Abstract

   Acute lung injury (ALI) causes lung inflammation and edema as well as
   resulting in gut microbiota disorder. Probiotics, however, can improve
   the gut microbiota composition and modulate its immune response,
   playing an important role in ALI pathogenesis. Therefore, our study
   aims to investigate the effect of Lactobacillus reuteri on
   Lipopolysaccharide (LPS)-induced ALI in mice and to probe the mechanism
   of its synergistic modulatory effect on the lungs and intestines. We
   assessed the therapeutic effects of L. reuteri in the ALI mouse model
   by histopathology, alveolar lavage fluid and serum inflammatory factor
   analysis and explored microbiome and transcriptome alterations. L.
   reuteri intervention effectively attenuated lung tissue injury and
   significantly reduced the LPS-induced inflammatory response and
   macrophage and neutrophil infiltration. Additionally, L. reuteri
   improved the intestinal barrier function and remodeled the disordered
   microbiota. In conclusion, our study showed that L. reuteri attenuated
   the inflammatory response, ameliorated the pulmonary edema, repaired
   the intestinal barrier, and remodeled the gut microbiota in ALI mice.
   This study provides new perspectives on the clinical treatment of ALI.

   Keywords: Lactobacillus reuteri, acute lung injury, gut microbiota,
   inflammation

1. Introduction

   Acute lung injury (ALI) is an acute respiratory failure due to diffuse
   interstitial lung lesions caused by damage to the pulmonary vascular
   endothelium and alveolar epithelium by noncardiogenic factors such as
   severe infections, shock, trauma, and burns. The Berlin definition
   published in 2012 defines ALI as mild or moderate acute respiratory
   distress syndrome (ARDS) [[38]1]. The main manifestations of ALI/ARDS
   include increased lung permeability, inflammatory cell infiltration and
   alveolar edema, which are highly susceptible to the development of
   critical illness [[39]2]. In addition, the mortality rate of ALI/ARDS
   is approximately 40% in numerous studies [[40]3,[41]4]. Currently,
   ALI/ARDS treatment is based on mechanical ventilation and fluid
   management without effective clinical therapies, resulting in a poor
   prognosis for most patients [[42]5].

   In recent years, the interactions between lung disease and gut
   microbiota has been frequently reported in studies [[43]6,[44]7,[45]8].
   One study showed that an increased permeability of the intestinal wall
   in patients with ARDS and enrichment of intestinal bacteria in the lung
   microbiota is closely related to the severity of acute systemic
   inflammation [[46]6]. Another study indicated that the gut microbiota
   composition was closely associated with inflammatory factor levels and
   inflammatory markers in COVID-19 patients, suggesting that there may be
   a link between the gut microbiota and host immune response [[47]9].
   These studies are evidence that the gut microbiota may be important in
   the treatment of ALI. Recently, numerous studies have noted the
   potential of probiotic supplementation in the clinical treatment of
   patients with ALI/ARDS, which can improve patient symptoms, enhance
   immunomodulation, and remodel the gut microbiota
   [[48]10,[49]11,[50]12,[51]13]. However, only a few probiotics have been
   proved to be protective in patients with ALI.

   Lactobacillus is widely used as an important probiotic that helps
   protect the intestinal mucosal barrier, improve the host’s immune
   function, and maintain the intestinal microbial balance
   [[52]14,[53]15,[54]16,[55]17]. Lactobacillus reuteri as an important
   member of Lactobacillus, has been shown to maintain intestinal
   epithelial homeostasis and repair pathological damage to the intestinal
   mucosa [[56]14]. In addition, in another study, L. reuteri was revealed
   to inhibit the production of inflammatory cytokines, increasing the
   survival rate of ADRS mice [[57]11]. Moreover, Milagros Griet et al.
   reported that L. reuteri CRL1098 soluble factors reduced inflammatory
   factors released by LPS-stimulated mouse macrophages [[58]18]. In
   conclusion, L. reuteri provides new approaches for ALI treatment.

   In our study, we intervened with L. reuteri in LPS-induced ALI model
   mice with the aim of exploring the effects of L. reuteri on mice with
   ALI and clarifying the possible mechanisms involved through multiomic
   analyses, including 16S rRNA sequencing, transcriptomics, and
   metabolomics.

2. Materials and Methods

2.1. Strain and Culture Conditions

   Following previous research, the probiotic strain L. reuteri was
   obtained from Protectis baby drops (Biogaia AB, Stockholm, Sweden)
   [[59]19]. The strain was incubated anaerobically in a Man–Rogosa–Sharpe
   (MRS) broth at 37 °C for 24 h. Afterward, the medium was centrifuged at
   4000× g for 10 min, and then washed and resuspended in phosphate
   buffered saline (PBS) for gavage.

2.2. Animal Experiments

   The model was induced by intratracheal administration of LPS
   (Sigma–Aldrich, Gillingham, UK). C57BL/6 male mice (6 weeks, 22 ± 0.5
   g) were housed in a specific pathogen-free room with a controlled 12-12
   h light-dark cycle and a constant temperature of 22–24 °C. After
   acclimatization feeding, the mice were randomly divided ([60]Figure
   1A): the NC (PBS + PBS) group, the LPS (PBS + LPS) group, and the LR
   (L. reuteri + LPS) group (n = eight per group). The LR group was
   gavaged daily with L. reuteri suspension, whereas the NC and LPS groups
   were treated with PBS as a placebo treatment for 21 days. On day 22,
   mice in the LPS and LR groups were induced with 0.5 mL of 5 mg/kg LPS,
   and the NC group received 0.5 mL of PBS. After 5 days, mice were
   euthanized by injection of sodium pentobarbital, the left lungs were
   washed with PBS to collect bronchoalveolar lavage fluid (BALF), and
   samples of blood, lung, and intestinal tissues were collected. All
   animal experiments were reviewed by the Ethics Committee (No.
   2023-969).

Figure 1.

   [61]Figure 1
   [62]Open in a new tab

   L. reuteri attenuates lung injury in ALI mice. Experimental flowchart
   (A); Changes in body weight (B); (C) Images of HE staining of lung
   tissue (magnification × 20, scale = 50 μm); (D) lung dry/wet ratio in
   mice; Concentration of total protein (F), total cell count (E), and LDH
   activity (G) in BALF; Data are presented as mean ± SEM. ** p < 0.01,
   and *** p < 0.001. NC, PBS + PBS group; LPS, PBS + LPS group; LR, L.
   reuteri + LPS group.

2.3. Histopathologic Evaluation

   Lung and colon tissues were fixed in 10% formalin and after 24 h we
   dehydrated them through a graded alcohol series and embedded them in
   paraffin. We cut the tissue wax block into 3 μm slices and stained them
   with hematoxylin and eosin (H&E). We stained the lung tissue with
   anti-F4/80 and anti-myeloperoxidase (MPO) antibodies; five
   representative fields of view were randomly selected at 40×
   magnification and quantified by calculating the percentage of
   positively stained areas in the field of view by ImageJ, and the
   pathology scores were assessed by two independent professors of
   pathology in a double-blind manner. Colon tissue sections were
   fluorescently stained with anti-ZO-1, anti-occludin and anti-MUC2
   antibodies to visualize the intestinal barrier. We scanned the images
   using a Pannoramic MIDI scanner (3DHISTECH, Budapest, Hungary).

2.4. Calculation of Lung Tissue W/D Ratio

   Mouse right middle lobe lung tissue was collected and its wet weight
   (W) was measured immediately after suction drying of the surface water.
   The tissues were then dried at 70 °C for 72 h to measure the dry weight
   (D). Tissue edema was assessed by calculating the W/D value.

2.5. BALF Assay Analysis

   The BALF was centrifuged at 800× g for 15 min, erythrocytes were lysed
   and resuspended in 1000 μL of PBS, and then the number of cells was
   measured using an automated cell counter (Thermo Fisher Scientific,
   Waltham MA, USA). Meanwhile, we used a BCA protein assay kit (P0011,
   Beyotime Biotechnology, Nantong, China) to test the total protein level
   in the supernatant. In addition, the levels of lactate dehydrogenase
   (LDH) activity, IL-1β and IL-6 in the BALF were measured using ELISA
   kits (Abcam, Cambridge, UK).

2.6. Hematological Assay

   The LPS binding protein (LBP) concentration was measured using an ELISA
   kit (ab269542, Abcam, Cambridge, UK), and a mouse Cytokine 23-Plex
   Assay (Kit Bio-Rad, Hercules, CA, USA) was used to measure the
   concentration of serum inflammatory factors. All procedures were
   performed according to the kit manuals.

2.7. Real-Time Fluorescent Quantitative PCR Analysis

   We used an RNeasy Plus Mini Kit (74134, Qiagen, Valencia, CA, USA) to
   extract RNA from colon tissues and converted it to cDNA (500 ng RNA per
   10 μL of cDNA system), and then the mRNA levels were detected in the
   ViiA7 real-time PCR system (Applied Biosystems, Waltham, MA, USA). We
   used the mRNA expression level of Gapdh as the control target gene. The
   sequences of gene primers are shown in [63]Supplementary Table S1.

2.8. Microbial Composition Analysis

   We collected mouse colon contents and extracted DNA from them by using
   a DNeasy PowerSoil pro kit (47016, Qiagen, Hilden, Germany). PCR
   amplification was then performed using primers for the variable regions
   V3 to V4 of the 16S rRNA gene ([64]Supplementary Table S1). After
   identification, libraries were constructed, and the PCR products were
   sequenced on an Illumina NovaSeq 6000 platform (Illumina Inc., San
   Diego, CA, USA).

   The data were imported into QIME2 for processing, clustered into
   amplicon sequence variant (ASV) groups by using the DADA2 plug-in and
   categorized according to the Silva 138 database in QIME2. Finally,
   α-diversity and β-diversity were assessed by QIME2 software.
   Differences between taxa were also analyzed using linear discriminant
   analysis (LDA) effect size (LEfSe). Raw data were uploaded to the SRA
   database (PRJNA1015362).

2.9. Metabolomic Analysis

   Liquid chromatography mass spectrometry (LC–MS) metabolic analysis was
   performed on the cecum contents. The samples preparation method was as
   before [[65]20] and the results were subsequently analyzed in an
   ACQUITY UPLC I-Class liquid chromatograph mass spectrometer (Waters
   Corporation, Milford, MA, USA).

2.10. Transcriptome Analysis

   Lung tissue RNA was extracted, and a library was constructed and
   sequenced as described previously [[66]21]. Subsequently, between-group
   differential transcript analysis was performed using the R package
   DESeq2, genes for which DESeq2 determined a p-value < 0.05 were
   considered differentially expressed. We used the Kyoto Encyclopedia of
   Genes and Genomes (KEGG) pathways for enrichment analysis of
   differential genes. The data were uploaded to the SRA database
   (PRJNA1015376).

2.11. Statistical Analysis

   We analysed the data using SPSS software (version 20.0; SPSS, Chicago,
   IL, USA). We tested the date normality by using the Kolmogorov Smirnov
   test, and then analyzed them using the Mann–Whitney U test or Student’s
   t test. The results were expressed using the mean ± standard error of
   the mean (SEM), and p < 0.05 was considered statistically significant.
   Images were prepared in a GraphPad Prism 9.0 and R (version 4.2).

3. Results

3.1. L. reuteri Attenuates Lung Injury in ALI Mice

   We established the ALI model by intratracheal injection of LPS (5
   mg/kg) ([67]Figure 1A). According to [68]Figure 1B, pregavage of the
   probiotic L. reuteri for 21 days had no effect on the body weight,
   whereas after LPS induction, the weight of the LPS group was
   significantly reduced, but the L. reuteri intervention alleviated this
   symptom.

   To assess the condition of the lungs of the mice, we stained the lung
   pathology sections with H&E. The alveolar wall in the LPS group was
   thickened and associated with diffuse interstitial infiltration. In
   contrast, the alveolar wall was significantly thinner in the LR group
   and the immune cell infiltration was reduced ([69]Figure 1C). In
   addition, we also collected the BALF to measure the total protein
   content ([70]Figure 1E), total cell count ([71]Figure 1F), and LDH
   activity ([72]Figure 1G) to further assess lung injury. Compared to the
   NC group, the total cell count, total protein level and LDH activity
   were increased in the LPS group, while the LR group showed a decrease
   in all of these parameters. In addition, L. reuteri treatment
   attenuated the degree of pulmonary oedema, with an increase in the W/D
   value of the lung tissue in the LPS group and a decrease in the LR
   group. ([73]Figure 1D).

   Overall, L. reuteri attenuated LPS-induced ALI in mice, especially in
   terms of pulmonary edema and alveolar epithelial permeability.

3.2. L. reuteri Reduces the Inflammatory Response Caused by LPS

   Inflammatory cell infiltration is an important feature of the
   LPS-induced ALI model, and we assessed the protective effect of L.
   reuteri by measuring inflammatory markers in serum and alveolar lavage
   fluid. As shown in [74]Figure 2C–F, we observed that serum levels of
   proinflammatory factors were elevated in the LPS group; however, these
   proinflammatory factors were significantly decreased after L. reuteri
   intervention. In addition, compared to the NC group, the levels of
   inflammatory factors in the BALF were elevated in the LPS group,
   whereas they were down-regulated in the LR group after the L. reuteri
   intervention ([75]Figure 2A,B).

Figure 2.

   [76]Figure 2
   [77]Open in a new tab

   L. reuteri reduces the inflammatory response caused by LPS. IL-1β (A)
   and IL-6 (B) concentrations in BALF; serum levels of IL-1β (C) IL-6
   (D), TNF-α (E), and MIP-1 (F). Representative images of F4/80 (G) and
   MPO (H) immunohistochemical staining (magnification × 40, scale = 20
   μm); n = three per group and five randomly selected visual fields. Data
   are presented as mean ± SEM. * p < 0.05, ** p < 0.01, and *** p <
   0.001. NC, PBS + PBS group; LPS, PBS + LPS group; LR, L. reuteri + LPS
   group.

   In addition, to further evaluate the effects of lung macrophages and
   neutrophils on the ALI model mice after LPS induction, we stained the
   lung tissues using anti-F4/80 and anti-MPO antibodies. The LPS group
   showed significant enhancement of macrophages and neutrophils, whereas
   the LR group showed reduced recruitment of macrophages and neutrophils
   ([78]Figure 2G,H). In conclusion, L. reuteri attenuated LPS-induced
   pulmonary and systemic inflammation in mice.

3.3. L. reuteri Reduces Intestinal Damage and Repairs Intestinal Function

   Systemic inflammation induced by lung injury disrupts the intestinal
   barrier integrity, which increases the permeability to intestinal
   microorganisms and their products and finally leads to exacerbation of
   the systemic inflammatory condition [[79]6]. As shown in [80]Figure 3A,
   colon HE staining indicated that villous atrophy and rupture, as well
   as epithelial barrier disruption, were clearly present in intestinal
   tissues of the LPS group, whereas L. reuteri markedly ameliorated the
   colonic epithelial damage. In addition, we further used
   immunofluorescence staining and q-PCR to assess the intestinal barrier
   function. The intestinal barrier was significantly impaired in the LPS
   group, with significantly lower mRNA expression levels of ZO-1 and
   occludin than those in the NC group, which was ameliorated by the L.
   reuteri intervention ([81]Figure 3C,D). Meanwhile, the
   immunofluorescence results showed similar trends ([82]Figure 3A).

Figure 3.

   [83]Figure 3
   [84]Open in a new tab

   L. reuteri reduces intestinal damage and repairs intestinal function.
   (A) Representative images of HE staining (magnification × 10, scale =
   100 μm) and immunofluorescence staining for ZO-1, occludin, and MUC2 of
   colon tissues (magnification × 15, scale = 100 μm). (B) Serum LBP
   levels were measured by ELISA. The mRNA levels of ZO-1 (C), occludin
   (D), and MUC2 (E) (red indicate positive areas). Data are presented as
   mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001. NC, PBS + PBS
   group; LPS, PBS + LPS group; LR, L. reuteri + LPS group.

   Whereas disruption of the intestinal barrier leads to the translocation
   of bacteria and their metabolites into the bloodstream, thereby
   affecting systemic inflammation, CD14 is a receptor for the LPS and LBP
   complex [[85]22]; therefore, we assessed bacterial translocation by
   measuring serum LBP concentrations. We observe that the serum LBP level
   in the LPS group was higher than in the NC and LR groups ([86]Figure
   3B). Meanwhile, we assessed intestinal permeability by detecting MUC2
   levels in colonic tissues, and immunofluorescence staining in the LPS
   group showed significantly fewer positive areas ([87]Figure 3A) and
   significantly lower levels of mRNA expression of MUC2 compared with
   that of the NC group, whereas the L. reuteri intervention markedly
   increased the level of MUC2 expression ([88]Figure 3E).

   In conclusion, LPS-induced intestinal mucosal damage in mice with ALI
   was severe; the intestinal barrier function was significantly
   disrupted, which may be closely related to the translocation of the
   intestinal flora, and L. reuteri was able to ameliorate the intestinal
   barrier dysfunction.

3.4. Effects of L. reuteri on the Composition of the Gut Microbiota

   To investigate the gut microbiota composition, we sequenced the 16S
   rRNA gene from mouse colon feces and obtained a total of 1,924,335 raw
   sequences from 24 samples. As shown in [89]Figure 4A–C, our study
   demonstrated that the richness (Chao1 and observed species indexes) and
   diversity (Shannon index) of the gut microbiota were decreased in the
   LPS group; however, the diversity index was slightly increased after L.
   reuteri intervention. In addition, we further assessed the β-diversity
   of the microbiota by performing the PCoA on the samples based on binary
   Jaccard distances. As shown in [90]Figure 4D, the microbiotas of the
   three groups were significantly separated (PERMANOVA, p = 0.001).

Figure 4.

   [91]Figure 4
   [92]Open in a new tab

   Effects of L. reuteri on the composition of the gut microbiota. Chao1
   (A), observed species (B) and Shannon indices (C) of intestinal flora.
   (D) The PCoA plot based on binary Jaccard distances of three groups.
   LEfSe analyses plot between the NC and LPS groups (E) and the LPS and
   LR groups (F). ** p < 0.01, and *** p < 0.001. NC, PBS + PBS group;
   LPS, PBS + LPS group; LR, L. reuteri + LPS group.

   To further explore the key bacteria in gut microbiota, we used LEfSe
   analysis. Compared to the NC group, the phylum Actinobacteriota, class
   Coriobacteriia, and orders Erysipelotrichales and Coriobacteriales were
   depleted in the LPS group. Furthermore, at the family level,
   Muribaculaceae, Rikenellaceae, Coriobacteriales_Incertae_Sedis,
   Eggerthellaceae, Mycoplasmataceae and Erysipelatoclostridiaceae were
   also depleted in the LPS group. However, the phylum Proteobacteria, the
   phylum Deferribacterota, the class Alphaproteobacteria, the order
   Rhodospirillales, the family Tannerellaceae, the genus Citrobacter and
   the genus Parabacteroides were enriched in the LPS group ([93]Figure
   4E).

   In addition, the phylum Verrucomicrobiota, the class Verrucomicrobiae,
   the order Verrucomicrobiales, and the family Akkermansiaceae were
   enriched in the LR group compared with those in the LPS group
   ([94]Supplementary Figure S1). Additionally, the LR group was enriched
   for the genus Clostridia_UCG_014, the family Oscillospiraceae, the
   genus Colidextribacter, the family Anaerofustaceae, the order
   Eubacteriales, the genus Eubacterium__ventriosum_group, the genus
   UCG_003, and the family Christensenellaceae. Moreover, the LR group was
   depleted for the genus Muribaculaceae, the genus Acetatifactoraceae the
   genus Acetatifactor, the genus Citrobacter, the genus
   Eubacterium__ruminantium_group, the genus Morganella, and the genus
   Dubosiella ([95]Figure 4F).

   In conclusion, the richness and diversity of the gut microbiota in mice
   with ALI were disrupted, and L. reuteri intervention was able to
   reshape the gut microbiota.

3.5. Effects of L. reuteri on the Metabolic Composition in Mice with ALI

   To investigate the effect of L. reuteri on intestinal metabolites, we
   used LC–MS to analyze the contents of the mouse cecum. As shown in
   [96]Figure 5A, metabolite level differences were observed in the
   partial least squares-discriminant analysis (PLS-DA) plot among the
   three groups, the NC, LPS and LR groups. The plot of orthogonal PLS-DA
   (OPLS-DA) between different groups also showed differences in
   metabolite levels between groups ([97]Figure 5B,C). To further explore
   the differences in metabolite levels between the LPS and LR groups, we
   screened the results according to the thresholds variable importance in
   projection (VIP) >1, and p values < 0.05. Compared to those in the LPS
   group, a total of 185 metabolites was enriched and 108 metabolites were
   depleted in the LR group ([98]Figure 5D).

Figure 5.

   [99]Figure 5
   [100]Open in a new tab

   Effects of L. reuteri on the metabolic composition in mice with ALI.
   (A) PLS-DA plot of three groups. (B) OPLS-DA plot of the NC and LPS
   group. (C) OPLS-DA plot of the LPS and LR group. (D) Volcano plot of
   differential metabolites between the LPS and LR groups. (E) KEGG
   enrichment analysis of different metabolites between the LPS and LR
   groups. NC, PBS + PBS group; LPS, PBS + LPS group; LR, L. reuteri + LPS
   group.

   In addition, we further examined the major metabolic pathways in the
   LPS and LR groups by KEGG pathway enrichment analysis, and identified a
   total of 26 downregulated metabolic pathways, including the FoxO
   signaling pathway, glutamatergic synapse, phospholipase D signaling
   pathway, ferroptosis, and inflammatory mediator regulation of TRP
   channels ([101]Figure 5E). These pathways may be important for the
   amelioration of inflammation by L. reuteri in mice with ALI.

3.6. L. reuteri Regulates Lung Transcription in Mice with ALI

   In an effort to comprehend the potential mechanism by which L. reuteri
   ameliorates LPS-induced lung injury, we conducted transcriptome
   profiling of lung tissue. Applying thresholds of |log2FC| ≥ 1 and
   p-value ≤ 0.05, we identified 27,088 differentially expressed genes
   (DEGs) between the LPS and NC groups. Specifically, there were 1698
   upregulated and 1451 downregulated DEGs in the LPS group compared to
   the NC group. Furthermore, when comparing the treated with L. reuteri
   group to the LPS group, we saw that there were 666 upregulation and 781
   downregulation DEGs ([102]Figure 6A,B). Moreover, it was observed that
   the gene expression profile of the mice treated with L. reuteri
   exhibited a greater similarity to that of the mice in the NC group, as
   opposed to the untreated ALI model mice ([103]Figure 6C). This finding
   suggests that L. reuteri has the ability to reverse the molecular
   changes associated with lung injury. Furthermore, we employed the KEGG
   pathway database to map the aforementioned genes, specifically
   emphasizing pathways that exhibited overlap. By applying an adj-p <
   0.05 threshold, we identified a total of 42 downregulated inhibitory
   pathways, which possess meaningful implications. Notably, these
   pathways encompass the Toll-like receptor signaling pathway,
   cytokine-cytokine receptor interaction and NF-kappa B signaling pathway
   ([104]Figure 6E). To further investigate the genes associated with
   protection, we further constructed a protein–protein interaction (PPI)
   network by categorizing the genes located within the downregulated
   pathways that displayed significant inhibition. Following the
   establishment of the PPI network, an examination of the cluster with
   the highest MCODE score was conducted to investigate the essential gene
   network. Furthermore, the identified key hub genes included Tlr8, Tlr1,
   Tlr9, Tlr6, Tlr2, Cd86, Cd14, Serpinb9b, Ccl2, Cxcl1, Cxcl10, Casp1,
   Fcgr1, Nlrp3, Socs3, Arg1, Ptgs2, and Jak2 ([105]Figure 6E). Activation
   of the Toll-like receptors (TLRs) pathway triggers the release of
   proinflammatory cytokines, which may be important for the development
   of ALI.

Figure 6.

   [106]Figure 6
   [107]Open in a new tab

   L. reuteri regulates lung transcription in mice with ALI. Volcano map
   of differentially expressed gene distribution between the NC group and
   LPS group (A) and the LPS group and LR group (B). (C) Heat cluster plot
   of the three groups. (D) Pathways in the lung were upregulated in the
   LPS group (compared with the NC group). (E) Pathways in the lung were
   downregulated by L. reuteri intervention (compared with the LPS group).
   (F) Visualization of the cluster network with the highest MCODE score.
   NC, PBS + PBS group; LPS, PBS + LPS group; LR, L. reuteri + LPS group.
   n = 3 in each group.

4. Discussion

   Numerous recent studies have mentioned that systemic inflammation
   caused by lung injury may lead to increased intestinal permeability and
   intestinal barrier damage, which increases intestinal permeability to
   the microbiota and its metabolites, resulting in the lungs accumulating
   intestinal bacteria, which can further contribute to lung disease
   exacerbation [[108]23,[109]24,[110]25,[111]26]. Crosstalk between the
   gut and the lungs is strongly correlated with lung disease severity,
   and the main direction of this crosstalk is from the gut to the lungs,
   although it cannot be ruled out that it occurs in the opposite
   direction [[112]6,[113]27,[114]28,[115]29]. Previous studies have shown
   that L. reuteri has significant potential for supporting intestinal
   epithelial regeneration, repairing the intestinal barrier, remodeling
   the intestinal microbiota, and inhibiting the production of
   proinflammatory factors, among many other activities
   [[116]14,[117]30,[118]31]. However, the molecular mechanisms by which
   L. reuteri ameliorates ALI symptoms remain unclear. In our study, we
   evaluated the effect of L. reuteri intervention on ALI model mice, and
   elucidated the possible related mechanisms based on a multiomics
   approach (transcriptome, microbiome, and metabolome)
   ([119]Supplementary Figure S2). The results indicated that L. reuteri
   ameliorated the symptoms of mice with LPS-induced ALI by suppressing
   lung inflammation, attenuating pulmonary edema, repairing the
   intestinal barrier, and remodeling the intestinal microbiota.

   Inflammatory mediators are important in ALI/ARDS pathogenesis,
   especially proinflammatory factors, which can cause diffuse damage to
   alveolar epithelial and vascular endothelial cells, increase vascular
   permeability, and promote protein leakage, which further aggravates
   lung edema [[120]32,[121]33]. In our study, the HE staining showed that
   under the influence of LPS, the lung tissue structure of mice exhibited
   alveolar cell and interstitial damage, and L. reuteri intervention
   alleviated alveolar and interstitial edema and reduced inflammatory
   cell infiltration. In addition, we observed that L. reuteri reduced the
   levels of IL-1β and IL-6 in the lungs and found that L. reuteri
   intervention reduced capillary permeability by measuring BCA and LDH
   levels and total cell counts in the BALF. This finding is consistent
   with previously reported results that inhibition of inflammation is
   beneficial in reducing lung tissue damage [[122]34]. Acute tissue
   injury or viral infection can activate immune cells, and IL-1β is
   mainly derived from activated macrophages [[123]35,[124]36], and can
   activate neutrophils, inducing a shock-like state in animal models
   [[125]37]. In addition, elevated production of IL-6 has been suggested
   to be stimulated by IL-1β and TNF-α, and IL-6 can also be produced by a
   variety of cells, including macrophages and endothelial cells
   [[126]38]; moreover, circulating levels of IL-6 have been considered by
   many studies to be a major predictive marker of ARDS severity due to
   different etiologies [[127]39,[128]40]. Therefore, we also performed
   immunohistochemical staining of lung tissues with anti-F4/80 and
   anti-MPO antibodies, which showed an increase in the amount of
   macrophages and neutrophils in the lung tissues of ALI mice; moreover,
   L. reuteri intervention reduced the levels of the above cells.

   Our study also showed in lung transcriptome analysis that regulation of
   inflammation-related pathways may be the mechanism involved in
   attenuating lung injury by L. reuteri intervention. By analyzing
   differential genes through the KEGG pathway database, L. reuteri may
   attenuate LPS-induced lung injury by downregulating the Toll-like
   receptor and NF-kappa B signaling pathway. Previous research has shown
   that in mice with ALI, activation of the TLR signaling pathway in
   alveolar endothelial and epithelial cells phosphorylates NF-kB
   downstream of the pathway [[129]41], which induces the release of
   inflammatory cytokines, leading to increased inflammation [[130]42]. We
   further categorized genes within the downregulated pathways that were
   significantly inhibited in the LR group that may be involved in the
   alleviation of lung injury by L. reuteri. For example, the NLRP3
   inflammatory vesicle induces caspase-1 activation and promotes IL-1β
   secretion, thereby affecting the inflammatory response [[131]43].
   Additionally, TLR2 is highly expressed in the lung macrophages and
   activated endothelial cells [[132]44], and TLR2/Myd88/NF-κB activation
   (phosphorylation of the p65 subunit) induces secretion of MCP-1, TNF-α,
   IL-6, and IL-1β [[133]45]. Our results for serum inflammatory factor
   levels were consistent with previous findings [[134]45,[135]46] and the
   L. reuteri intervention reduced these levels. Toll-like receptors are
   expressed on immune cells, which can activate the immune system,
   increase inflammatory response, disrupt the intestinal mucosal barrier
   and cause bacterial translocation [[136]47,[137]48]. Our results showed
   that L. reuteri intervention decreased serum LBP concentrations,
   increased the mRNA expression levels of intestinal ZO-1, occludin and
   MUC2, reduced intestinal permeability and repaired the intestinal
   barrier function.

   Intestinal epithelial cells are arranged via tight junctions to form
   the first line of defense of the intestinal tract against infection,
   which forms a physical barrier, and both it and the microbial barrier
   are important intestinal barriers [[138]49]. The microbial barrier is
   mainly composed of the intestinal microbiota, which can maintain the
   homeostasis of the intestinal mucosa and prevent the invasion of
   pathogenic bacteria, and the intestinal microbiota is relatively stable
   under normal conditions [[139]50]. However, previous studies have shown
   that inflammation, immunity, and other factors may lead to disruption
   of and a decrease in the diversity of the intestinal flora, resulting
   in an increased susceptibility to harmful bacteria [[140]51].
   Consistent with previous findings, the 16S rRNA sequencing results
   showed a significant decrease in the abundance and diversity of the gut
   microbiome in ALI model mice. In addition, we observed that the LPS
   group was enriched in Proteobacteria at the phylum level, which has
   been shown in previous studies to produce endotoxin LPS and induce a
   strong inflammatory response favoring the invasion of harmful pathogens
   [[141]52,[142]53]. Moreover, the LPS group showed enrichment of the
   harmful bacteria Parabacteroides and Citrobacter. Previous studies
   showed that Parabacteroides was enriched in septic lung-injured rats
   [[143]54], whereas Citrobacter was significantly enriched in mice
   coinfected with IAV–MRSA [[144]55]. The LR group showed a decreased
   abundance of the harmful bacteria Morganella [[145]56] and Citrobacter
   but an increased abundance of the beneficial bacteria Akkermansia,
   Colidextribacter and the Eubacterium__ventriosum_group. One study has
   shown that Colidextribacte reduces LPS-induced liver injury and
   inflammation by modulating hepatic TLR4 and NF-κB signaling [[146]57].
   Akkermansia has been suggested to ameliorate LPS-induced lung injury
   and inflammation in numerous studies [[147]54,[148]58]. Notably, the LR
   group, although not enriched for L. reuteri, showed an increased
   abundance of beneficial bacteria capable of ameliorating inflammation
   upon L. reuteri intervention.

   Furthermore, L. reuteri intervention downregulated the expression of
   inflammatory mediators that regulate the TRP channel. Many studies have
   shown that TRP channels are expressed in various immune cells,
   including macrophages and T cells, and activation of TRPV1 in T cells
   may enhance inflammatory responses [[149]59], but activation of TRPV4
   leads to the release of the chemokine MCP-1, which may activate
   signaling pathways by inducing macrophage recruitment [[150]60]. Thus,
   the gut microbiota is involved in various metabolic processes and these
   metabolites are important in the gut barrier function and the immune
   system [[151]61,[152]62].

5. Conclusions

   In conclusion, our study explored the effects of L. reuteri on an
   LPS-induced ALI mouse model by means of multiomic analysis. The results
   showed that lung and systemic inflammation caused by LPS-induced ALI
   may result in a damaged gut barrier and a disturbed gut microbiota,
   while L. reuteri can repair the damaged gut barrier and remodel the
   microbiota, thereby reducing the inflammatory response. This study
   explores the potential mechanism by which L. reuteri reduces lung
   inflammation during LPS exposure, providing a theoretical basis for the
   ameliorative effects of probiotics on ALI symptoms and thus offering
   new perspectives on ALI/ARDS treatment in the clinic.

Supplementary Materials

   The following supporting information can be downloaded at:
   [153]https://www.mdpi.com/article/10.3390/nu15194256/s1, Figure S1: (A)
   The differential species branching diagram between the NC group and the
   LPS group. (B) The differential species branching diagram between the
   LPS group and the LR group. Figure S2: Schematic diagram of L. reuteri
   intervention in LPS-induced acute lung injury. Table S1: Primer
   sequences used for real-time PCR analysis and 16S rRNA. Table S2: List
   of antibodies we used. Table S3: List of hub genes in PPI network of LR
   vs. LPS.
   [154]Click here for additional data file.^ (347.9KB, zip)

Author Contributions

   Conceptualization, J.S.; methodology, S.W.; software, H.X.; data
   analysis, H.C., Y.H., Z.W. and S.H.; original draft preparation, J.S.;
   review and editing manuscript, L.L. All authors have read and agreed to
   the published version of the manuscript.

Institutional Review Board Statement

   All procedures were approved by the Ethical Inspection of Animal
   Experiments of the First Affiliated Hospital of Zhejiang University
   School of Medicine (2023-969).

Informed Consent Statement

   Not applicable.

Data Availability Statement

   Not applicable.

Conflicts of Interest

   The authors declare no conflict of interest.

Funding Statement

   This work was supported by the Fundamental Research Funds for the
   Central Universities (2022ZFJH003), the National Key R&D Program of
   China (2021YFC2301805, 2022YFC3602003) and the Research Project of
   Jinan Microecological Biomedicine Shandong Laboratory (JNL-2022045D).

Footnotes

   Disclaimer/Publisher’s Note: The statements, opinions and data
   contained in all publications are solely those of the individual
   author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI
   and/or the editor(s) disclaim responsibility for any injury to people
   or property resulting from any ideas, methods, instructions or products
   referred to in the content.

References