Lysates of Methylococcus capsulatus Bath induce a lean-like microbiota, intestinal FoxP3+RORγt+IL-17+ Tregs and improve metabolism

Interactions between host and gut microbial communities are modulated by diets and play pivotal roles in immunological homeostasis and health. We show that exchanging the protein source in a high fat, high sugar, westernized diet from casein to whole-cell lysates of the non-commensal bacterium Methylococcus capsulatus Bath is sufficient to reverse western diet-induced changes in the gut microbiota to a state resembling that of lean, low fat diet-fed mice, both under mild thermal stress (T22 °C) and at thermoneutrality (T30 °C). Concomitant with microbiota changes, mice fed the Methylococcus-based western diet exhibit improved glucose regulation, reduced body and liver fat, and diminished hepatic immune infiltration. Intake of the Methylococcu-based diet markedly boosts Parabacteroides abundances in a manner depending on adaptive immunity, and upregulates triple positive (Foxp3+RORγt+IL-17+) regulatory T cells in the small and large intestine. Collectively, these data point to the potential for leveraging the use of McB lysates to improve immunometabolic homeostasis.


Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging 16S sequencing data is uploaded to a public repository with study accession number: PRJEB41983 and available at the following link: http://www.ebi.ac.uk/ena/ data/view/PRJEB41983. All non-omics datasets included in the current study are available in the figshare repository Jensen, B. A. H. et al. Data underlying the manuscript: Lysates of Methylococcus capsulatus Bath induce a lean-like microbiota, intestinal FoxP3+ROR"t+IL-17+ Tregs and improve metabolism. figshare https://doi.org/10. 6084/m9.figshare.13522283 (2021). Code availability: Standard codes from the cited R-packages were used in this manuscript. Specific scripts are available upon reasonable request We did not perform specific power calculations in this study, but instead used a sufficiently large n-size as per experience. CMT studies were limited by available amount of cecal content to transfer. The n-size was thus restricted accordingly.
Three WT mice (two WD-REF-fed and 1 WD-MvcB-fed) were excluded for immunological assessment by flow cytometry in the third experiment on hepatic and gastrointestinal immune profiling ( Figure 2O-R, 5I-K, S4C-F, S5J-K), due to visual wounds on their legs and/or back. These wounds were a result of hierarchical dominance within the cages and only evident for the last few days of the experiment for which reason, they were included in all other analysis. One LFD-fed mouse were excluded from the hepatic immune assessment (also by flow cytometry), due to mishandling of the single cell suspension. The following samples were excluded and/or missing from insulin analyzes during oral glucose tolerance test: one WD-REF fed RAG2 mouse were excluded at T15 by pre-established outlier criteria (~10-fold increased compared to all other mice at T15) and one WD-McB fed mouse were excluded at T60 due to hemolysis (pre-established exclusion criteria). Lastly, several T0 samples from the CMT experiments were below LOD, just as some time points were missing sample material, as indicted in the figure legends and also evident from the raw data deposited at figshare (link in the data availability section).
As specified throughout the manuscript, the general findings reported here has been replicated in several mouse strains, at different experimental duration and at different temperatures. All replication attempts were successfully achieved. Data supporting the main conclusion has been replicated at least twice. All data from such replication studies are explicitly shown and discussed. When applicable, data from replication studies are represented in the same bar plots, where the shape of each dote are experiment-dependent. This was deliberately done to corroborate and visualize the extraordinary level of reproducibility in the reported phenotype.
For all therapeutic intervention studies, mice were stratified into treatment groups based on their 'baseline' body weight and glucoregulatory capacity, as described in the manuscript. For all prophylactic experiments, mice were randomly assigned to experimental groups.
All mice were assigned a random number as mouse ID. Staff handling the mice and/or performing specific analyzes only knew this ID and were thus blinded for group ID during experimental handling (e.g. OGTT and GSIS) and data processing (e.g. SCFAs, FLow cytometry etc.). Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.
This study did not involve wild animals.
This study did not involve samples collected from the field.
Ex vivo stimulation of SI-LP, LI-LP and liver cells SI-LP, LI-LP and liver cells were re-stimulated in vitro as previously described27. Briefly, cells were simulated in R-10 media in the presence of either 20 ng/mL IL-23 (R&D Systems) or 250 ng/mL PMA (Sigma-Aldrich) in combination with 0.5 µg/mL ionomycin (Sigma-Aldrich) for 4 hrs at 37 !C. After 1 hr incubation, 10 µg/mL brefeldin A (BioLegend) was added.
Flow cytometry Flow cytometry was performed according to standard procedures. Cell aggregates (identified in FSC-H or FSC-W vs. FSC-A scatter plots) and dead cells identified by using Zombie Aqua or Zombie UV Fixable Viability Kit (BioLegend) were excluded from analyses. Intracellular staining was performed using the eBioscience FoxP3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer's instructions.
Data was acquired on a LSRII (BD Biosciences).