findings obtained with the aspergillosis model. However, it is not clear what is the actual relevance of adding this information, particularly in light of a few conflicting findings between the models, e.g., the levels of CXCL1 in the BAL. I would therefore recommend streamlining the manuscript to focus on the relevant findings on Aspergillus and removing the information on the Staphylococcus infection as it does not strengthen the main message of the manuscript.

3) The in vivo experiments look very rigorous, however, additional information should be provided before infection to see how ethanol-fed mice are affected by the pre-treatment period alone. This is information can also be vital to mechanistically understand the observed neutrophil defects

• Do the mice have systemic inflammation (elevated CRP/IL-1/TNF/CXCL1 levels)?

• Can endotoxin be detected systemically?

• Do the mice lose bodyweight as a result of chronic alcohol consumption?

• Are their differences in the absolute numbers of circulating immune cells (neutrophils/monocytes/lymphocytes) or ratio's between the circulating leukocytes?

• Were there differences in water/food consumption of the animals?

These data of the pre-treated alone should be presented at the beginning of the study.

4) It is unclear why the authors chose such an unusual model of experimental aspergillosis using a remarkably high inoculum of 3x10^8 conidia. Immunocompetent mice are typically resistant to infection with A. fumigatus even when using high doses. Could this somehow explain the partial loss of survival observed in control mice? The rationale for the use of such as aggressive model should be clearly explained.

5) Are the differences in lung CFUs sustained after 48 hr or do the surviving mice resolve the infection?

6) Is there a role for the P-selectin ligand interaction with its receptor in this model? This process is critical in leukocyte adhesion to the vessels and cooperative signaling between P-selectin and CXCR2 in neutrophils has been shown to increase adhesion and the effector functions of neutrophils in several lung injury models. This point should be at least addressed in the Discussion.

7) The fact that the functional impairments of neutrophils are also present in cells derived from the bone marrow implies that the alcohol treatment may have a direct effect on the neutrophil precursors in the bone marrow. Have the authors looked at metamyelocytes and promyelocytes in the bone marrow?

eLife. 2020 Jul 23;9:e58855. doi: 10.7554/eLife.58855.sa2

Author response

Essential revisions:

1) The rationale to study aspergillosis in relation to chronic alcohol consumption is poorly justified in the paper. Indeed the authors cite the literature that ARDS is more common in chronic alcohol abuse and list a number of pathogens, but not Aspergillus. The rationale to study aspergillosis specifically remains unclear.

Thank you for addressing this Discussion. Aspergillosis is well known to be an important cause of morbidity and mortality to immunocompromised patients such as in hematological malignancies and transplants. However, there are several other risk factors that are not considered as traditional risks for developing aspergillosis. Kousha et al., 2011, published an extensive clinical review for pulmonary aspergillosis (PA). These authors discuss that chronic obstructive pulmonary disease (COPD) is an important risk factor for PA due to some reasons such as antibiotics, frequent hospitalization and comorbidities, such as alcoholism, among others. Moreover, PA can also affect patients who are mildly immunocompromised due to alcoholism, diabetes mellitus, chronic liver disease and low-dose corticosteroid therapy. Indeed, alcoholism is considered a risk factor for different clinical forms of aspergillosis since the decade of 1970 (Blum et al., 1978, Baddley, 2011 and Gustot et al., 2013). In fact, the rationale of this study is to understand the underlying mechanisms involved in increased susceptibility to Aspergillus pulmonary infection in chronic alcohol consumers. We also included some of that information in the Introduction section.

2) The use of an infection model with Streptococcal is used to validate the findings obtained with the aspergillosis model. However, it is not clear what is the actual relevance of adding this information, particularly in light of a few conflicting findings between the models, e.g., the levels of CXCL1 in the BAL. I would therefore recommend streamlining the manuscript to focus on the relevant findings on Aspergillus and removing the information on the Staphylococcus infection as it does not strengthen the main message of the manuscript.

Thank you for this suggestion. Indeed, our purpose in including the results with the S. pneumoniae model was to validate the mechanisms previously characterized for A. fumigatus infection. However, we also agree that this set of results do not strengthen the manuscript. We removed all data related to the S. pneumoniae model.

3) The in vivo experiments look very rigorous, however, additional information should be provided before infection to see how ethanol-fed mice are affected by the pre-treatment period alone. This is information can also be vital to mechanistically understand the observed neutrophil defects

• Do the mice have systemic inflammation (elevated CRP/IL-1/TNF/CXCL1 levels)?

Thank you for this suggestion. Actually, we have performed a set of experiments to understand better the effects of alcohol consumption and collected the majority of the requested data. In case of systemic inflammation due ethanol consumption, we did not observe any increase in TNF-α, IL-1β and CXCL1 levels in serum from mice treated with ethanol, compared to control mice. We added this information in the manuscript, please see Figure 1.

• Can endotoxin be detected systemically?

We did not measure endotoxin in alcohol-treated mice. However, our results do not demonstrate a systemic inflammation that could indicate the presence of circulating endotoxins. Serum cytokines and chemokines levels, such as TNF-α, IL-1β and CXCL1 are in basal levels. TNF-α is one of the cytokines considered to play a pivotal role as a mediator of host’s response to LPS. Thus, whenever there is a presence of LPS, the release of systemic TNF-α is induced in high levels and can cause even lethality in mice or human subjects (Ruggiero et al., 1993 – Mediat of inflam 2:S43-S50; Wang et al. World J Gastroenterol 2002 Jun;8(3):531-6 and Hamilton, et al. – Scan J Infect Dis, 1992;24(3):361-8; Copeland et al., Clin Diagnot Lab Immunol, 2005 12:60-67). Moreover, LPS, together with ATP are also capable of induce inflammasome mediated IL-1β release and our model do not demonstrate this rise of IL-1β levels in serum (Nee LE et al., Kidney Int. 2004;66(4):1376-1386; Dinarello CA. Blood. 2011;117(14):3720-3732; Wang et al., Pharmazie – An Internat Jl of Pharmac Sci, 2014 69(9), pp. 680-684(5)).

• Do the mice lose bodyweight as a result of chronic alcohol consumption?

That is a very good question. We actually made a set of experiments prior Aspergillus infection and we collected data related to body weight change in mice weekly. In our work we did not observe significant changes in weight from ethanol-fed mice compared to control mice. We added this information in the manuscript, please see Figure 1.

• Are their differences in the absolute numbers of circulating immune cells (neutrophils/monocytes/lymphocytes) or ratio's between the circulating leukocytes?

Thank you for this question. Actually, in the first version of the manuscript we showed a graph with total cell count in mice after treatment. Now, we present a detailed graph with differential cell count in blood prior Aspergillus infection, please check Figure 1.

• Were there differences in water/food consumption of the animals?

These data of the pre-treated alone should be presented at the beginning of the study.

Thank you for this comment. We actually collected data related to weekly food and liquid consumption in mice during ethanol treatment. Indeed, in our work we observed that the introduction of a diet based on ethanol consumption decreased the amount of food and liquid intake in mice, compared to control mice. However, the decreased consumption of food and liquids did not affect the overall weight of animals. We added this information in the manuscript, please see Figure 1.

4) It is unclear why the authors chose such an unusual model of experimental aspergillosis using a remarkably high inoculum of 3x10^8 conidia. Immunocompetent mice are typically resistant to infection with A. fumigatus even when using high doses. Could this somehow explain the partial loss of survival observed in control mice? The rationale for the use of such as aggressive model should be clearly explained.

Thank you so much for this comment. Indeed, we have addressed this question during the development of this work. In order to standardize inocula and extensively characterize the aspergillus model of pulmonary infection, our group previously demonstrated the infection profile in C57BL/6 mice in two different inocula: 1x10^8 and 3x10^8 conidia. In the lower inoculum, we observed lethality about 25% while in the higher inoculum was about 52% lethality. We attributed the increased lethality to the huge inflammatory response triggered by the high inoculum, since we have characterized the course of acute A. fumigatus lung infection in immunocompetent mice, investigating the immunological, pathological and tissue functional modifications (Malacco et al., 2020). This previous article paved the way for the choice of inoculum in the present work. We have also seen BALB/C mice mortality in immunocompetent model of Aspergillus infection with 1x10^8 inoculum (Malacco, et al., 2019. Front Cell Infect Microbiol Jan 11;8:453. doi: 10.3389/fcimb.2018.00453. eCollection 2018). Other works have used similar inocula and still showed different lethality rates in immunocompetent mice. Guerra et al. (PLoS Pathog. 2017 Jan; 13(1): e1006175) using BALB/C mice verified about 30% lethality with a 5x10^7 inoculum.

5) Are the differences in lung CFUs sustained after 48 hr or do the surviving mice resolve the infection?

Thank you so much this question. Again, we addressed some aspects of this question in another publication (Malacco et al., 2020). Using the same immunocompetent model for pulmonary aspergillosis, we demonstrated an intense neutrophil influx into the airways after 24 hours of infection, reaching a peak at 72 hours of infection. After that, neutrophils numbers progressively decreased until 168 hours of infection, when the complete spontaneous resolution of inflammation occurs, with a resolution index of 36 hours. Moreover, the high production profile of inflammatory mediators change after 3 days of infection with decrease of pro-inflammatory and up-regulation of anti-inflammatory cytokines and chemokines. These previous results suggest that, after 3 days post infection, lung CFU levels should decrease, at least in control (water-fed) animals. Unfortunately, we are not able to state if this is happening in ethanol-fed mice, nor if the differences between groups are sustained after that. However, taking into account our results: (i) lethality (Figure 2B); (ii) weight loss (Figure 2C) and (iii) inflammatory mediators (Figure 3A-F), we can assume that the inflammatory process is gradually diminishing in both groups (ethanol and water-fed mice), even with differences among them.

6) Is there a role for the P-selectin ligand interaction with its receptor in this model? This process is critical in leukocyte adhesion to the vessels and cooperative signaling between P-selectin and CXCR2 in neutrophils has been shown to increase adhesion and the effector functions of neutrophils in several lung injury models. This point should be at least addressed in the Discussion.

Thank you for your comment. Indeed, we agreed about the relevance of the selectin ligand and CXCR2 cooperation in neutrophil migration. The switch of β2 integrin from a low-affinity to a high-affinity state is an essential step for neutrophil crawling. We have added this point in the Discussion.

7) The fact that the functional impairments of neutrophils are also present in cells derived from the bone marrow implies that the alcohol treatment may have a direct effect on the neutrophil precursors in the bone marrow. Have the authors looked at metamyelocytes and promyelocytes in the bone marrow?

That is a very good question and represents one important point we are interested to explore. We already had collected samples of bone marrow and a hematologist analyzed them. Results showed no differences between neutrophils precursors in ethanol-fed mice compared to control mice. The results are presented as Supplementary file 1 and in the subsection “Chronic ethanol consumption did not induce systemic inflammation”.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    Figure 1—source data 1. Values for the outcome of ethanol treatment in mice.
    elife-58855-fig1-data1.xlsx (11KB, xlsx)
    Figure 3—source data 1. Values for inflammatory mediators in BALF after A. fumigatus infection.
    elife-58855-fig3-data1.xlsx (12.9KB, xlsx)
    Figure 4—source data 1. Values for indirect measurement of cell infiltration in lung tissue and CD3+CD4+IL-17 cells in BALF after A. fumigatus infection.
    elife-58855-fig4-data1.xlsx (12.2KB, xlsx)
    Figure 6—source data 1. Values for bone marrow neutrophil chemotaxis.
    elife-58855-fig6-data1.xlsx (10.6KB, xlsx)
    Figure 7—source data 1. Values of flow cytometry data.
    elife-58855-fig7-data1.xlsx (11.2KB, xlsx)
    Supplementary file 1. Percentage of neutrophil precursors in bone marrow after ethanol treatment.
    elife-58855-supp1.docx (13.2KB, docx)

    Data Availability Statement

    All data generated or analysed during this study are included in the manuscript and supporting files.

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