Porphyromonas gingivalis Strain W83 Infection Induces Liver Injury in Experimental Alcohol-Associated Liver Disease (ALD) in Mice

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. If the authors would like to assess the effects of porphyromonas gingivalis strain on ALD in mice, germ-free mouse should be used. 

2. Porphyromonas gingivalis strain should be oral administration to mice. Intraperitoneal injection would induce severe inflammation in mice. Therefore, your obsereved results might attributed to inlammatation but not porphyromonas gingivalis strain itself. 

Comments on the Quality of English Language

Moderate editing of English language required.

Author Response

Reviewer 1.

Q1.  If the authors would like to assess the effects of porphyromonas gingivalis strain on ALD in mice, germ-free mouse should be used. 

Thank you for your suggestion. Germ free mice are of choices to dissect the role of P. gingivalis on gut microbiota change-related regulation.  We will include several gut microbiota related methods, such as germ free (your suggestion), antibiotic treatment and fecal microbiota transplant in the future study.

 

Q2. Porphyromonas gingivalis strain should be oral administration to mice. Intraperitoneal injection would induce severe inflammation in mice. Therefore, your obsereved results might attributed to inlammatation but not porphyromonas gingivalis strain itself. 

Yes, P. gingivalis I.P. injection causes severe inflammation, but with P. gingivalis bacteremia as well. P. gingivalis could reach organs through transient bacteremia after oral procedures in patients with periodontal disease, which could be mimicked by I.P. injection (PMID: 16677328). Our data showed that liver P. gingivalis bacteria (at least segment of Pg) occurred in the liver, which were more in the mice treated with alcohol. 

Reviewer 2 Report

Comments and Suggestions for Authors

1.   Literature showed that pair-feeding of control animals with the liquid diet can also affect the immune system, this paper showed that Pg can affect immune response, so additional control group should be used.

2.   Authors checked ALT and AST, how about alkaline phosphatase, serum albumin and total bilirubin?

3.   Authors should check the mRNA level of MCP-1, CXC chemokines (such as CXCL8, CXCL9), TLR4, and TRIF.

4.   Authors need to do the quantification for the Fig 2I.

5.   In Fig 3I, the western blot band looks not good. Authors should modify the condition.

6.   Authors should do the quantification for the TUNEL staining.

7.   Authors mentioned that the mRNA level of lipogenic gene, how about the protein level?

Comments on the Quality of English Language

Minor editing of English language required

Author Response

Reviewer 2.

Q1.  Literature showed that pair-feeding of control animals with the liquid diet can also affect the immune system, this paper showed that Pg can affect immune response, so additional control group should be used.

Thank you for the suggestion. The purpose of this study was to demonstrate whether P. gingivalis infection exacerbates the alcohol-associated liver disease. We have 4 groups of mice under pair-feeding, alcohol-feeding and P. gingivalis challenging of each. We will add additional groups in the future study to dissect the role of P. gingivalis more precisely in immune response.

Q2.  Authors checked ALT and AST, how about alkaline phosphatase, serum albumin and total bilirubin?

We did not check ALP, albumin and Bilirubin, since we believe ALT and AST levels are sufficient to show the hepatocyte injury. We could measure ALP and bilirubin etc. (more for cholangiacyte markers) in the future study for better understanding other cell type injury such as cholangiacyte.

Q3. Authors should check the mRNA level of MCP-1, CXC chemokines (such as CXCL8, CXCL9), TLR4, and TRIF.

We do checked mRNA expression levels of MCP-1, CXCL8 (il8)and TLR4 etc, and we did not find they were significantly different between groups with and without P. gingivalis.

Q4. 4.   Authors need to do the quantification for the Fig 2I.

Yes, Fig 2I was quantified and added to Figure 2.

Q5.  In Fig 3I, the western blot band looks not good. Authors should modify the condition.

This is our original blot. Changes are significant. We quantified and added the quantification in the revision.

Q6. Authors should do the quantification for the TUNEL staining.

Yes, Fig 4G TUNEL image was quantified and added.

Q7. Authors mentioned that the mRNA level of lipogenic gene, how about the protein level?

We have not yet performed protein assay for lipogenic genes, but it could be done in future studies.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors focused on the role of Porphyromonas gingivalis strain W83 infection in inducing liver injury in experimental alcohol-associated liver disease (ALD) in mice

 Porphyromonas gingivalis is considered a major pathogen in adult periodontitis and is also associated with multiple systemic diseases, for example, cardiovascular diseases. One of its most important virulence factors is invasion of host cells.

 P. gingivalis accelerates the pathogenesis of ALD via the oral-gut-liver axis, necessitating a new treatment strategy for patients with ALD complicated by periodontitis.

Therefore, studying the specific mechanism and shedding light on the direct role of P. gingivalis in the progress of ALD  will provide a reference and basis for prevention and treatment of this condition.

Q1. It is helpful for a better understanding of the role of P. gingivalis to add to the manuscript the factors of virulence and pathogenicity of P.gingivalis , adding references  10.1016/j.jmb.2021.166836; 10.3390/pathogens11101173.

 

Q2. P.gingivalis is also closely related to systemic diseases including rheumatoid arthritis  cardiovascular disease  diabetes  and Alzheimer’s disease. The authors should provide evidence regarding these pathologies 10.1016/j.intimp.2023.109936; 10.3389/fimmu.2023.1103592; 10.1002/JLB.3MA0121-045R.

Comments on the Quality of English Language

Minor English Editing required

Author Response

Reviewer 3.

Q1.  It is helpful for a better understanding of the role of P. gingivalis to add to the manuscript the factors of virulence and pathogenicity of P.gingivalis , adding references  10.1016/j.jmb.2021.166836; 10.3390/pathogens11101173.

Thank you for the suggestion. We have added the references (PMID 33539891and 36297228) in the revision for better understanding the virulence of P. gingivalis.

Q2. P.gingivalis is also closely related to systemic diseases including rheumatoid arthritis  cardiovascular disease  diabetes  and Alzheimer’s disease. The authors should provide evidence regarding these pathologies 10.1016/j.intimp.2023.109936; 10.3389/fimmu.2023.1103592; 10.1002/JLB.3MA0121-045R.   

Thank you again. We have added the citations (PMID: 37098654, 3699040 and 34057740) which describe the association of P. gingivalis to systemic diseases such as diabetes, Alzheimer’s disease etc. 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Following the present methods provided by the authors, many variables affect the experimental results, which makes all results unrelieable. 

Comments on the Quality of English Language

No