Introduction: After tissue injury, infiltrating monocytes differentiate into CD86^hiLy6c^hi pro-inflammatory and CD206⁺CD301⁺ reparative macrophages (MΦs) that complete repair. MΦs differentiation is coordinated by tissue injury signals, often referred to as damage-associated molecular patterns (DAMPs), and local cytokines. For example, after heart transplant (HTx) IL-33 is released from graft fibroblasts and acts as a reparative/regulatory DAMP that metabolically reprograms MΦs to differentiate into reparative subsets that protect against chronic HTx rejection. Transplanting allogeneic materials is a novel situation where innate allorecognition of mismatched MHCI may disrupt normal MΦ responses to damaged tissues. For instance, paired immunoglobulin-like receptor (PIR)-A3 on C57BL/6 (B6) monocytes recognition of  BALB/c H2-D^d triggers their differentiation into pro-inflammatory dendritic cells that promotes HTx rejection. Monocyte PIR-B binding to self MHCI negatively regulates PIR-A signals. How PIRs shapes DAMP-mediated MΦ differentiation after Tx is not understood, so we hypothesize that in addition to DAMPs, recognition of self versus allogeneic MHCI by PIRs modulates reparative or inflammatory MΦ differentiation.

Methods: Monocyte-derived MΦ differentiation in response to donor materials was tested by administration of 20x10⁶ irradiated BALB/c allogeneic (H-2^d; Allo) or (B6) syngeneic (H-2^b; Syn) splenocytes intraperitoneally alone or with recombinant IL-33. Recipients of donor cellular materials were B6 wildtype (WT), Rag2^-/-γc^-/-, Pira^-/- and Pirb^-/- mice. The precise impact of H-2D^d (Allo) or H-2D^b (Syn) recombinant MHCI on MΦ differentiation was determined using B6 WT and Pira^-/- bone marrow-derived MΦs (BMDMs). Flow cytometry was utilized to assess MΦ differentiation.

Results: Administration of Syn cells predominantly stimulated monocyte differentiation into CD206⁺CD301⁺ reparative MΦs. Allo cells instead drove monocyte differentiation to pro-inflammatory Ly6c^hiCD86^hi MΦs. A similar outcome was observed in Rag2^-/-γc^-/- recipients, suggesting these changes are not driven by adaptive immunity. Incubation of BMDMs with recombinant allo MHCI, but not syn MHCI, triggered inflammatory Ly6c^hiCD86^hi MΦs in both WT and Pira^-/- BMDMs. Co-administration of allo cells with recombinant IL-33 supported differentiation into CD206⁺CD301⁺ reparative MΦs. Interestingly, Pirb^-/- mice display reduced C206⁺CD301⁺ MΦs, which correlated with decreased IL-33 receptor expression.

Conclusions: These data indicate allorecognition by monocytes or MΦs disrupts the typical generation of reparative MΦs in response to injury. We show that monocyte-derived MΦs PIR-B recognition of self MHCI may trigger expression of the receptor needed for IL-33-mediated reparative MΦ differentiation. These new data provide important insights into how innate allorecognition and repair pathways intersect to direct the differentiation of reparative or inflammatory MΦs that shape HTx outcomes.