Oral administration of Lactobacillus delbrueckii UFV-H2b20 protects mice against Aspergillus fumigatus lung infection

doi:10.21203/rs.3.rs-3999628/v1

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Research Article

Oral administration of Lactobacillus delbrueckii UFV-H2b20 protects mice against Aspergillus fumigatus lung infection

https://doi.org/10.21203/rs.3.rs-3999628/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 28 Aug, 2024

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Methods

we investigated survival, respiratory mechanics, histopathology, colony forming units, cytokines in bronchoalveolar lavage, IgA in feces, efferocytosis, production of reactive oxygen species and the cell population in the mesenteric lymph nodes.

Results

L. delbrueckii induces tolerogenic dendritic cells, IL-10⁺macrophages and FoxP3⁺regulatory T cells in mesenteric lymph nodes and increased IgA levels in feces; after infection with A. fumigatus, increased survival and decreased fungal burden. There was decreased lung vascular permeability without changes in the leukocyte profile. There was enhanced neutrophilic response and increased macrophage efferocytosis. L. delbrueckii-treated mice displayed more of FoxP3⁺Treg cells, TGF-β and IL-10 levels in lungs, and concomitant decreased IL-1β, IL-17A, and CXCL1 production.

Conclusion

our results indicate that L. delbrueckii UFV H2b20 ingestion improves immune responses, controlling pulmonary A. fumigatus infection. L. delbrueckii seems to play a role in pathogenesis control by promoting immune regulation.

Lactobacillus delbrueckii UFV H2b20

probiotic

regulatory T cell

Aspergillus fumigatus

neutrophilic response

Probiotics are “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” [1]. Health benefits include prevention of infection or growth restriction of pathogenic microorganisms, epithelial barrier protection, expression of genes that modulate inflammation in the gut [1, 2], and stimulation of host immunity through a wide range of mechanisms [3].

Probiotics interact in different ways with macrophages, mucosal dendritic cells (DCs), and intestinal epithelial cells. Pattern recognition receptors such as Toll-like receptors [4] and NOD-type receptors [5] play a crucial role in recognition and transmission of signals that lead to different types of responses. It is well established that probiotics can also affect T helper (Th) response, either directly or indirectly, via regulatory T lymphocytes (Tregs) [4]. Additionally, lactic acid bacteria can induce the production of IL-12, further enhancing Th1 cell proliferation and IFN-γ production (reviewed by Yadav et al. [6]). Thus, probiotics play a significant role in modulation of the immune system and protection against pathogens.

Recent literature suggests an axis between gut health and vital organs, including the lungs [7, 8]. The gut-lung axis is essential for lung immune response and airway homeostasis, and there is increasing evidence that probiotics may regulate this interaction (reviewed by Yadav et al. [6]. There is still much to explore in understanding the gut-lung axis and many strategies regarding the gut microbiota have already been proposed to treat and prevent lung diseases [9]. Recently, we have shown that L. delbrueckii UFV-H2b20 promotes protective effects on OVA-induced allergic pulmonary inflammation in mice by stimulating regulatory responses through increased numbers of FoxP3⁺ Treg cells [10].

Aspergillus fumigatus is a ubiquitous fungus with environmental and clinical impacts. Infection with A. fumigatus occurs after inhalation of air-borne conidia, followed by their deposition into the alveoli or bronchioles [11]. In immunocompetent individuals, inhaled conidia are rapidly eliminated by physical barriers and innate cells [12] (reviewed by [13]. However, in immunocompromised patients, host defenses are not enough to eliminate conidia [13–15]. When not efficiently eliminated by alveolar macrophages, these conidia can germinate into hyphae and invade lung tissue [16]. The innate immune system is the first line of defense against conidia [12]. Neutrophils, which are rapidly recruited to the alveoli, phagocyte both conidia and germ tubes and are considered one of the main defense cells against hyphae, due to their ability to produce reactive oxygen species (ROS), release of granular content and formation of neutrophil extracellular traps [12, 14, 17].

Most studies relating to probiotics and lung infections have focused on viral and bacterial infections [18, 19] and there are no studies associating the benefits of administering oral probiotic bacteria to ameliorate fungal pulmonary infection.

Lactobacillus delbrueckii UFV-H2b20 is a typical intestinal tract inhabitant probiotic [20]. Studies from our group have already shown L. delbrueckii UFV-H2b20 ability to stimulate their host immune system by enhancing phagocytic capacity by macrophages and improving gram-negative bacteria elimination [20, 21], inducing inflammatory cytokine production in vitro and in vivo [22, 23]. Here, we demonstrate that oral administration of L. delbrueckii UFV H2b20 can protect mice against A. fumigatus lung infection by increasing cytokine production and numbers of cells involved in immune regulation and improving mechanisms of fungal clearance.

Experimental animals

All animal experiments were conducted and approved according to Brazilian national guidelines on animal work (CONCEA) and UFMG ethics committee on the use of animals (CEUA/UFMG) protocol number 46/2018. Specific-pathogen-free (SPF) male BALB/c AnNCrl mice weighing 20–26 grams and eight to ten weeks of age were obtained from the UFMG Central Animal Facility. Mice were grouped into three to five animals per cage in ventilated racks and kept in the Laboratory of Gnotobiology and Immunology animal facility (UFMG) under controlled light conditions. Animals were given autoclaved water. The availability of food, water and probiotic was monitored daily.

Cultivation and treatment with Lactobacillus delbrueckii UFV-H2b20

L. delbrueckii UFV-H2b20 was isolated at the Federal University of Viçosa (UFV) from newborn feces and brought to UFMG in 12% reconstituted skimmed milk (RSM) medium as previously described [20]. This material was expanded in 1 L of 12% RSM and cultivated for 18 hours at 37°C. This culture was aliquoted, and these aliquots were used as stock culture in our experiments. For association of mice with L. delbrueckii UFV-H2b20, 100µL of this preparation were inoculated in 10 mL of MRS broth (Merck, São Paulo, Brazil) and cultured for 18 to 20 hours at 37°C. A culture containing 10⁹ CFU/mL was obtained. Cultures were centrifuged at 2500xg at 4°C, washed once in sterile saline, placed entirely in the bottle with additional 150 mL of sterile water and offered to mice. Control mice received only sterile water. Treatment was given 10 days before infection with A. fumigatus and was maintained until euthanasia. Bottles were changed daily, and treatment was maintained with viable bacteria throughout the experiment.

Infection with Aspergilllus fumigatus

To determine the impact of probiotic intake on fungal pulmonary infection, male BALB/c AnNCrl mice were treated and infected intranasally with Aspergillus fumigatus A1163 strain [24]. The fungus was grown in Aspergillus complete solid medium (YAG) for 48 hours at 37˚C [25]. Fungal conidia were harvested by washing the medium with sterile phosphate-buffered saline, pH 7.4 (PBS). After filtering, conidia were centrifuged at 1400xg, resuspended, and counted in a Neubauer chamber. Mice were infected with 1x10⁸ conidia/animal, prepared in PBS.

Survival Analysis

For survival analysis, mice were inoculated with 1x10⁸ A. fumigatus viable conidia, survival was monitored for 10 days. Death in different groups was recorded daily and survival curve determined. Survival analysis was made by Log Rank test.

Respiratory mechanics function test

For invasive in vivo spirometry test assessment, mice were tracheostomized and placed in a whole-body plethysmograph (Forced Pulmonary Maneuver System, Buxco Research Systems, New Brighton, MN, USA) to maintain spontaneous breathing connected to a computer-controlled ventilator (Buxco Research Systems), as we previously described [10, 25, 26]. Under mechanical ventilation, total lung capacity (TLC), compliance (Cchord), forced vital capacity (FVC), and baseline pulmonary resistance were determined by RC resistance and compliance test. Pressure-volume maneuvers were performed to measure compliance, inflate the lungs to a standard pressure of + 30 cm H₂O, and then slowly exhale until a negative pressure of -30 cm H₂O was reached. Fast flow volume maneuvers were performed to measure forced expiratory volume in 50 milliseconds (FEV50), and the flow-volume curve was recorded during this maneuver.

Histopathological analysis

After euthanasia, lungs were immediately removed. The left lung was fixed in 10% formalin solution (PBS formalin) until the samples were processed. Samples were dehydrated in different concentrations of ethanol, cleared in xylene, and embedded in paraffin blocks. With the aid of a microtome, histological sections of 4 µm were obtained from lung sections arranged on microscope slides. Sections were stained with hematoxylin-eosin (HE) to assess the inflammatory infiltrate. The slides were scanned using a digital microscope 3D Slide Scanner (Histech, Budapeste, Hungary) and analyzed by CaseViewer 3DHistech software, version 2.4 (Histech, Budapeste, Hungary). The total histopathology score considered inflammatory infiltrate, interstitial and alveolar edema, and hemorrhage [27].

Colony forming units (CFU) determination

To assess fungal clearance capacity in mice, mice were euthanized with a solution of 150 mg/kg of ketamine (Syntec, Cotia, Brazil) and 10 mg/kg of xylazine (Syntec) intraperitoneally and the right lung was removed 24 hours after infection. The collected lung was homogenized in 1 mL of sterile PBS. After serial dilution, 100 µL aliquot suspensions were seeded, in duplicates, with glass rods, into Petri dishes containing YAG fungal medium [28]. CFU were counted after 20 hours of growth at 37 ºC and the result was expressed as the number of CFU x10⁶ per right lung.

BAL and tissue extraction

After 12, 24, or 48 hours of infection, mice were anesthetized with ketamine (100 mg/kg) and xylazine (6 mg/ kg), and blood smear and serum or plasma were collected. After that, mice were euthanized, and bronchoalveolar lavage (BAL) was harvested as previously described [25]. BAL total cell counts were determined by counting leukocytes in the Neubauer chamber. Differential cell count and in vivo phagocytosis were obtained from cytospin preparations (Shandon III, Gothenburg, Sweden). BAL supernatants were used for cytokine, chemokine, and protein measurements. The Bradford assay quantified Protein amounts in BAL samples [29]. Lungs were harvested for myeloperoxidase (MPO) [30], N-acetyglucosaminidase (NAG) [31], or eosinophil peroxidase (EPO) [32] determination.

Cytokine, chemokine and IgA quantification

Cytokine and chemokine concentrations were quantified in BAL supernatant 24 hours after infection and total IgA concentrations were quantified in mouse feces, by ELISA, following the manufacturer’s protocol R&D Systems (Minneapolis, Minnesota, USA) for cytokines and chemokines and Southern Biotech (Birmingham, Alabama, USA) for IgA. BAL supernatant was obtained by centrifuging samples 300xg for 10 minutes at 4°C.

Obtaining macrophages and neutrophils from the bone marrow

Male BALB/c AnNCrl mice aged 8 to 10 weeks untreated or treated with L. delbrueckii UFV H2b20 were euthanized and had their femur and tibia bones removed for bone marrow collection. Epiphyses were cut and the bones were flushed with 3 mL of PBS supplemented with 100 U/mL of penicillin (GIBCO, New York, NY, USA) and 100 µg/mL of streptomycin (GIBCO). Cell suspensions were centrifuged at 400xg for 8 minutes at 4°C. For macrophages, the supernatant was discarded and cells were resuspended in DMEM F12 medium (GIBCO), incubated in a Petri dish (JProlab, São José dos Pinhais, Brazil) for seven days in DMEM F12 medium supplemented with 10% inactivated fetal bovine serum (GIBCO), 100 U/mL of penicillin, 100 µg/mL of streptomycin, 2 mM of l-glutamine (GIBCO) and 20% of L929 fibroblast culture supernatant, which produce the factor GM-CSF responsible for the differentiation of precursor cells into macrophages [33]. Cells were kept in 10 ml of DMEM F12 medium for four days, after which another 5 ml of DMEM F12 medium were added. Finally, after seven days in culture, macrophages were removed from the plate with ice-cold PBS. During the differentiation process and for all assays, cells were incubated in a humidified atmosphere oven at 37°C and 5% CO₂. For neutrophils, supernatant was discarded, and cells were resuspended in RPMI (GIBCO). Neutrophil separation was performed by centrifugation at 400xg for 30 minutes at 4°C, in a density gradient using Histopaque® 1077. Red blood cells were lysed by adding water followed by adding 10x PBS in a 9:1 ratio. Neutrophils were counted in a Neubauer chamber. The purity of neutrophils in all experiments was above 80%.

Efferocytosis assay

Efferocytosis was performed ex vivo with macrophages and neutrophils from L. delbrueckii UFV H2b20-treated and not-treated mice. Apoptotic neutrophils were obtained after marrow-derived neutrophils were exposed to ultraviolet irradiation for 3 hours. Afterwards, macrophages and neutrophils were incubated for 3 hours at 37°C with 5% CO₂ in a 3:1 ratio. After that, cytospin slides were prepared and stained with rapid Panoptic kit (Laborclin, São José do Rio Preto, Brazil). Macrophage phagocytic capacity was determined by calculating the percentage of macrophages that phagocytosed neutrophils in relation to the total amount of macrophages.

Measurement of reactive oxygen species (ROS)

Bone marrow-derived neutrophils from treated and untreated with L. delbrueckii UFV H2b20 mice, at 5x10⁵ cells per 200µL, were incubated for 30 minutes with the dihydroethidium (DHE) probe (1µM) in a humidified atmosphere oven at 37°C and 5% CO₂, washed three times with PBS and incubated again for another 30 min. An immunofluorescence assay was performed by incubating these neutrophils with fluorescent zymozan at a ratio of 1:5 for 1 hour at 37°C with 5% CO2. Afterwards, neutrophil ROS production was evaluated by fluorescence microscopy. Briefly, neutrophils from animals treated and untreated with L. delbrueckii UFV H2b20 and stimulated with zymosan were placed on cytospin slide preparations and fixed with 4% formaldehyde. Samples were permeabilized with 1% triton X-100. Subsequently, the cells were labeled with DAPI (Thermo Fisher, Waltham, MA, USA). The preparations were visualized on a Nikon Ti A1R Confocal microscope with a spectral detector and XYZ motorized stage (Nikon, Tokyo, Japan). The images were acquired using a 20x objective and with a definition of 1024x1024.

Flow Cytometry

Mesenteric lymph node cells from uninfected mice treated and not treated with L. delbrueckii UFV H2b20, after 10 days of treatment, were collected, processed, centrifuged at 300xg for 10 minutes at 4°C and the supernatant discarded. For mice infected with A. fumigatus, BAL cells were submitted to analysis by flow cytometry. Samples were centrifuged at 300xg for 10 minutes at 4°C. BAL or mesenteric lymph node cells were then labeled for 20 minutes with different combinations of antibodies: anti-CD45 (clone 30-F11); anti-CD11b (clone M1/70); anti-CD11c (HL3 clone); anti-F4/80 (clone BM8); anti-Ly6C (clone HK1.4); anti-CD103 (clone 2E7) anti-Ly6G (clone 1A8-Ly6g) anti-TCRβ (clone H57-597), anti-CD4 (clone GK1.5), anti-CD16/32 (clone 2.4G2) and appropriate negative or isotype controls. Then, 1 ml of PBS was added, and the samples were centrifuged at 400 g for 10 minutes at 4°C to remove excess antibodies. This procedure was repeated three times. Then, cells were fixed and permeabilized with the eBioscienceTM transcription factor labeling kit (ThermoFisher, cidade, MA, USA) and stained for 45 minutes at 4ºC with anti-FOXP3 (clone MF23) antibody. All antibodies used were purchased from BD (BD Biosciences Pharmingen, San Diego, CA, USA). The LIVE/DEAD Aqua labeling kit (Thermofisher) was used to exclude dead cells. At the end of labeling, cells were washed twice and resuspended in PBS containing 10% fetal bovine serum. The acquisition was performed on an LSRFortessaTM (BD) flow cytometer. The analyses were performed using the FlowJo software (B&D).

Statistical Analysis

Results were presented as mean ± standard error of each group's mean (SEM). Cytokine production kinetics assays were analyzed by One-Way ANOVA, followed by post-hoc test Holm-Sidak to determine differences between the times and the evaluated groups. To determine differences between groups, unpaired Student's t test was used using two-tailed distribution, after applying a normality test and verifying the symmetry of the data. All statistical analyses were performed using the GraphPad Prism software, version 8.00 (GraphPad, San Diego, CA, USA). Differences were considered statistically significant when p < 0.05.

Lactobacillus delbrueckii UFV H2b20 ingestion modulates gut immunity

To assess whether treatment with probiotics modulates the gut immune response before pathogen challenge, we evaluated mesenteric lymph node leukocyte profiles of mice treated or not with L. delbrueckii UFV H2b20. Figures 1A and B show the DC profile. Treatment with L. delbrueckii increased some DC subset profiles: total DCs (Fig. 1A), CD11b⁻CD103⁺ DCs, and a tolerogenic subset of DC. There was a tendency of increase in the CD11b⁺CD103⁺ DC subset, a plastic subtype of DC (Fig. 1B). We also found that probiotic intake increased the number of macrophages (Fig. 1C), IL-10⁺ macrophages (Fig. 1D), and Tregs (Fig. 1E) in mesenteric lymph nodes. We could not observe any differences in IL-10⁺ Treg cells (Fig. 1F). Furthermore, we analyzed IgA levels in feces of treated and non-treated mice and identified a significant increase in immunoglobulin levels in the treated group (Fig. 1G).

Lactobacillus delbrueckii UFV H2b20 intake decreases the susceptibility to Aspergillus fumigatus infection

To evaluate the effect of L. delbrueckii UFV H2b20 administration on the host susceptibility during fungus infection, we verified the survival rate and weight change of treated and non-treated mice after intranasal infection with A. fumigatus. The treated infected group showed significantly lower mortality rate (50% up to the tenth day after infection) when compared to non-treated infected group (80% on the sixth day after infection) (Fig. 2A). Regarding weight change, both groups experienced loss of body mass until the third day. Both groups lost weight similarly until the third days post-infection, but L. delbrueckii UFV-H2b20-treated animals progressively recovered body mass, almost recovering the initial weight after 10 days of infection. (Fig. 2B).

Lung histopathological analysis was performed to assess the damage caused by infection. Lung tissue of uninfected mice treated or not with L. delbrueckii UFV-H2b20 remained intact, and its morphological characteristics were preserved. On the other hand, infection by A. fumigatus caused alterations in lung parenchyma of both treated and non-treated mice after 1 day of infection, presenting similar degrees of inflammatory infiltrate, with neutrophils predominance, especially in the perivascular and peribronchiolar regions (Fig. 2C), which the Inflammatory Score confirmed. However, we did see differences between groups in other lung parameters analyzed. Vascular permeability, measured as protein extravasation in BAL, was lower in treated mice compared to non-treated mice 1 day after infection (Fig. 2D). In addition, we assessed lung function, namely, total lung capacity, compliance, forced expired volume in 50 milliseconds, and basal lung resistance. We observed that treatment significantly restores the functional deficit caused by infection. Infected treated group recovered total lung capacity, compliance and forced expired volume in 50 milliseconds compared to infected non-treated group. Both groups had similar performances in Basal Lung Resistance (Fig. 2E). Interestingly, all improvements comfered by treatment were associated with decreased fungal load into the lungs 12, 24 and 48 hours after infection (Fig. 2F), indicating that administration of L. delbrueckii UFV-H2b20 led to a better control of fungal infection by the host.

Oral administration of Lactobacillus delbrueckii UFV H2b20 influences cell influx into the lungs after Aspergillus fumigatus infection

To verify whether there is a correlation between infection susceptibility improvement and the leukocyte profile at the site of infection, we evaluated cellular recruitment into the alveoli. Results showed that infected treated mice had decreased total leukocyte influx into the airways 24 hours after infection compared to infected non-treated mice (Fig. 3A). In order to identify the predominant cell type in the alveolar spaces, differential cell-counting was performed, demonstrating that neutrophil numbers are lower in the infected treated mice group (Fig. 3B), corroborating MPO levels in the lung tissue (Fig. 3C). Interestingly, infected treated mice showed increased numbers of macrophages in BAL compared to infected untreated group (Fig. 3D), despite the same levels of NAG in the lungs (Fig. 3E). Eosinophil counts in BAL (Fig. 3F), EPO amounts in lung tissue (Fig. 3G), and lymphoid cell counts in BAL (Fig. 3H) were not different between groups.

Mice treated with Lactobacillus delbrueckii UFV H2b20 have higher influx of ROS-producing neutrophils and decreased frequency of apoptotic neutrophils after infection

Since we observed decreased susceptibility to A. fumigatus infection and alteration in cell influx to the lungs from treated mice, we aimed to investigate a possible improvement in neutrophilic functions in this group. At first, we performed an ex vivo experiment with DHE probe labeling neutrophils taken from bone marrow of treated or not-treated mice incubated with FITC-labeled zymosan. We could see the differences in DHE probe fluorescence between the groups mainly when zymosan activated by zymosan. The treated group presented higher and more intense fluorescence, indicating more ROS production (Fig. 4A). We also performed flow cytometry to assess ROS-producing neutrophils in BAL 12 hours after infection and we confirmed that the quantity and proportion of these cells were higher in the treated group (Fig. 4B). These data indicate that L. delbrueckii UFV-H2b20 treatment improved neutrophilic response, confirming its protective potential.

Apoptosis of neutrophils and the removal of these apoptotic cells contribute to control inflammation. To verify if probiotic treatment had an effect in these anti-inflammatory processes, the amount and frequency of apoptotic neutrophils in BAL was verified through the expression of annexin V. We observed a significant decrease in the frequency of apoptotic neutrophils (expressing annexin V) in the treated group compared to non-treated group (Fig. 4C). To verify if this difference could be due to the removal of apoptotic cells, we analyzed whether treatment with the probiotic would optimize efferocytosis by bone marrow-derived macrophages. We observed that macrophages from the L. delbrueckii-treated group showed a significantly higher efferocytosis capacity than the non-treated group. However, after stimulation with IFN-γ and LPS, this difference is no longer present. In addition, there is also no difference between stimulated and non-stimulated treated groups, suggesting a possible priming of the macrophages by the probiotic treatment (Fig. 4D). These results confirm that L. delbrueckii UFV-H2b20 treatment can improve the removal of apoptotic neutrophils.

Lactobacillus delbrueckii UFV-H2b20 intake promotes regulation of inflammation after Aspergillus fumigatus infection

The next step was to verify the mechanisms by which L. delbrueckii UFV-H2b20 regulates the immune response during infection. Previous studies by our group showed that L. delbrueckii UFV-H2b20 modulates host cytokine production [21–23]. Therefore, we verified the profile of inflammatory mediators in the airways. Initially, we analyzed the regulatory cytokines IL-10 (Fig. 5A) and TGF-β (Fig. 5B) and observedthat L. delbrueckii treatment could not increase these cytokines in non-infected groups. However, after infection, there was an increase in IL-10 levels in both groups, but this increase was more conspicuous in the L. delbrueckii-treated group. TGF-β levels did not change. We then analyzed the inflammatory cytokines IL-17A, IL-1β, and TNF and the chemotactic chemokines for neutrophils CXCL1 and CXCL2 and observed a decrease in IL-17A (Fig. 5C), IL-1β (Fig. 5D) and CXCL1 (Fig. 5F) in treated versus untreated infected mice. The increase in regulatory cytokines and decrease in inflammatory mediators indicate that the probiotic stimulates greater regulation of inflammation in treated animals. There were no differences in TNF and CXCL2 levels between groups (Figs. 5E and G). Afterwards, we quantified Treg cells in BAL and observed a significant increase of these cells in the airways of treated infected mice compared to untreated infected ones (Fig. 5H), indicating that the regulation of inflammation promoted by oral treatment with L. delbrueckii UFV-H2b20 occurs through the increase of Treg cells quantity in the lungs of infected mice.

Probiotics can provide therapeutic benefits, directly acting in the gut and preventing or treating lung diseases, such as respiratory infections [19]. Here, we demonstrated that the probiotic Lactobacillus delbrueckii UFV-H2b20 promoted immune system modulation in uninfected mice, as indicated by an increase in numbers of conventional dendritic cells, regulatory dendritic cells, macrophages, IL-10-producing macrophages, and regulatory T lymphocytes in the mesenteric lymph nodes, in addition to higher levels of IgA in feces. Our data showed that mice that ingested L. delbrueckii UFV-H2b20 had lower lethality and plasma leakage in BAL, indicating less edema. Treated mice also presentedsmaller fungal load in lungs at 12, 24 and 48 hours after infection, attenuated lung dysfunction related to decreased neutrophilic influx, together with increased macrophage numbers in lung tissue 24 hours after infection. This improvement is likely promoted by enhanced neutrophil functions, such as increased ROS production by neutrophils and in the ability to remove apoptotic neutrophils at the site of infection. In addition, our results also showed that the probiotic promoted control of dysregulated inflammation, as indicated by elevated IL-10 production and increase in Treg lymphocytes, culminating in the decrease of pro-inflammatory mediators such as IL-17A, IL-1β and CXCL1 in lung tissue after 24 hours of infection.

Studies by our group had already shown that L. delbrueckii UFV-H2b20 modulates the immune system and protects hosts against infections by Escherichia coli and Salmonella enterica serovar Typhimurium [21], Listeria monocytogenes [23] and against OVA-induced allergic pulmonary inflammation [10]. However, none of these works had evaluated the immunomodulatory effect promoted by the probiotic before challenge. Here, we observed that probiotic ingestion promotes differences in cellular populations in mesenteric lymph nodes, such as higher numbers of regulatory T lymphocytes, macrophages, IL-10-producing macrophages, conventional dendritic cells, CD11b⁻CD103⁺ and CD11b⁺CD103⁺ dendritic cells (DCs). DCs, especially CD103⁺ DCs in mouse mucosal sites, are responsible for the uptake of orally administered antigens in the intestine and promote the differentiation of naive T cells into Treg cells. For this reason, they are considered tolerogenic [34, 35]. The CD103⁺CD11b⁻DC subset is involved in the induction of FOXP3⁺ Treg cells. It is the only subset of mucosal DCs that can activate TGF-β via αvβ8 integrin expression [34]. Our data are in accordance with these results, as we observed an increase in both cell types in the lymph nodes of mice treated with L. delbrueckii UFV-H2b20. The increase in this cell population suggests that the pre-treatment with probiotics, before the challenge with A. fumigatus, changes the host immune system, dampening the uncontrolled inflammation caused by the infection since the lethality in this model is mainly caused due to an intense inflammatory response in immunocompetent mice [25]. In addition to the changes in cell profiles, we also observed increasing total IgA levels in mouse feces. IgA is the main immunoglobulin found in mucosal secretions and is crucial in protecting this tissue type [36]. These data reinforce the probiotic modulatory role since what we are observing is an increase in the number of dendritic cells that migrate to the mesenteric lymph node and induce the differentiation of regulatory T cells, as already described in the literature for other commensal bacteria, thus assisting in intestinal homeostasis [37]. Considering previous literature which has shown that intestinal dendritic cells carrying live commensal bacteria induce class switching to IgA when they reach the mesenteric lymph nodes [38], we have in this increase of IgA a suggestion that L. delbrueckii can be mucosal protective.

After challenging mice with A. fumigatus, we observed a decreased susceptibility to infection in treated mice, as indicated by reduced lethality and weight loss. These effects were associated with a decreased lung fungal load. Our results corroborate other studies that also indicate protection induced by probiotics during respiratory infections. For example, Kawahara et al and Zolnikova et al showed that oral or intranasal administration of various Lactobacillus spp. and Bifidobacterium spp. strains suppress symptoms of several viral infections, including Influenza [39, 40]. Mortaz et al. and Shimizu et al. showed that probiotics reduce the incidence of ventilator-associated pneumonia [41, 42]. In our work, we observed no histopathological differences in lungs between the probiotic-treated and non-treated groups, probably because the time of infection evaluated is associated with an initial and acute phase. However, when we evaluated vascular permeability, we identified that the amount of total protein in BAL of treated infected mice was lower than untreated, indicating the presence of lower edema in the lungs of treated mice. Moreover, we observed that the treated infected group significantly restored some of lung functional deficits caused by the infection. Tissue injury triggered by inflammation in response to infection causes loss of volume, elasticity, and decreased lung airflow [25]. Here, we saw that the administration of probiotic L. delbrueckii UFV H2b20 promoted the preservation of essential lung functions such as total lung capacity, compliance, and forced expired volume.

While evaluating the cellular response pattern at the infection site, we observed a significant decrease in recruited cells, mainly neutrophils, 24 hours after infection. We could also observe a higher proportion of macrophages in BAL of treated infected mice compared to the untreated infected mice at this time of infection, which would probably be helping to regulate the excessive neutrophilic response. Thus, we also investigated whether the probiotic would improve neutrophil function during A. fumigatus infection. Neutrophils recruited to the site of inflammation play an essential role against the growth of conidia and hyphae by several mechanisms [43], and it is known that failure in the production of ROS by these cells leads to higher susceptibility to infection by A. fumigatus [44]. In fact, we observed in vitro that the probiotic promoted increased production of ROS by neutrophils. We also observed in vivo that, at initial times of infection, such as 12 hours, the treated animals had a significant amount of ROS producer neutrophils at the site of infection, indicating that L. delbrueckii UFV H2b20 enhances the initial bactericidal neutrophilic response against the fungus 12 hours after infection, and faster control of this response since, 24 hours after infection, these treated mice presented lower amounts of neutrophils in BAL. These results confirm the influence of the probiotic treatment on induction and regulation of the neutrophilic response.

Quick removal of apoptotic neutrophils at the site of infection is essential for the resolution of acute inflammation [45]. Therefore, we also analyzed efferocytosis in our model. We observed that efferocytosis by non-stimulated macrophages from treated mice was higher when compared to macrophages from untreated mice. In addition, non-stimulated macrophage efferocytosis from treated mice was equal to the stimulated macrophage efferocytosis, indicating that treatment with the probiotic can act as a macrophage stimulus. Corroborating these data, we also showed by flow cytometry that BAL from infected treated mice contains lower frequency of apoptotic neutrophils. These data confirm that, in addition to neutrophil response, treatment with L. delbrueckii also helps to rapidly remove neutrophils at the site of infection and consequently promote resolution of the inflammatory process.

We also investigated the mechanisms involved in attenuated inflammation promoted by L. delbrueckii UFV H2b20. There is evidence that probiotics can regulate the immune system against respiratory infections. For example, Harata G. et al [46] showed that L. rhamnosus GG administration protects mice against H1N1 infection by regulating the immune response in the airways. Here, we observed that BAL from treated infected mice presented higher numbers of Foxp3 + regulatory T cells than untreated infected mice. Treg cells use a variety of immunosuppressive mechanisms to regulate immune response by targeting effector cells, including secretion of immunoregulatory cytokines, granzyme/perforin-mediated cell cytolysis, metabolic perturbation, directing the maturation and function of antigen presenting cells (APC) and secretion of extracellular vesicles for the development of immunological tolerance (reviewed by Grover et al. [47]). Here, we show that BAL from infected treated mice showed higher amounts of regulatory cytokines IL-10 and TGF-β, which were associated with lower amounts of pro-inflammatory cytokines IL-17 and IL-1β, suggest that the increase in these regulatory cytokines, with a consequent decrease in CXCL1 and inflammatory cytokines, is one of the mechanisms of inflammation regulation induced by the probiotic.

Collectively, our results allow us to conclude that Lactobacillus delbrueckii UFV-H2b20 promotes a protective effect against A. fumigatus experimental infection. Thus, we suggest that L. delbrueckii UFV H2b20 ingestion modulates the immune response against A. fumigatus early infection, leading to a lower susceptibility of the host, by enhancing ROS production by neutrophils and improving clearance of A. fumigatus while increasing the numbers of tolerogenic dendritic cells (DCs), IL-10⁺ macrophages, FoxP3⁺ regulatory T cells (Tregs) that are important for counterbalancing the excessive inflammatory response and tissue damage. We believe that our study contributes new insights into the mechanisms of protection of probiotics during lung infections.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

This work was supported by Pró-Reitoria de Pesquisa - Universidade Federal de Minas Gerais, Fundação de Amparo à Pesquisa do Estado de Minas Gerais - APQ-03950-17; APQ-01899-18 and CNPq 312954/2021-2. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001 and Instituto Nacional de Ciência e Tecnologia em Dengue e Interação Microrganismo Hospedeiro (INCT em Dengue). FMS and LQV are CNPq fellows, and ACMMA was a FAPEMIG fellow. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Contribution

Conception of the study: A.C.M.M.A., L.Q.V. and F.M.S. Designed the experiments: A.C.M.M.A., L.Q.V., L.M.S., R.C.R. and F.M.S. Performed the experiments: A.C.M.M.A., N.L.O., A.E.N.S, F.R.B.M., M.E.M.L, L. T. and C.M.Q.J. Interpretation of the results and data analysis: A.C.M.M.A., L.Q.V., L.M.S., R.C.R., C.M.Q.J. and F.M.S. Wrote the manuscript: A.C.M.M.A., L.Q.V. and F.M.S. All authors read and approved the final manuscript.

Acknowledgments

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

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No competing interests reported.

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Oral administration of Lactobacillus delbrueckii UFV-H2b20 protects mice against Aspergillus fumigatus lung infection

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Respiratory mechanics function test

Histopathological analysis

Colony forming units (CFU) determination

BAL and tissue extraction

Cytokine, chemokine and IgA quantification

Obtaining macrophages and neutrophils from the bone marrow

Efferocytosis assay

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