Intestinal microbiome dysbiosis increases Mycobacteria pulmonary colonization in mice by regulating the Nos2-associated pathways

Reviewer #1 (Public Review):

Summary:

This work sought to demonstrate that gut microbiota dysbiosis may promote the colonization of mycobacteria, and they tried to prove that Nos2 down-regulation was a key mediator of such gut-lung pathogenesis transition.

Strengths:

They did large-scale analysis of RNAs in lungs to analyze the gene expression of mice upon gut dysbiosis in MS-infected mice. This might help provide an overview of gene pathways and critical genes for lung pathology in gut dysbiosis. This data is somewhat useful and important for the TB field.

Weaknesses:

(1) They did not use wide-type Mtb strain (e.g. H37Rv) to develop mouse TB infection models, and this may lead to the failure of the establishment of TB granuloma and other TB pathology icons.

(2) The usage of in vitro assays based on A542 to examine the regulation function of Nos2 expression on NO and ROS may not be enough. A542 is not the primary Mtb infection target in the lungs.

(3) They did not examine the lung pathology upon gut dysbiosis to examine the true significance of increased colonization of Mtb.

(4) Most of the studies are based on MS-infected mouse models with a lack of clinical significance.

Reviewer #2 (Public Review):

The manuscript entitled "Intestinal microbiome dysbiosis increases Mycobacteria pulmonary colonization in mice by regulating the Nos2-associated pathways" by Han et al reported that using clindamycin, an antibiotic to selectively disorder anaerobic Bacteriodetes, intestinal microbiome dysbiosis resulted in Mycobacterium smegmatis (MS) colonization in the mice lungs. The authors found that clindamycin induced damage of the enterocytes and gut permeability and also enhanced the fermentation of cecum contents, which finally increased MS colonization in the mice's lungs. The study showed that gut microbiota dysbiosis up-regulated the Nos2 gene-associated pathways, leading to increased nitric oxide (NO) levels and decreased reactive oxygen species (ROS) and β-defensin 1 (Defb1) levels. These changes in the host's immune response created an antimicrobial and anti-inflammatory environment that favored MS colonization in the lungs. The findings suggest that gut microbiota dysbiosis can modulate the host's immune response and increase susceptibility to pulmonary infections by altering the expression of key genes and pathways involved in innate immunity. The authors reasonably provided experimental data and subsequent gene profiles to support their conclusion. Although the overall outcomes are convincing, there are several issues that need to be addressed:

(1) In Figure S1, the reviewer suggests checking the image sizes of the pathological sections of intestinal tissue from the control group and the CL-treatment group. When compared to the same intestinal tissue images in Figure S4, they do not appear to be consistently magnified at 40x. The numerical scale bars should be presented instead of just magnification such as "40x".

(2) In Figure 4d, the ratio of Firmicutes in the CL-FMT group decreased compared to the CON-FMT group, whereas the CL-treatment group showed an increase in Firmicutes compared to the Control group in Figure 3b. The author should explain this discrepancy and discuss its potential implications on the study's findings.

(3) In Figure 6, did the authors have a specific reason for selecting Nos2 but not Tnf for further investigation? The expression level of the Tnf gene appears to be the most significant in both RT-qPCR and RNA-sequencing results in Figure 5f. Tnf is an important cytokine involved in immune responses to bacterial infections, so it is also a factor that can influence NO, ROS, and Defb1 levels.