IL-33: a key player in the development of iron-recycling red pulp macrophages

Splenic red pulp macrophages (RPMs) are tissue-resident macrophages that are responsible for erythrocyte homeostasis, iron recycling, and defense against blood-borne infections,^1,2 but how RPMs are developed remains elusive. A recent study by Lu et al. uncovered a critical role of IL-33 in the maturation of RPMs,³ adding a new dimension to the existing knowledge of RPM development and expanding the diversity of IL-33 functions.

The immune system is endowed with an intricate network of immune regulation, wherein macrophages are one of the prominent cell types controlling host defense and tissue homeostasis. Macrophages are localized in almost every tissue and possess diverse tissue-specific homeostatic functions,¹ and local microenvironment-associated factors impact macrophage differentiation and function. As an example, the development of splenic RPMs is dependent on the transcription factor SpiC,^2,4 but the molecular basis and mechanisms through which the phenotypic and functional diversity of RPMs are regulated remain to be defined. RPMs, by definition, are located in the splenic red pulp where the bloodstream slows, trapping red blood cells (RBCs) and blood-borne pathogens, which allows RPMs to recognize and remove damaged or aging RBCs and pathogens by phagocytosis.^1,2 The function of erythrocytes is oxygen delivery via iron-containing hemoglobin and an iron-recycling pathway, and RPMs are intimately linked to erythropoiesis and iron homeostasis.^2,5 Iron is an important metal that is implicated in a wide range of biological processes, including oxygen transport, DNA synthesis, and enzymatic activities. Iron deficiency can result in anemia,⁵ and excessive free iron can cause tissue damage by enhancing free radical production and the oxidative stress response.

How RPMs are developed in the spleen and what microenvironmental factors in the red pulp drive their development are still unknown. Pioneering studies have suggested that RPMs are derived from either embryonic or hemopoietic origins, possibly through fetal monocytes.^1,2,4 Monocytes, when receiving signals from local mediators, particularly hemin from RBCs, differentiate into pre-RPMs with distinguishable markers and functions from those of mature RPMs (Ref. ^1,4 and Fig. 1). One of the missing pieces in the RPM puzzle is what drives the pre-RPM development into the mature RPMs.

Fig. 1.

Splenic red pulp macrophages (RPMs) are critical for host iron recycling and protection against blood-borne infection in the spleen. RPMs are derived primarily from embryonic hematopoietic stem cells (HSCs) via a sequential maturation process from fetal monocytes to pre-RPMs to mature RPMs. The study by Lu et al. illustrates a role of IL-33 receptor signaling combined with hemin from red pulp RBCs in promoting the development of mature RPMs via receptor-dependent ERK activation and the transcriptional activity of GATA2. IL-33 signaling is also critical for maintaining mature RPM levels throughout adult life

IL-33, a member of the IL-1 family and a pleotropic cytokine, has attracted significant attention owing to its profound effects on homeostasis and diseases (for review, see ref. ⁶ and the refs therein). IL-33 is expressed by innate and tissue-resident cells, and intriguingly, IL-33 is stored in the cell nucleus and released as an alarmin when the cell undergoes necrosis. Once released, IL-33 signals via a receptor complex composed of IL-1RL1 (also known as ST2) and IL-1RAcP, which are expressed on a wide range of immune and tissue-resident cells, and IL-33 profoundly impacts cell differentiation, proliferation, and activation. The IL-33 signal selectively polarizes type 2 (M2) but not M1 macrophages and plays a critical role in tissue repair and inflammation resolution. IL-33 also promotes the maturation of erythrocyte precursors into mature RBCs.⁷ A recent study by Lu et al. broadened the role of IL-33 as a tissue-specific microenvironmental factor that is critical to the maturation of RPMs, and surprisingly, IL-33 was associated with RBCs themselves.³

In the study by Lu et al.,³ it was noted first that among a battery of cytokines, the combination of IL-33 and hemin but not IL-33 alone increased the number of RPMs differentiated from mouse and human monocytes. Whether the IL-33-enhanced numbers of RPMs were due to the effect of IL-33 on the proliferation of mature RPMs or the maturation of pre-RPMs was assessed, and it was found that while hemin alone induced the development of pre-RPMs but not mature RPMs, hemin and IL-33 together enhanced the number of mature RPMs but not pre-RPMs, suggesting that IL-33 is required for the maturation of RPMs from pre-RPMs. In a kinetic study using neonatal, adult and elderly WT and IL-33 receptor (IL1RL1)-deficient mice, it was noted that RPMs increased with age, and IL-33 receptor signaling was critical for the maintenance of RPMs in adult life, particularly during aging. Remarkably, IL-33 signaling had no impact on the other cell populations in the spleen or Kupffer cells in the liver, suggesting a selective effect of IL-33 on RPM maturation. Since the primary function of RPMs is iron recycling, the authors then investigated the implications of IL-33 signaling on iron recycling in mice. Indeed, the reduced numbers of RPMs in IL-33- or IL-33 receptor-deficient mice were accompanied by markedly enhanced iron levels in the spleen, suggesting that IL-33 signaling is essential for the development of iron-recycling RPMs.

Regarding the source of IL-33 in the spleen, it was suspected that RBCs or other cell types colocalized with pre-RPMs as the source of IL-33 in the red pulp. In fact, it has been demonstrated that RBCs can store cytokines and chemokines.⁸ Indeed, a considerable amount of IL-33 has been measured in the RBC lysates, suggesting that RBCs may store or carry IL-33 for reasons that are currently unclear. Nevertheless, the capability of RBCs-derived IL-33 in promoting RPM development was confirmed in vivo, as reconstitution of IL-33-deficient mice with RBCs from WT but not IL-33-deficient mice restored RPM development. Thus, RBCs are indeed a plausible source of IL-33 and hemin for RPM development (Fig. 1).

Moreover, a series of molecular and biochemical analyses, including phosphokinase assays, showed that among the various kinases examined, only ERK1/2 was selectively activated by both hemin and IL-33. In addition, IL-33 failed to induce RPMs in MyD88-deficient mice, and an ERK1/2 inhibitor impaired IL-33-induced RPM development in mice. Mechanistically, the lineage-specific transcription factor GATA2, which is downstream of IL-33 signaling in pre-RPMs, was found to mediate the maturation of pre-RPMs into RPMs. Incidentally, GATA1 and 2 have been shown to play roles in the generation of RBCs and in iron metabolism, while GATA1-deficient mice develop severe anemia.^9,10 Collectively, the authors elegantly demonstrated that IL-33 and hemin released by RBCs in the spleen microenvironment induce the maturation of RPMs that are essential for iron recycling (Fig. 1).

The findings by Lu et al.³ provide much needed molecular insights and have significant ramifications on RPM development and function, IL-33 biology and iron metabolism. It would be worthwhile to further elucidate the detailed mechanisms, particularly the involvement of GATA2 in the maturation of RPMs. This study also identified a previously unknown function of RBC-associated IL-33 in the development of RPMs. While IL-33 is widely implicated in many immune responses, the serum level of IL-33 is relatively low and unstable, and RBCs may provide a novel means for the safe storage and delivery of IL-33, although the actual source of IL-33 in tissues remains to be determined. Since erythrocyte precursors express both IL-33 and its receptor,⁷ it is possible that IL-33 in RBCs may be inherited from precursor cells during differentiation. Future investigation is needed to ascertain how IL-33 is associated with RBCs and its functional correlates in resting and inflammatory conditions.

Balanced iron recycling is important for health and in disease.⁵ Notably, IL-33-deficient mice did not develop anemia, and serum iron levels were unaffected, while considerable amounts of recycled iron were lost. The potential clinical importance of the IL-33/RPM-mediated iron-recycling pathway in humans is currently unknown and requires further investigation. The discovery of IL-33-mediated RPM maturation may also open a new avenue in the study of IL-33 and RPM functions beyond iron recycling, such as in infectious diseases and immunoregulation,^1,2 as RPMs may exert their functions via IFN-α in parasitic infections and may also regulate the development of Tregs.² Collectively, this recent work may also open up new therapeutic opportunities to target macrophage functions in various diseases.

Competing interests

The authors declare no competing interests.