Thymosin β4-sulfoxide attenuates inflammatory cell infiltration and promotes cardiac wound healing

The downstream consequences of inflammation in the adult mammalian heart are formation of a non-functional scar, pathological remodelling and heart failure. In zebrafish, hydrogen peroxide released from a wound is the initial instructive chemotactic cue for the infiltration of inflammatory cells, however, the identity of a subsequent resolution signal(s), to attenuate chronic inflammation, remains unknown. Here we reveal that thymosin b4-sulfoxide lies downstream of hydrogen peroxide in the wounded fish and triggers depletion of inflammatory macrophages at the injury site. This function is conserved in the mouse and observed after cardiac injury, where it promotes wound healing and reduced scarring. In human T-cell/ CD14 þ monocyte co-cultures, thymosin b4-sulfoxide inhibits interferon-g, and increases monocyte dispersal and cell death, likely by stimulating superoxide production. Thus, thymosin b4-sulfoxide is a putative target for therapeutic modulation of the immune response, resolution of fibrosis and cardiac repair.

ardiac injury activates an innate immune mechanism (reviewed in Frangogiannis 1 ) underpinned by sequential recruitment of inflammatory neutrophils and macrophages (leukocytes) to the site of injury. A direct consequence of immune cell infiltration is activation of fibroblasts and their differentiation to myofibroblasts, the principle source of collagen deposition, fibrosis and scarring. Recently, a study in the zebrafish revealed that the earliest 'damage' signal that instigates rapid neutrophil and macrophage influx to the wound is hydrogen peroxide (H 2 O 2 ) 2 . The identity of subsequent downstream signalling molecules or cues that encourage the resolution of immune cells away from the wound, once the acute phase is complete, remains unknown 3 .

Thymosin b4 (Tb4) is an actin-binding peptide, which exerts both anti-inflammatory and anti-fibrotic effects in the context of tissue wounding; notably following alkali eye injury 4 and blebomyocin-induced damage in the lung 5 . Tb4 is one of the first proteins to be upregulated after wounding 6 and has been shown to modulate oxidative stress, targeting cardiac fibroblasts and cardiomyocyte survival following myocardial infarction 7, 8 . There are two naturally occurring derivatives of Tb4, the tetrapeptide Ac-SDKP, an N-terminal cleavage product known to have anti-fibrotic effects during hypertension 9 and which can reverse inflammation and fibrosis in rodents post MI 10 and Tb4-sulfoxide (Tb4-SO), formed by oxidation of the methionine-6 residue and thought to be the biologically active, secreted form of the parent peptide. Tb4-SO is largely unstudied to-date, but has been shown to inhibit neutrophil chemotaxis in vitro and attenuate inflammation in carrageenan-injected mice 11 . We sought to investigate whether Tb4-SO might act as the immune cell dispersal signal during cardiac wound healing to prevent chronic inflammation.

Here we identify Tb4-SO as downstream of H 2 O 2 acting to disperse inflammatory macrophages and promote healing following tail, body flank or heart injury in zebrafish. This function is conserved in mice, where Tb4-SO, via a direct effect on leukocytes at key time points within healing myocardium, contributes to reduced fibrosis and scarring in the injured heart. Extrapolating these findings to humans, we show that Tb4-SO blocks the production of pro-inflammatory IFN-g to reduce the adhesion of activated monocytes and increases cell death downstream of elevated reactive oxygen species (ROS) production.