Preprint Club
A cross-institutional Journal Club Initiative

Keywords

• Intestinal macrophage diversity and function

• Mucosal immunology

• TGF-β signaling

Main Findings

In their study, Prise et al. investigate and challenge established beliefs on the defined roles and characteristics of macrophages in the small intestine. Central to this study are two key areas of macrophage biology: their longevity and specific sub-tissular location, alongside their diverse functional responses. Based on previous evidence, it was assumed that lamina propria (LP) macrophages constitute a transient population that is continually replenished by monocytes via the CCR2-CCL2 axis. Extending this view to other intestinal compartments, macrophages within the submucosa/muscularis (S/M) were shown to be long-lived and self-sustaining, identifiable by their expression of CD4 and Tim4.

Within this preprint, the authors’ analyses introduce a new perspective on this spatial divergence in macrophages. Using single-cell RNA sequencing, they discovered that macrophages labeled as “long-lived” possess gene signatures indicative of localization within the LP or the S/M, disrupting the idea that long-lived macrophages are exclusively associated with the submucosa/muscularis. The authors’ investigation reveals that CD163 exclusively labels macrophages in the S/M region. This marker in combination with CD4 and Tim4, allows the identification of six distinct macrophage populations, categorized by the presence or absence of CD163 and the expression pattern of Tim4 and CD4 (i.e. Tim4-CD4-, Tim4-CD4+, or Tim4+CD4+).

The use of these markers, CD4 for maturity, Tim4 for longevity, and CD163 for localization, led to the significant finding that Tim4+ macrophages are present in both LP and S/M niches, and that macrophages within these distinct regions exhibit diverse functional thresholds that are influenced by their location and lifespan. The authors delve deeper to reveal unique transcriptional responses to TGF-β signaling based on their location, underscoring the nuances of macrophage functionality in the gut.

Notably, by employing a novel Timd4-cre Tgfbr2-fl/fl mouse model, they reveal that the absence of TGF-β signaling, while not producing evident defects at steady state, led to an increase in CD163+ macrophages and a disruption in their distribution in the gut. Instead of retaining their location in the S/M, Tgbr2-deficient CD163+ macrophages are now detectable within the LP. Moreover, the investigators show loss of TGF-βR signaling impacts the function of LP and S/M macrophages, particularly affecting iNOS production and their phagocytic activity.

Collectively, this study identifies a region-specific signal that determines microanatomic localization and functional adaptations, paving the way towards a better understanding of macrophages within the body’s largest mucosal surface.

Limitations

• While the author focuses on the steady state, it remains unknown if acute or chronic inflammation or repair/resolution would actively affect the distribution of S/M macrophages into the LP.

• It remains unclear if a similar zonation mechanism is seen in the healthy human gut as the markers used to identify macrophages in mice are not directly translatable to human macrophages. Confirming a similar regional patterning or perturbed pattern during tissue repair or fibrosis would strengthen the authors’ claim.

• The specific roles of CD163 expression on macrophages remain unclear. Is CD163 expression directly regulated by TGF-β? Does TGF-b regulated ADAM10/17 expression regulate CD163 surface expression?

• The authors nicely demonstrate changes in the distribution of macrophages, but their study falls short on demonstrating how loss of CD163+ macrophage zonation impacts tissue homeostasis or the microenvironment. This would ideally be paired by tackling effector functions the authors have assigned  to the respective macrophage subsets (e.g. IL-1, TNF-a, iNOS, phagocytosis)

• Questions still remain  and should focus on gut leakiness or impact of timing the loss of TGF-b signaling to a period prior to birth. Additionally, it should be addressed whether TGF-b expression or the availability of bioactive TGF-b is restricted to the S/M.

Significance/Novelty

This preprint provides a paradigm shift in mucosal macrophage biology by highlighting the presence and functional diversity of long-lived macrophages in both the LP and S/M. The insights into the role of TGF-β signalling in macrophage zonation and function have broad implications for understanding intestinal immunity, disease and repair.

For general immunologists, this study provides new perspectives for exploring macrophage biology in mucosal immunity and provides a framework on using location specific pathways to direct macrophages to new locations. For patients, especially those with gastrointestinal disorders, these findings could pave the way for defining detrimental and beneficial roles for macrophages and explore targeted therapies modulating macrophage function and location.

Credit

Reviewed by Louis Ngai (University of Toronto, Department of Immunology) as part of a cross-institutional journal club between the Icahn School of Medicine at Mount Sinai, the University of Oxford, the Karolinska Institute and the University of Toronto.

The author declares no conflict of interest in relation to their involvement in the review.