Citrobacter rodentium infection activates colonic lamina propria group 2 innate lymphoid cells

Background to the preprint

Citrobacter rodentium (CR) is a Gram-negative enteric pathogen that infects the colonic epithelium of mice, serving as a widely used model for studying human infections caused by attaching and effacing (A/E) pathogens such as Enteropathogenic Escherichia coli (EPEC) and Enterohemorrhagic E. coli (EHEC) [1]. Upon infection, C. rodentium adheres to intestinal epithelial cells, causing epithelial damage, crypt hyperplasia, and inflammation [2]. It is well known that Group 3 Innate Lymphoid Cells (ILC3) are recruited during the first days of CR infection [3], but some studies have shown that ILC2-derived cytokines (IL-4, IL-5, and IL-13) are found in the mucosa of infected mice and that this population may perform an important role in local immunity [6-7]

This preprint shows that ILC2 cells are expanded upon recognition of CR and suggests that their derived cytokines play a protective role in the mucosa with bacterial load control and epithelial barrier reinforcement, apparently through IL-18 signaling.

Key findings

• ILC2 from lamina propria respond to C. rodentium

In female mice that received the CR inoculum, after 4 days, there were ILC2 cells in the colonic lamina propria, an important effector site within the gut-associated immune system (Figure 1 of the preprint). This result infers the potential role of ILC2 cells in maintaining immunity against enteric pathogens.

• Type 2 cytokines are released after a CR infection.

The authors measured the concentration of IL-4, IL-5, and IL-13, the cytokines that are responsible for type 2 immunity, in the supernatant of isolated cells (Figure 2 of the preprint). All three mediators were increased, showing that in fact CR induces a type 2 response.

• ILC2-activating cytokines are released by epithelial cells infected with CR

Lastly, in figure 3 of the preprint, the authors show that the intestinal epithelial layer, after CR recognition, releases IL-18 and IL-33, cytokines that activate ILC2 cells, suggesting that CR has an indirect effect on promoting ILC2 recruitment through an IEC-dependent signaling pathway.

What I like about the preprint/why I think this new work is important

When the immune system is unable to regulate the progression of infectious colitis, the bacteria burst, making it difficult to treat the disease. On the other hand, treatment with antibiotics has driven antimicrobial resistance with an increase in mortality and hospitalization. Describing tricks to manipulate the immune response to contain bacterial dissemination in susceptible individuals is important for public health strategies. This research provides interesting evidence that type 2 immunity is important to fight against enteric pathogens. It also unveils the cellular targets that can be manipulated when aiming for pathogen clearance and tissue protection.

Future directions and questions for the authors

Major questions

Q1: Although the authors have shown that ILC2 cells are responsible for containing C. rodentium during the infection course, the functional role of these cells is still unexplored. Could the authors explain, in the discussion section, the mechanisms underlying the recruitment and differentiation of the ILC2 cells in the context of bacterial infection?

Q2: Given that normal distribution of data is a necessity for statistical parametric tests, the authors could state whether normal distribution tests were performed.

Q3: The authors might consider including the sample size calculation in the method section.

Q4: Also, did the authors consider performing the proof of concept using RORα knockout mice? This would show that ILC2 depletion would affect CR infection.

Q5: Assuming that type 2 immunity is significant against intestinal parasites, did the authors use a deworming protocol prior to the experiments? If this is the case, including it in the supplemental materials would strengthen the hypothesis.

Q6: To ensure optimal consistency across experiments, did the author synchronize the estrous cycle of female mice during the experiments?  Researchers frequently utilize male mice, especially when describing something novel about intestinal infection models.

Q7: The role of the gut in lung homeostasis and the migratory behavior of ILC2 in this context are known [4]. The authors could discuss the lung inflammatory status of the CR-infected mice.

Minor questions

Q1: Clostridium difficile: this species was rearranged to a new genus, named Clostridioides difficile [5]. Please correct the abstract and introduction.

Q2: In M&M section, please name the agar medium used to culture the bacteria after stool isolation and the time of Percoll gradient centrifugation.

References

[1] Bouladoux, N., Harrison, O. J., & Belkaid, Y. (2017). The mouse model of infection with Citrobacter rodentium. Current protocols in immunology, 119(1), 19-15.

[2] Vallance, B. A., Deng, W., Jacobson, K., & Finlay, B. B. (2003). Host susceptibility to the attaching and effacing bacterial pathogen Citrobacter rodentium. Infection and immunity, 71(6), 3443-3453.

[3] Mullineaux-Sanders, C., Sanchez-Garrido, J., Hopkins, E. G., Shenoy, A. R., Barry, R., & Frankel, G. (2019). Citrobacter rodentium–host–microbiota interactions: immunity, bioenergetics and metabolism. Nature Reviews Microbiology, 17(11), 701-715.

[4] Pu, Q., Lin, P., Gao, P., Wang, Z., Guo, K., Qin, S., … & Wu, M. (2021). Gut microbiota regulate gut–lung axis inflammatory responses by mediating ILC2 compartmental migration. The Journal of Immunology, 207(1), 257-267.

[5] Lawson, P. A., Citron, D. M., Tyrrell, K. L., & Finegold, S. M. (2016). Reclassification of clostridium difficile as clostridioides difficile (Hall and O’Toole 1935) Prévot 1938. Anaerobe, 40, 95-99.

[6] Frisbee, A. L., Saleh, M. M., Young, M. K., Leslie, J. L., Simpson, M. E., Abhyankar, M. M., … & Petri Jr, W. A. (2019). IL-33 drives group 2 innate lymphoid cell-mediated protection during Clostridium difficile infection. Nature communications, 10(1), 2712.

[7] Uddin, M. J., Thompson, B., Leslie, J. L., Fishman, C., Sol-Church, K., Kumar, P., & Petri Jr, W. A. (2024). Investigating the impact of antibiotic-induced dysbiosis on protection from Clostridium difficile colitis by mouse colonic innate lymphoid cells. Mbio, 15(3), e03338-23.

Disclaimer

Artificial intelligence tools (Quillbot) were used to assist in language editing and improving clarity during the revision of this report.

Tags: citrobacter rodentium, colitis, ilc2

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