High cellulose dietary intake relieves asthma inflammation through the intestinal microbiome in a mouse model.

Numerous epidemiological studies have shown that a high dietary fiber intake is associated inversely with the incidence of asthma in the population. There have been many studies on the role of soluble dietary fiber, but the mechanism of action for insoluble dietary fiber, such as cellulose-the most widely existing dietary fiber, in asthma is still unclear. The current study investigated the outcomes of a high-cellulose diet in a mouse model of asthma and detected pathological manifestations within the lungs, changes in the intestinal microbiome, and changes in intestinal short-chain fatty acids (SCFAs) in mice. A high-cellulose diet can reduce lung inflammation and asthma symptoms in asthmatic mice. Furthermore, it dramatically changes the composition of the intestinal microbiome. At the family level, a new dominant fungus family Peptostreptococcaceae is produced, and at the genus level, the unique genus Romboutsla, [Ruminococcus]_torques_group was generated. These genera and families of bacteria are closely correlated with lipid metabolism in vivo. Many studies have proposed that the mechanism of dietary fiber regulating asthma may involve the intestinal microbiome producing SCFAs, but the current research shows that a high-cellulose diet cannot increase the content of SCFAs in the intestine. These data suggest that a high-cellulose diet decreases asthma symptoms by altering the composition of the intestinal microbiome, however, this mechanism is thought to be independent of SCFAs and may involve the regulation of lipid metabolism.

Introduction

Allergic asthma is a disease caused by allergens entering the respiratory tract and stimulating B lymphocytes to produce specific antibodies, mainly IgE, and stimulate mast cells, basophils, and other immune cells to release inflammatory mediators [1]. The main pathophysiological changes include chronic airway inflammation, airway hyper-responsiveness to various stimulus factors, namely Airway Hyper Reactivity (AHR) [2], infiltration of eosinophils and lymphocytes in the airway, degranulation of mast cells and goblet cell hyperplasia, the expression of Interleukin-4 (IL-4), IL-5, IL-13, Th2-dominated TNF, and the up-regulation of other cytokines [3].

Recently, many epidemiological studies have shown that the incidence of asthma is related to diet. A diet that is high in fat and low in fiber increases the risk of developing asthma [4]. Additionally, giving clinical asthma patients a diet high in fruits and vegetables can relieve their respiratory symptoms [5]. However, the specific mechanism of this beneficial clinical outcome is currently not understood.

Cellulose is an insoluble dietary fiber formed by the dehydration of many glucose molecules connected by β-1,4 glycosidic bonds. It is widely present in plant cell walls and is the most common dietary fiber in grains, vegetables, and fruits. Bacteria that can decompose cellulose are found in both the human [6] and mouse intestines [7]. Changes in cellulose intake can alter the composition of the intestinal microbiome of rats and the production of SCFAs [8], regulating the occurrence of intestinal inflammatory diseases by regulating lipid metabolism [9]. A low-cellulose diet has been reported to aggravate pathological symptoms in asthmatic mice [10]. Dietary cellulose can also reduce ozone-induced airway hyperreactivity [11].

Lipid metabolism can influence the polarization of immune cells and the development of inflammatory responses [12]. Abnormal lipid metabolism has been associated with the development of asthma [13, 14]. A high-fiber diet can reduce body weight and regulate dyslipidemia [15, 16] and is involved in the regulation of inflammation [17]. The hypothesis of microbial diet interaction [18] and related research [19] suggests that high-fiber diets can affect the occurrence and development of allergic diseases and other inflammatory diseases by changing the composition and metabolites of the intestinal microbiome. Most fiber-containing foods include approximately one-third of soluble fiber and two-thirds of insoluble fiber [20]. It is currently believed that most soluble dietary fiber is fermented and broken down by intestinal bacteria to produce short-chain fatty acids (SCFAs), which can participate in the initiation and development of asthma by reducing inflammation within the respiratory tract in asthmatic mice [21] and inhibit the migration of eosinophils [22]. However, the biological outcomes of dietary insoluble fibers such as cellulose are still unclear.

Therefore, to further explore the role of cellulose in the occurrence and development of asthma, we established a mouse model of asthma and intervened by giving high cellulose (30%) diet. After that, we determined the lung inflammation of mice by ELISA and H&E staining. The changes of intestinal microbiome and short-chain fatty acids in the feces of mice were detected. The results showed that the lung inflammation symptoms of asthmatic mice treated with high cellulose diet were significantly improved, and different dominant intestinal microbiome concerning lipid metabolism was produced, but this change may not be achieved by increasing the amount of short-chain fatty acids.

Materials and methods

Animals

A total of 30 SPF C57BL/6J mice, 8w, were provided by the Experimental Animal Center of Chongqing Medical University (SCXK-(Chongqing) 2018–0003, Chongqing, China). All experimental protocols were performed according to the Guide for the Care and Use of Laboratory Animals, and they were approved by the Institutional Ethics Committee of Chongqing Medical University. After one week of adaptive feeding under standard conditions, the 30 mice were divided into the normal group (N1-N10), asthma group (AS1-AS10), and high-fiber diet group (HF1-HF10). The normal and asthma groups were both fed with clean-grade feed and purified water, while the high-fiber group was fed with high-fiber feed (Jiangsu Xietong Pharmaceutical Bio-engineering Co., Ltd.) and purified water. All three groups were free to eat and drink. The feed of the normal group contained 5% cellulose and the high-fiber feed contained 30% cellulose. The increased percentage of cellulose was achieved by reducing the amount of corn starch. The formula of the feed can be found in the S1 File.

The asthma group and the high-fiber diet group were injected intraperitoneally with the mixed preparation of V-grade OVA (Sigma, USA) and Al(OH)3 (Alladin, CHINA), and stimulated by aerosol inhalation of grade II OVA (Sigma, USA) to prepare the mouse asthma model. On the 0th, 7th, and 14th days, mice were injected intraperitoneally with 0.2ml of the sensitizing solution, and on the 21st day, the challenge solution was inhaled by aerosol for 30 minutes each time for one week. The sensitization solution is a normal saline suspension with V-grade OVA 1mg/ml and Al (OH)3 200mg/ml. The stimulating fluid is a physiological saline solution of grade II OVA 10mg/ml. The normal group is not processed. All groups were sampled within 24h after the end of nebulization on the 28th day.

Determination of IgE, IL-4 levels, and H&E staining

Enzyme-linked immunosorbent assay (ELISA) kits were used to determine IgE and IL-4 levels (MultiSciences Biotech Co., Ltd. Hangzhou, China), and the kit instructions were; ‘take out the serum samples from the refrigerator and gradually return to room temperature, and then measure the serum IgE and IL-4 levels. After fixing the lung tissue with 10% formalin solution for 24 hours, it was embedded in paraffin, sectioned (3μm), and stained with HE. The infiltration of inflammatory cells in the lungs of mice was observed under a light microscope.

DNA extraction, PCR Amplification, and sequencing

The frozen tissue samples were thawed at room temperature and then homogenized via bead beating (FastPrep bead matrix E, MP Biomedicals, Santa Ana, CA, USA) with 500 μl of aseptic saline. Aseptic saline was added until a volume of 1000 μl was obtained, and bacterial DNA was extracted with a bacterial genomic DNA extraction kit as per the manufacturer’s instructions. After DNA concentration was determined, 1 μl of DNA was diluted to 100 ng/μl with ultra-pure water and then stored at −20°C.

Universal primers 338F/806R were synthesized to amplify the 16s rRNAV4–V5 region. The sequences of the forward primer and reversed primer were 5′-ACTCCTACGGGAGGCAGCAG-3′ and 5′-GGACTACHVGGGTWTCTAAT-3′, respectively. In brief, PCR consisted of 4 μl of 5× FastPfu Buffer, 2 μl of dNTPs (2.5 mM), 0.8 μl of forwarding primer (5μM), 0.8 μl of reverse primer (5 μM), 0.4 μl of FastPfu polymerase, 10 ng of template DNA, and ultra-pure water added to obtain a volume of 20 μl. The reaction conditions were as follows: 5 min at 95°C, 27 cycles of 30 s each at 95°C and 53°C and 45 s at 72°C, and 10 min at 72°C. The amplification products were analyzed through Illumina high-throughput sequencing (Illumina PE250). Finally, microbial diversity was analyzed based on the sequencing results.

Short chain fatty acids extraction and analysis

Place an approximately 50 mg sample of feces into a 2 ml grinding tube, add a steel ball, 450 μl of methanol, and 50 μl of internal standard (1000 μg/ml of 2-ethyl-butyric acid, methanol configuration), and grind it in a freezing grinder 50HZ 3 twice per minute. Then ultrasonic the sample in an ice-water bath for 30 minutes, stand at -20°C for 30 minutes, and centrifuge at 13000g for 15 minutes (4°C). Transfer the supernatant to a 1.5 ml centrifuge tube. Add 50mg of anhydrous sodium sulfate, vortex, centrifuge at 13000g for 15min (4°C), and take the supernatant solution on the machine for gas chromatographic analysis. The analytical instrument used is Agilent Technologies Inc. (CA, USA) 8890B-5977B GC/MSD GC/MSD. HP FFAP capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA, USA). The protocol used included a carrier gas that is high-purity helium (purity not less than 99.999%), the flow rate is 1.0 ml/min, the inlet temperature is 260°C, an injection volume of 1μl, split injection, split ratio 10:1, and solvent extension 3min. Program temperature rise: the initial temperature of the column oven is 80°C, the temperature is programmed to increase to 120°C at 40°C/min increments, and then increased to 200°C at 10°C/min steps, and finally runs at 230°C for 6 minutes. Mass spectra were collected using an electron impact ion source (EI), ion source temperature 230°C, quadrupole temperature 150°C, transmission line temperature 230°C, and electron energy 70eV. The scanning mode is the full scan mode (SCAN), and the quality scan range: m/z: 30–300. The obtained data were assessed using Masshunter quantitative software (Agilent, USA, version number: v10.0.707.0) to automatically identify and integrate each ion fragment with default parameters, and assist manual inspection. Linear regression standard curve lines were drawn with the mass spectrum peak area of the analyte as the ordinate and the concentration of the analyte as the abscissa. Sample concentrations were calculated by substituting the mass spectrum peak area of the sample analyte into the linear equation to calculate the concentration results.

Statistical analysis

The Prism software (GraphPad) was used for statistical analysis of the data. The data of cytokine were statistically analyzed by one-way ANOVA, and for the microbiome data, the data between different groups were detected by Student`s-T test method and Tukey method. (*, P <0.05; **, P <0 .01; ***, P <0 .001).

Results

High-fiber diet can reduce lung inflammation and the production of IL-4 and IgE

Pathological sections of the lung showed that the walls and smooth muscles of the small bronchioles in group N were thin, with no foreign bodies in the cavities and alveoli. There was no obvious inflammatory cell infiltration around the bronchial tube or the blood vessel walls. In the AS group, the walls of small and medium bronchioles and blood vessels were thickened, smooth muscles were proliferated, and showed hypertrophy and the lumen was more narrow. Many eosinophils and lymphocytes were infiltrated around the bronchi and blood vessels. The lung interstitium and alveolar cavity are filled with exudate, and the alveolar space is widened. Compared with the AS group, the HF group had a relatively complete airway epithelial structure, and the degree of inflammatory cell infiltration around the bronchus and blood vessels was significantly reduced. Exudates in the lung interstitium and alveolar cavity are also significantly reduced (see Fig 1).

Fig 1

High-fiber diet can reduce lung inflammation and related cytokine production.

A: The pulmonary inflammation in the AS group was significantly worse than that in the N group, but the situation was alleviated in the HF group. B: The IL-4 level in the AS group was significantly higher than that in the N group (P<0.05), while the IL-4 level in the HF group was significantly reduced (P<0.01). C: The IgE level in the AS group was higher than that in the N group (P<0.05), while the IL-4 level in the HF group was significantly reduced (P<0.05).

The levels of IL-4 and IgE in the AS group were statistically significantly higher than those in the N group (P<0.05, P<0.05), but the levels of IL-4 and IgE in the HF group were significantly lower than those in the AS group (P<0.01, 0<0.05).

Microbial diversity analysis

Alpha-diversity

The student`s T-Test analysis was applied to bacterial DNA and genomic data. The Shannon index, Simpson index, Ace index, Chao index, and Coverage index of each sample are shown in Table 1. The results showed that the Shannon index of the AS and HF groups were significantly different (P<0.05), however no difference in the Simpson indexes for each group was found. This indicates that there is a difference in community diversity between the AS and HF groups, while no difference between the N group and the other two groups was found. Both the Ace index and Chao indexes yielded significant differences between the N and HF groups (P <0.05, P <0.05), and between the AS and HF groups (P <0.001, P <0.001). These results confirm that there are significant differences in community richness between the HF group and those on lower nonsoluble fiber diets.

Table 1

The Alpha diversity analysis of microbiome in feces.

	Nmean±sd	ASmean±sd	HFmean±sd	PAS-HF	PHF-N	PAS-N
SHANNON	3.7070±0.5590	3.7659±0.3632	3.2486±0.5736	0.0265	0.0860	0.7820
SIMPSON	0.0837±0.0649	0.0746±0.0279	0.1037±0.0695	0.2338	0.5123	0.6883
ACE	473.1872±74.7466	528.2429±77.3340	379.1904±78.4053	0.0001	0.0033	0.1033
CHAO	482.7851±84.5991	533.6014±80.4786	374.7840±66.6396	0.0001	0.0065	0.0846
COVERAGE	0.9979±0.0004	0.9978±0.0004	0.99855±0.0003

Community-composition and beta-diversity

Community barplot analysis showed that on the family level (see Fig 2), the dominant families in the AS group were Prevotellaceae, Lachnospiraceae, and Muribaculaceae; the dominant families in the HF group were Lachnospiraceae, Peptostreptococcaceae, and Desulfovibrionaceae and the dominant families in the N group were Muribaculaceae, Prevotellaceae, and Bacteroidaceae; The abundance of Peptostreptococcaceae in the HF group increased significantly, resulting in the emergence of a newly dominant family of bacteria.

Fig 2

Fecal microbial composition analysis and beta-diversity analysis.

A, B: analysis of the fecal microbial composition of the three groups on the family level and genus level; B, C: PCA analysis is used to explore the correlation of the composition of the microbiome on the family level and genus level of each group; C, D: ANOSIM analysis compares differences between groups and within groups (C:P <0.001, D: P<0.001).

At the Genus level (Fig 2B), compared with the N group, the proportion of norank_f__Muribaculaceae in the AS group decreased, while the proportion in the HF group decreased significantly. Again, compared to the N group, the proportion of Lactobacillus and Lachnospiraceae_NK4A136_group in the AS group slightly increased, while the proportion in the HF group decreased. Additionally, some uniquebacterial groups emerged in the HF group, such as Romboutsla, and the [Ruminococcus]_torques_group.

PCA analysis and ANOSIM methods were used to analyze bacterial populations at the family and genus levels, respectively. The PCA analysis showed that at the family level, the composition of the microbiome of the N group and the AS group overlapped, but the composition of the HF microbiome was significantly different (Fig 2C). The results of PC1 and PC2 were 54.98% and 19.49%. At the genus level, the N group and AS group also showed an inevitable overlap, while the HF group has a unique microbiome composition (Fig 2D). The data for PC1 and PC2 are 41.7% and 21.18%. ANOSIM results showed that at the family level (Fig 2E), the difference between groups was more significant than the differences within the groups (Weighted_Unifrac R = 0.7145, P<0.001). At the genus level (Fig 2F), the difference between the groups was also more significant than the difference within the groups (Weighted_Unifrac R = 0.7271, P<0.001)). These data show that three groups have statistically significant differences, regardless of whether the analysis is at the family level or the genus level.

Analysis of significant differences between the groups at both the family and genus levels

As Fig 3 shows, at the family level, there are significant differences between the three groups in populations of Lachnospiraceae, Muribaculaceae, Prevotellaceae, Peptostreptococcaceae, Rikenellaceae, and Lactobacillaceae families. At the genus level, populations of norank_f__Muribaculaceae, Prevotellaceae_UCG-001, Romboutsia, [Ruminococcus]_torques_group, Helicobacter, and norank_f__Desulfovibrionaceae are significantly different in the three groups (P<0.001).

Fig 3

Significantly different species analysis on the family level and genus level.

A: Analysis of significantly different species at the family level; B: Analysis of significantly different species at the genus level (*, P < 0.05; **, P <0 .01; ***, P <0 .001).

The Kruskal-Wallis test was then applied to conduct a multi-group analysis between Romboutsia, [Ruminococcus]_torques_group, and Helicobacter, which had noticeable compositional changes in the HF group at the genus level. Romboutsia is significantly different between the HF and AS groups, and the HF and N groups (P<0.01 and P<0.01 respectively), while [Ruminococcus]_torques_group shows significant differences between the HF and AS groups, and HF and N groups (P<0.001 and P<0.001 respectively). Finally, the composition of Helicobacter bacteria is also significantly different between the HF group and AS groups and between the HF and N groups (P<0.01 and P<0.05 respectively).

Short-chain fatty acid (SCFAs) analysis

One proposed mechanism that the intestinal microbiome affects human health and disease is through the production of short-chain fatty acids in the intestine. We used GC-MS to detect the SCFAs content in mouse feces (Fig ). Surprisingly, the contents of various SCFAs in feces from the HF group were lower than the other two groups. To analyze the correlation between the distribution of short-chain fatty acids and the intestinal microbiome, we performed a Variance inflation factor (VIF) analysis. The results showed that at the genus level, the distribution of the bacterial microbiome showed a strong positive correlation with acetic acid, isobutyric acid, and butyric acid, and a weak correlation with concentrations of isohexanoic acid, but no significant correlation with hexanoic acid.

Analysis of short-chain fatty acid content in feces.

A-H: Quantitative analysis of short-chain fatty acids in mouse feces.

Discussion

Cellulose is the most common insoluble dietary fiber. In the current study, feeds containing different concentrations of cellulose were fed to asthmatic mice to study the effect of a high-fiber diet on lung inflammation in asthmatic mouse models. Also, the influence that varying amounts of dietary cellulose had on the microbiome of the mice intestinal tracts was delineated and analyzed.

The hypothesis of microbial diet interactions [18] proposes that cellulose may participate in the progression of allergic diseases by affecting the composition of the intestinal microbiome, which is closely related to pulmonary inflammation. Disrupting the composition of the intestinal microbiome with antibiotics can intensify the reaction of Th2 cells [21, 23], thereby intensifying the production of lung inflammation. Alterations in intestinal bacteria caused by the administration of antibiotics during the perinatal period have been reported to increase susceptibility to developing asthma in the future [24]. The data presented from the current study shows that a high-cellulose diet reduces the total OUT (Operational Taxonomic Units) number of intestinal bacteria, while also producing more unique OTU sequences. In contrast, the HF group is lower than the AS group in community richness and community diversity. This is consistent with Kim Y’s research results [9], however, contrasts with the conclusions from Peng X et al. study [8].

A principal mechanism that the intestinal microbiome affects remote organs is through the metabolism of SCFA’s [25, 26]. Soluble dietary fiber can produce large quantities of SCFA’s which then participate in the progression of inflammation and allergic diseases. However, our study has shown that insoluble cellulose cannot be fermented by intestinal bacteria to produce larger amounts of SCFAs. The concentrations of almost all SCFAs were significantly reduced (except for hexanoic acid). Similarly, Kim et al. (2020) showed that increased dietary cellulose intake did not result in changes to the intestinal acetic acid, propionic acid, and butyric acid contents.

Romboutsla and [Ruminococcus]_torques_group merits special attention. They are a unique microbiome induced by a high-fiber diet, present only in very small quantities in the N and AS groups. Romboutsla is classified as a member of the Peptostreptococcaceae family [27]. These bacteria primarily produce both acetic and lactic acids but also produce C16:0 saturated straight-chained fatty acids. It does not, however, use cellulose as a fermentation substrate. [Ruminococcus]_torques_group is classified as being in the Lachnospiraceae family [28], and its main fermentation products are acetic acid, propionic acid, and lactic acid. It is involved in the regulation of GPR41 stimulated by SCFAs, which induces adipocytes to produce leptin and influences the lipid profile. The Lachnospiraceae family is also significantly increased in the HF group and these bacteria activate the oxidation and new synthesis of fatty acids, inhibits lipid decomposition, thereby reducing circulating plasma lipid levels and body weight [29]. The data from Kim et al. (2020) showed that the intake of antibiotics can reduce the beneficial effects of cellulose on colitis. This study also demonstrated that cellulose regulates colon inflammation by regulating the metabolic spectrum of intestinal bacteria. The linoleic acid, nicotinate and nicotinamide, glycerophospholipid, glutathione, and sphingolipid metabolic pathways were all significantly modified by cellulose intake [9]. Tibbitt [30] performed single-cell RNA sequencing on TH2 cells in the airway and found that fatty acid oxidation and synthesis genes are highly enriched in TH2 cells. Simultaneously, blocking lipid metabolism pathways in vivo can inhibit T helper cell differentiation and the development of airway inflammation. These findings all support the proposal that the regulation of cellulose on the intestinal microbiome may not be through fermentation pathways that produce increased concentrations of SCFA’s but through the regulation of lipid metabolism and other metabolic pathways. Importantly, this study has demonstrated that the mechanisms and outcomes that increased dietary insoluble fiber exert on the gut microbiome and overall inflammatory processes require further exploration and characterization.

Presently, most research investigating the effects of cellulose on inflammation focuses on intestinal inflammation. Nagy-Szakal et al. (2013) showed that supplementing cellulose in early life can alleviate the symptoms of colitis in mice [31]. This early study also demonstrated changes in intestinal bacteria but did not fully characterize or explain these results. Fischer et al. (2020) reported that cellulose may participate in the occurrence of colitis by regulating the function of intestinal immune and epithelial cells [7]. Whilst investigating cellulose and respiratory diseases, Zhang et al. (2016) reported that the intake of cellulose can improve OVA-induced pulmonary inflammation [10], focusing primarily on changes in cytokines in the alveolar lavage fluid. Tashiro et al. (2020) reported that cellulose can improve ozone-induced airway hyperresponsiveness, which is one of the main pathological features of asthma [11].

Generally, this research showed that a high dietary intake of insoluble cellulose was able to reduce lung inflammation in asthmatic mice. This outcome may not be induced by increased intestinal SCFAs, but rather by regulating the composition of the intestinal flora to regulate the body’s lipid metabolism, thereby participating in the inflammatory response of remote organs.

Formula of high cellulose feed.

(DOCX)

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Formula of regular feed.

(DOCX)

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(RAR)

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19 May 2021

PONE-D-21-07750

High Cellulose dietary intake relieves asthma inflammation through the intestinal microbiome in a mouse model

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Reviewer #1: Overall, the paper is methodologically sound, the statistical analyses that are presented seem adequate, and the interpretations reasonable. For some reason, messages such as "(see Fig Error! Reference source not found.)" are scattered throughout the text, please edit. Were the mice used male,female, both? If only male or female, why was one sex chosen, and how may that affect results? Why was the number of mice chosen used? Was a power analysis or other statistical analysis done to ensure proper numbers of mice?

Reviewer #2: The paper “High Cellulose dietary intake relieves asthma inflammation through the intestinal microbiome in a mouse model”, by Song Wen and colleagues, aimed to evaluate the role of a high-cellulose diet in a mouse model of asthma, detecting pathological manifestations in lungs, changes in the gut microbiome, and changes in intestinal SCFA in a murine model.

The paper is well written and the results clear to the reader.

Here you find my suggestions for this paper:

• Table 1: I think that the readability of this table could be increased as follow: mean and SD could be put together (mean�  SD) and you should add the p-value. Moreover, you can add a legend for this table.

• Lines 165-167: you could also provide data for differences between N and HF.

• Line 48: the term “asth ation” is unclear. Please amend it.

• Many times (i.e. line 164, 181, 193, 206, etc.), in the paper there is reported the following “Error! Reference source not found”, in correspondence to references to Figures and Tables citations. Please amend it.

Reviewer #3: SEE THE FOLLOWING COMMENTS BELOW

Introduction:

a. Line 48, the reviewer is not familiar with asth ation, can the authors clarify what this word is and its meaning. Perhaps it is a typo.

b. The introduction provides information about some background, but it is jumbled and would benefit from a reorganization and rewrite. Further there does not seem to be an overt purpose or hypothesis established for the study.

Methods:

c. The mice need a more complete description and location of the vendor purchased from. Microbiome/microbiota can vary greatly between vendors and thus influence results of the study.

d. Given the perceived purpose is to examine the influence of fiber and asthma, the experimental design seems incomplete, there is no asthma-fiber group. Only normal, asthma and high fiber.

e. There does not appear to the tables for dietary composition. How were diets altered to account for the high cellulose content? Protein, fat? There was a lot of time spent on the importance of fat in the introduction. This would be critical for understanding interpretation of the results.

f. If all animals were challenged with OVA, then should the groups be renamed?

g. The authors do not appear to have a scoring outline in place to understand how the IHC data was analyzed to note differences in staining.

h. The authors have 16S rDNA in the V4-V5 regions, this should be rRNA. While one must extract the DNA it then must be prepared for rRNA analysis.

i. SCFA in animal models are analyzed using cecal contents so that fecal pellets can be used for microbiota.

j. The statistical analysis is very vague and does not employ appropriate techniques to analyze m data of this fashion.

Based on these concerns any results that followed would be impossible to interpret given the methodological fatal flaws.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

2 Jun 2021

Dear Editor and Reviewers:

Thank you for your letter dated May 19. We were pleased to know that our work was rated as potentially acceptable for publication in Journal, subject to adequate revision. We thank the reviewers for the time and effort that they have put into reviewing the previous version of our manuscript entitled “High Cellulose dietary intake relieves asthma inflammation through the intestinal microbiome in a mouse model”. Their suggestions have enabled us to improve our work. Based on the instructions provided in your letter, we uploaded the file of the revised manuscript. Accordingly, we have uploaded a copy of the original manuscript with all the changes highlighted by using the track changes mode in MS Word. Appended to this letter is our point-by-point response to the comments raised by the reviewers. The comments are reproduced and our responses are given directly afterward in a different color (red). We would like also to thank you for allowing us to resubmit a revised copy of the manuscript.

Responds to the reviewers' comments:

Reviewer #1:

a: Response to comments: Messages such as "(see Fig Error! Reference source not found.)" are scattered throughout the text, please edit.

Response: Thanks for your comment. We apologize for the incorrect writing errors in the manuscript, and we have corrected some of the spelling and formatting errors that have already been mentioned.

b: Response to comments: Were the mice used male, female, both? If only male or female, why was one sex chosen, and how may that affect results?

Response: Thanks for your comment. All the mice used in our experiments are male mice. The reason for this decision is that many studies have pointed out the influence of gender differences on the intestinal microbiome and our focus of research is not to explore the impact of gender differences on the results of the experiment. Therefore, to rule out the possible influence of sex difference, we all selected male mice as experimental subjects.

c: Response to comments : Why was the number of mice chosen used? Was a power analysis or other statistical analysis done to ensure proper numbers of mice?

Response: Thanks for your comment. For the number of mice, we did not do a power analysis to determine it before the experiment. However, we fully considered that only enough samples can reflect the overall level of change, and considering the ability of our laboratory staff, we finally chose the scheme of 10 mice in each group, a total of 30 mice.

Special thanks to you for your good comments.

Reviewer #2:

a: Response to comments: Table 1: I think that the readability of this table could be increased as follow: mean and SD could be put together (mean�  SD) and you should add the p-value. Moreover, you can add a legend for this table.

Response: Thank you for your advice on our Table, which makes it easier to read and clearer. We revised the expression of mean and SD and added P-values between the groups in the revised manuscript.

b: Response to comments: Lines 165-167: you could also provide data for differences between N and HF.

Response: Thanks for your suggestion. We added the P-value between groups at the end of the sentence.

c: Response to comments: Line 48: the term “asth ation” is unclear. Please amend it.

Many times (i.e. line 164, 181, 193, 206, etc.), in the paper there is reported the following “Error! Reference source not found”, in correspondence to references to Figures and Tables citations. Please amend it.

Response: Thanks for your comment. We apologize for the spelling and formatting errors in the original version, all of which have been corrected in the new version.

Special thanks to you for your good comments.

Reviewer #3:

a: Response to comments: Line 48, the reviewer is not familiar with asth ation, can the authors clarify what this word is and its meaning. Perhaps it is a typo.

Response: We apologize for the spelling and formatting errors in the original version, all of which have been corrected in the new version.

b: Response to comments: The introduction provides information about some background, but it is jumbled and would benefit from a reorganization and rewrite. Further there does not seem to be an overt purpose or hypothesis established for the study.

Response: Thank you for your comments. We rearrange the paragraphs in introduction, and describe the purpose and results of our experiment.

c: Response to comments: The mice need a more complete description and location of the vendor purchased from. Microbiome/microbiota can vary greatly between vendors and thus influence results of the study.

Response: Thank you for your comments. We clarified the mice strains and the location of the vendor in the revised manuscript. The laboratory animal center of Chongqing Medical university is the experimental animal center of Chongqing National Biological Industry Base and the experimental animal service platform of National Innovative Drug Incubation (Chongqing) Base. By the Chongqing Laboratory Animal Quality Inspection Center and the Chongqing Science and Technology Commission, the center have obtained the production and animal use license of SPF-grade rats and mice, and the animal use license of rabbits, guinea pigs, pigs, dogs, sheep, monkeys, and birds, etc. The quality of experimental animals can be fully guaranteed.

d: Response to comments: Given the perceived purpose is to examine the influence of fiber and asthma, the experimental design seems incomplete, there is no asthma-fiber group. Only normal, asthma and high fiber.

Response: Thank you for your comments. We mentioned the grouping of animals in line 91-92, “the 30 mice were divided into the normal group (N1-N10), asthma group (AS1-AS10), and high-fiber diet group (HF1-HF10).”, and the establishment of models in different groups in line 99-100, “All animals except normal mice were sensitized and challenged with ovalbumin (OVA) to induce airway allergic inflammation.” The HF group was established by high fiber diet intervention in the establishment period of the asthma model.

e: Response to comments: There does not appear to the tables for dietary composition. How were diets altered to account for the high cellulose content? Protein, fat? There was a lot of time spent on the importance of fat in the introduction. This would be critical for understanding interpretation of the results.

Response: Thank you for your suggestion. For the explanation of the feed formula, we put it in lines 96-97, but it lacks the explanation of other important ingredients in feed. Therefore, we decided to upload the complete formula of feed to the journal as a supplement material in the revised manuscript.

f: Response to comments: If all animals were challenged with OVA, then should the groups be renamed?

Response: Thanks for your comment. The grouping and modeling have been explained before. In order to make it easier for readers to understand different groups, we named the normal group N group, the asthma group AS group, and the asthma group with high fiber diet intervention, and we named it HF group based on the main intervention measures.

g: Response to comments: The authors do not appear to have a scoring outline in place to understand how the IHC data was analyzed to note differences in staining.

Response: Thanks for your comment. In order to determine the infiltration of inflammatory cells in the lungs of mice, we used H&E staining instead of IHC, and then observed the morphology and structure of bronchioles and the changes of infiltration of inflammatory cells around them under light microscope.

h: Response to comments: The authors have 16S rDNA in the V4-V5 regions, this should be rRNA. While one must extract the DNA it then must be prepared for rRNA analysis.

Response: Thanks for your comment. We corrected the inaccurate expression in the method section

i: Response to comments: SCFA in animal models are analyzed using cecal contents so that fecal pellets can be used for microbiota.

Response: Thanks for your comment. We know that SCFAs are mainly absorbed in the cecum and enter the blood circulation. The content of cecum can reflect the changes of SCFAs in mice more directly. In our experiment, considering that SCFAs may be a potential biomarker in human body, and the most convenient way to detect SCFAs in human body is to detect the content of SCFAs in fresh feces. So in order to simulate this situation, we collected fresh feces of mice in the experiment, and immediately put them into the refrigerator at - 80 ℃ for storage, to minimize the possible volatilization of SCFAs.

j: Response to comments: The statistical analysis is very vague and does not employ appropriate techniques to analyze m data of this fashion.

Response: Thanks for your comment. We apologize for the vague expression in the data analysis section. In the revised manuscript, we redescribed different data analysis methods for different experimental contents.

Special thanks to you for your good comments.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

7 Jul 2021

PONE-D-21-07750R1

High Cellulose dietary intake relieves asthma inflammation through the intestinal microbiome in a mouse model

PLOS ONE

Dear Dr. Li,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

More specifically, small changes are still needed.

Please submit your revised manuscript by the 2nd of August 2021. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see:  http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at  https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Catherine Mounier

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #4: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #4: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #4: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #4: There are many small problems in the article which need to be carefully revised by the author.Authors should check their manuscripts carefully before submitting them

(1)The author still hasn't corrected the "See Fig Error!".

(2)The methods are not very detailed, such as how long it takes to build the mouse model and how long the mice will be intervening.

(3)The picture is very blurry.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #4: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

12 Jul 2021

Responds to the reviewer`s comments:

Reviewer #4: There are many small problems in the article which need to be carefully revised by the author. Authors should check their manuscripts carefully before submitting them

(1)The author still hasn't corrected the "See Fig Error!".

Response: Thanks for your comment. We apologize for the errors in the manuscript, and we have corrected the formatting errors that have already been mentioned.

(2)The methods are not very detailed, such as how long it takes to build the mouse model and how long the mice will be intervening.

Response: Thanks for your comment. We added a detailed description of the steps to establish the mouse model in the method section.

(3)The picture is very blurry.

Response: Thanks for your comment. We replaced the unclear figures and adjusted them by Preflight Analysis and Conversion Engine (PACE).

Special thanks to you for your good comments.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

25 Nov 2021

15 Dec 2021

Based on the instructions provided in your letter, we replaced all the figure files of the manuscript. We sincerely hope that the images uploaded this time will meet the requirements for publication in your magazine.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

27 Jan 2022

High Cellulose dietary intake relieves asthma inflammation through the intestinal microbiome in a mouse model

PONE-D-21-07750R3

Dear Dr. Li,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Catherine Mounier

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

1 Mar 2022

PONE-D-21-07750R3

High Cellulose dietary intake relieves asthma inflammation through the intestinal microbiome in a mouse model

Dear Dr. Li:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Catherine Mounier

Academic Editor

PLOS ONE