Research in the last decade has led to the discovery of molecules that engage in unanticipated secondary activities, so called “molecular multitasking.” Alarmins and certain cytokines and chemokines are prototypical examples of biomolecules serving additional nonhomeostatic extracellular functions. Specifically, the term “alarmin” or “danger-associated molecular pattern” refers to those molecules that are released by dead cells during trauma to signal danger and tissue damage.1 The alarmin category of molecules encompasses proteins, chemokines, and cytokines (eg, HMGB-1, S100A8/9, macrophage migration inhibitory factors [MIFs], defensins), as well as small molecule metabolites (eg, ATP), nucleic acids (eg, DNA, RNA), and porphyrins (eg, heme). Alarmins are recognized at the cell level via receptor-mediated detection and are mainly responsible for the activation of signaling pathways which alert the innate immune system and elicit proinflammatory responses (eg, TLR4 pathway).1,2 However, recent evidence led to the identification of alarmins involved in anti-inflammatory and restorative functions which stimulate tissue repair and support immune regulation.1,2
A recent J Clin Invest article describes the unique “restorative alarmin” function of interleukin 33 (IL-33) in transplant tolerance.3 IL-33 is an IL-1 family member with dual function: on the one hand, under steady-state condition, intracellular IL-33 participates in the regulation of gene expression as a nuclear protein in stromal cells; on the other one, it serves as a stored alarmin that is released after tissue damage and signals to immune cells via the IL-33 receptor IL-1R–like 1, commonly known as serum stimulation-2.1,2
Using a mouse model of chronic rejection following heart transplant, Li et al3 showed that the lack of IL-33 expression in the transplanted heart accelerates graft rejection, associated with increased T-cell infiltration. In light of these observations IL-33 emerges as a stromal cell-derived alarmin which limits the local inflammatory response early after transplantation (Figure). Indeed, IL-33 is upregulated in allografts and its absence significantly aggravates vascular occlusion and subsequent fibrosis in the transplanted tissue.3
The ability of IL-33 to induce graft tolerance has been attributed to its potential to shape the functional phenotype of macrophages recruited to the transplanted heart. Myeloid cells are a major component of the infiltrating immune cells on graft and are usually abundant in samples from recipients with acutely and chronically rejected heart grafts.3,4 Loss of graft IL-33 results in increased recruitment of proinflammatory macrophages expressing the inflammatory marker inducible nitric oxide synthase (iNOS).3,5 Blockage of monocyte recruitment as well as hydrogel-based local IL-33 delivery to the graft reduced the number of infiltrating proinflammatory macrophages and protected against vasculopathy and fibrosis in transplanted IL33-deficient mice.3,4,6 Importantly, these effects are achieved via local—and not systemic—IL-33 action.
Mechanistically, IL-33 drives macrophage metabolic reprogramming which triggers a tolerogenic immune response and underlies the reparative and regulatory properties of this alarmin. In the presence of IL-33 macrophages undergo a metabolic skewing toward oxidative phosphorylation through an intact tricarboxylic acid cycle.3,5,7 Although similar to IL-4–induced macrophages, IL-33–triggered macrophages reflect a unique phenotype hallmarked by a specific metabolic remodeling with aspartate-arginosuccinate shunt features, increased fatty acid uptake and reduced iNOS expression.3,7 In vitro experiments demonstrated that IL-33 prime macrophages toward an “M2-like” anti-inflammatory phenotype through fatty acid uptake while limiting the induction of iNOS, which is required for a glycolytic and proinflammatory macrophage shift.3,5,7 Consistent with these findings, fatty acid uptake in macrophages from heart transplants is significantly reduced in the absence of IL-33, supporting an important role for graft IL-33 in stimulating fatty acid–driven metabolic switch in infiltrating myeloid cells and triggering a tolerogenic response to the transplant. The crucial and specific role of IL-33 in driving monocyte recruitment and macrophage differentiation after transplant was definitely proven in LysM-Cre conditional mice deficient for the expression of the IL-33 receptor serum stimulation-2 in myeloid cells. The lack of IL-33 signaling in macrophages at early time points after transplant exacerbates the recruitment and differentiation of graft-infiltrating myeloid cells into proinflammatory iNOS-positive macrophages defective in fatty acid uptake.3
Overall, these studies illustrate the uniqueness of IL-33 among alarmins due to its regulatory properties which temper myeloid cell proinflammatory responses after tissue injury and drive a metabolic program that supports homeostatic and reparative functions. This provides evidence how an alarmin can contribute to a protective anti-inflammatory rather than detrimental proinflammatory function after tissue injury. In a transplant setting, by limiting the proinflammatory switch of graft-infiltrating monocyte-derived macrophages, IL-33 prevents chronic rejection–associated vasculopathy and accelerated graft loss (Figure).
This study suggests that IL-33 may be part of a restoration system on tissue injury and opens the novel concept of stimulating natural reparative pathways rather than blocking detrimental alarmin-triggered inflammatory ones (eg, TLR4 pathways). A major advantage of such approach is the absence of any interference with pathways related to pathogen detection and responses. Open questions are whether IL-33 serves as restorative alarmin in injury settings different from transplant and whether additional alarmins exist which play similar homeostatic roles. The possibility to potentiate reparative and prevent inflammatory pathways for therapeutic purposes offers novel therapeutic opportunities, with the potential that combination therapies provide a synergic and more effective action applicable to multiple conditions ranging from transplants to chronic inflammation and trauma conditions.
1. Bertheloot D, Latz E. HMGB1, IL-1α, IL-33 and S100 proteins: dual-function alarmins. Cell Mol Immunol. 2017; 14:43–64
2. Martin NT, Martin MU. Interleukin 33 is a guardian of barriers and a local alarmin. Nat Immunol. 2016; 17:122–131
3. Li T, Zhang Z, Bartolacci JG, , et al. Graft IL-33 regulates infiltrating macrophages to protect against chronic rejection. J Clin Invest. 2020; 130:5397–5412
4. Kitchens WH, Chase CM, Uehara S, et al. Macrophage depletion suppresses cardiac allograft vasculopathy in mice. Am J Transplant. 2007; 7:2675–2682
5. Bailey JD, Diotallevi M, Nicol T, et al. Nitric oxide modulates metabolic remodeling in inflammatory macrophages through TCA cycle regulation and itaconate accumulation. Cell Rep. 2019; 28:218–230.e7
6. Hussey GS, Dziki JL, Lee YC, et al. Matrix bound nanovesicle-associated IL-33 activates a pro-remodeling macrophage phenotype via a non-canonical, ST2-independent pathway. J Immunol Regen Med. 2019; 3:26–35
7. Nomura M, Liu J, Rovira II, et al. Fatty acid oxidation in macrophage polarization. Nat Immunol. 2016; 17:216–217