Microbial Energy Metabolism Fuels a CSF2-dependent Intestinal Macrophage Niche within Tertiary Lymphoid Organs

Chiaranunt, P. et al. (BioRxiv) doi: 10.1101/2022.03.23.485563


  • Macrophages 

  • Microbiota 

  • Tertiary Lymphoid Organ (TLO)


Main Findings

Chiaranunt and collegues tried to address an important question that it has not been fully addressed so far: deciphering the host/microbiota signals that are affecting/controlling Macrophages development and plasticity. They showed that ILC3-enriched tertiary lymphoid organs (TLOs) are organized as intestinal niche for the development of monocyte-derived MPs. MPs’s heterogeneity and plasticity are skewed by ILC3 - derived colony stimulating factor 2 (CSF2). Further, MP turnover seems to be regulated by microbe-derived extracellular adenosine 5’-triphosphate (ATP). In conclusions, the authors, using Single cell RNA-sequencing, observed an unique transcriptomic profile of TLO-associated MPs critical for anti-microbial defense against enteric infection. 


​The authors extensively used different in-vivo and in-silico techniques to address their questions. The paper is quite easy to read, and their conclusions are overall supported by their data. Having said this, the manuscripts have some limitations that might be considered and possibly further addressed to improve the study.

  1. One of the biggest concerns is related to the ATP. Not only bacteria are producing ATP but also host cells, in particular epithelial cells. Indeed, in the GF mice (Fig. 3A) the level of ATP is not zero even in the absence of the microbiota. Thus, I’m wondering whether the phenotype that they are describing it’s coming from the bacteria that are producing ATP and then affecting the host or it is the host-derived ATP that it’s released upon bacterial colonization? In Fig 3, the authors show that an ATPex-deficient E. coli stain ( atpA-atpG) is impairing the accumulation of Ly6C+ MPs, CCR2+ MPs, ILC3 expansion and, consequently, CSF2-ILC3 production. One wonders whether this phenotype is directly driven by the absence of ATPex or it can be also due to the fact that a bacterium (especially an aerobic one) unable to “produce” ATP then it is “metabolically” different? Any metabolomic data comparing WT and  atpA-atpG E. coli would be helpful to understand this issue. What are the ATP levels in GF mice colonized with WT and  atpA-atpG E. coli? How about imapring ATP production in another bacterium, for instance anaerobic and/or gram positive? Would this have similar effects?

  2. Is the absence of Cfs2-/- per se affecting the microbiota? 

  3. Another limitation is that all the microbiological analyses were performed on faeces so the information related to the mucosal-associated bacteria is missing. Mucosal-associated bacteria are the one that are more involved in MPs development and TLO maturation since they are in close contact with the tissue, thus I would suggest the author to include these data.

  4. What is the concentration of ATPex before and after weaning?

  5. Have the authors tried to colonized mice with faeces and/or luminal content from mice pre and post weaning to see if you can see a differential skewing of MPs?

  6. The authors use CX3CR1 as a marker for macrophages. Did the authors try to use another marker to label and confirm these macrophages? Fig 4 panel C, 40% of CCR2 cells are also CX3CR1+, it would be great to see the % of CX3CR1 that are expressing CCR2 because, from the picture, it seems that the majority of CX3CR1 mac are not CCR2. Are the TLO also differentially enriched along different part of the colon?

  7. The RNA-seq analysis was well performed but I think that the take home message is a bit confusing and could be clarified. Moreover, I would suggest linking this part more with the rest of the paper. Moreover, it will be helpful if some of one of the main findings collected from the RNA seq in GF pre/post weaning and/or in mice infected with WT and mutated E. coli would be further validated. 

  8. The last figure related to C. rod is not yet well connected with the rest of the paper. Reading the preprint, it appear to be a “big jump” from the scRNAseq to the C. rod data (since C rod wasn’t mentioned before). 
    Additionally, there are some more open question related to this part: what is the level of ATPex in C. rod infected mice? What about the TLO composition in C. rod infected mice? Can you represent the liver CFUs/cell number? Because since the csf2-/- + C. rod are losing weight, one would assume that they have also a lighter liver compared to csf2+/+. If this is the case and total count of bacteria is identical between the two groups, what you are measuring in panel C is an increased bacteria density rather than an increased bacterial translocation. Moreover, to strengthen the point about bacteria translocation, it would be helpful to see results from the intestinal barriers (i.e. mucus, epithelium and vascular barriers). Just for curiosity, since it has been shown that bacteria translocation from the gut to the liver is not only linked to Intestinal barriers breakdown but also Immune cells migration (i.e. macrophages) from the gut to the liver, could it be that the specific subset of Mac that the authors are studying in the TLO, affected by the microbiota, are also the one that, in specific conditions, are migrating carrying specific bacteria (i.e. C. rod) to distant organs?



The potential idea that a bacterial metabolite can modulate TLO development and composition is fascinating. 

How does the result of the preprint matter for general immunologists and/or patients?

The data coming from the RNAseq would be a great source of data for all the immunologists that are studying TLO modifications upon bacterial infections. It would be great to understand human-derived TLO composition, comparing healthy vs disease status (i.e. infectious diseases); the ultimate goal would be to understand any genetic modification is affecting TLO development then causing disease. 



Reviewed by Alice Bertocchi as part of the cross-institutional journal club of the Immunology Institute of the Icahn School of Medicine, Mount Sinai, the Kennedy Institute of Rheumatology and the Oxford Centre for Immuno-Oncology  (OXCIO) (University of Oxford, GB) and Karolinska Institute’s Center for Infectious Medicine (CIM) & Center for Molecular Medicine (CMM).