Macrophages Are Required for Adult Salamander Limb Regeneration
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Macrophages are required for adult salamander limb regeneration James W. Godwina,1, Alexander R. Pintoa, and Nadia A. Rosenthala,b aAustralian Regenerative Medicine Institute, Monash University, Clayton, VIC 3800, Australia; and bNational Heart and Lung Institute, Imperial College London, London W12 0NN, United Kingdom Edited* by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved April 24, 2013 (received for review January 8, 2013) The failure to replace damaged body parts in adult mammals evidence for the immunological control of successful regeneration results from a muted growth response and fibrotic scarring. in an adult vertebrate. Although infiltrating immune cells play a major role in de- termining the variable outcome of mammalian wound repair, Results little is known about the modulation of immune cell signaling in Pro- and Anti-Inflammatory Signals Are Simultaneously Induced Following efficiently regenerating species such as the salamander, which can Axolotl Limb Amputation and Sustained Through Regeneration. We regrow complete body structures as adults. Here we present a screened the blastema of resected axolotl limbs with a cross-reactive mouse cytokine protein detection array, revealing a rapid induc- comprehensive analysis of immune signaling during limb regener- fl ation in axolotl, an aquatic salamander, and reveal a temporally tion of cytokines, chemokines, and in ammatory markers within 1 d defined requirement for macrophage infiltration in the regenerative of limb resection. Unexpectedly, we detected high levels of anti- inflammatory (Th2) cytokines (Fig. 1; Fig. S1), which are normally process. Although many features of mammalian cytokine/chemokine induced later in mammalian wound healing (20). The fibrotic signaling are retained in the axolotl, they are more dynamically responses normally associated with IL-4 and IL-13 induction were fl deployed, with simultaneous induction of in ammatory and anti- countered by increased expression of the antifibrogenic (Th1) cy- inflammatory markers within the first 24 h after limb amputation. tokine IFN-γ (21). All cytokines and chemokines returned to nor- Systemic macrophage depletion during this period resulted in mal baseline levels by 15 d postamputation (dpa; late blastema). wound closure but permanent failure of limb regeneration, as- Myeloid chemotactic molecules peaked within 1 dpa (Fig. 1), in- sociated with extensive fibrosis and disregulation of extracellular dicating that robust monocyte recruitment precedes limb regen- matrix component gene expression. Full limb regenerative capacity eration. A comparative analysis of the inflammatory response in- of failed stumps was restored by reamputation once endogenous duced by crush injury (necrotic model) and LPS injection (modeling macrophage populations had been replenished. Promotion of a bacterial infection) confirmed that many of the inflammatory regeneration-permissive environment by identification of macro- cytokines and chemokines induced in mammalian wounding exhibit similar patterns of induction in the axolotl (Fig. 1; Fig. S1), as ex- phage-derived therapeutic molecules may therefore aid in the fi β regeneration of damaged body parts in adult mammals. empli ed by the strong induction of IL-1 to LPS exposure (40- fold). Thus, we focused attention on the unusually early activation of anti-inflammatory signaling in the regenerating axolotl limb. developmental biology | immunology | limb development | wound healing Myeloid Cell Recruitment Is a Major Feature of Limb Regeneration. alamanders have the remarkable ability to regenerate complex To monitor leukocyte subsets entering the regenerating limb blas- Sstructures such as limbs, tails, retina, and spinal cord, along with tema, we used enzyme cytochemistry to detect monocytes, macro- some sections of the heart and brain, during any stage of their life phages, and granulocytes in regenerating axolotl tissue as early as cycle (1–7). Salamanders are also able to perform scar-free repair of 1 dpa, subsequently peaking between 4 and 6 dpa (Fig. 2; Fig. S2), deep tissue wounds after injury (8). Although the cellular mediators before returning to baseline levels by 15 dpa (late blastema). Using and immunological signaling necessary for regeneration in the naphthol AS-D chloroacetate (CA) esterase chemistry, neutrophil salamander have not been described, recent reports suggest granulocytes were also specifically detected within the wound 1 dpa that inflammation may influence the initiation and completion and persisted for up to 4–6d(Fig. S2). Unexpectedly, macrophages of wound healing and regeneration (9–14), as the cytokine detected with α-naphthyl acetate (NSE) staining were also in- microenvironment directly influences the time course of leu- creased within the 1-dpa blastema, located immediately around and kocyte infiltration, cellular proliferation, angiogenesis, and colla- within the developing wound epithelium. Macrophage numbers gen remodeling of damaged tissues. peaked at around 4–6 d and persisted in the regenerate up to the In mammals, fibrotic scarring is a major impediment to tissue early redevelopment phase before returning to baseline numbers regeneration (15), where cellular infiltration and immune signaling (Fig. 2; Fig. S3). fi play a key role (16). Macrophages are an important source of both To con rm the phagocytic capacity of macrophages detected inflammatory and anti-inflammatory signals, arriving in mammalian within regenerating limb and deep tissue injuries, three in- wounds 48–96 h after injury, where they clear dead cells, release dependent labeling methods were used (Fig. S4). First, carbon- proinflammatory cytokines, and subsequently produce factors that labeled phagocytes stained with Giemsa exhibited typical mono- dampen inflammation and stimulate angiogenesis, fibroblast mi- cyte morphology within the blastema and wound epithelium (Fig. S4 A–C). Second, neutral red staining, shown to be highly selective gration, and replication (17). In mice, macrophage depletion or fi disruption of macrophage transcriptional regulation after skeletal for macrophages in young zebra sh (22), revealed positive cells muscle injury results in incomplete muscle repair and induction of fibrotic scarring (18, 19). To investigate the role of macrophages during scar-free tissue Author contributions: J.W.G. designed research; J.W.G. performed research; A.R.P. con- repair in an efficiently regenerating model, we dissected the in- tributed new reagents/analytic tools; J.W.G. and A.R.P. analyzed data; and J.W.G., A.R.P., and N.A.R. wrote the paper. flammatory response to injury in regenerating limbs of the axolotl fl aquatic salamander (Ambystoma mexicanum)atthecellularand The authors declare no con ict of interest. molecular level. A distinctively dynamic signaling microenvironment *This Direct Submission article had a prearranged editor. during the early stages of axolotl limb regeneration was accompanied Freely available online through the PNAS open access option. by early myeloid cell recruitment. We documented an absolute re- 1To whom correspondence should be addressed. E-mail: [email protected]. quirement for macrophages in the orchestration of postamputation This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. IMMUNOLOGY immune microenvironment and blastema formation, providing direct 1073/pnas.1300290110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1300290110 PNAS | June 4, 2013 | vol. 110 | no. 23 | 9415–9420 Downloaded by guest on October 1, 2021 Macrophage Chemoattractants Th2 Cytokines expected macrophage/monocyte morphology, staining positive for CCL2/JE/MCP-1 IL-4 20 IL-5 NSE chemistry (Fig. S5). 20 CCL12/MCP-5 CCL3/MIP-1 alpha IL-13 IL-10 CCL4/MIP-1 beta 15 15 CXCL12/SDF-1 Mononuclear Phagocytes Regulate Regenerative Gene Expression 10 10 Patterns. Phagocytic uptake of liposome-encapsulated clodronate Fold induction Fold induction fi 5 5 (Clo-Lipo) is a well-established method for speci c in vivo ablation of macrophages (24). This reagent was effective in specifically de- 0 0 NL1235715CrushLPS NL 1 2 3 5 7 15 Crush LPS pleting circulating axolotl monocytes and tissue resident macro- phages on i.v. injection, leaving the neutrophil numbers relatively Dpa Treatment Dpa Treatment unaffected (Figs. S5 and S6). Replenishment of mononuclear – Th1 Cytokines Pleiotropic Cytokines phagocytes begins 5 10 d after complete ablation (Fig. 3A; Fig. S5). TNF-alpha IL-6 fi fi IL-17 50 By examining molecular pro les in the rst week after amputation 20 IL-23 IL-1 alpha IL-12 p70 IL-1 beta before macrophages return, we detected changes in macrophage IL-16 40 IL-1ra 15 IFN-gamma IL-27 chemoattractants (Fig. 3B), increased levels of inflammatory cyto- 30 IL-2 IL-3 fl 10 kines, and reduced levels of anti-in ammatory cytokines (Fig. 3C). IL-7 20 A concomitant decrease in the expression of the ECM-degrading Fold induction 5 Fold induction 10 enzymes matrix metalloproteinase 9 and 3 (MMP9 and MMP3, 0 0 respectively) was accompanied by a significant reduction in the ex- NL 1 2 3 5 7 15 Crush LPS NL 1 2 3 5 7 15 Crush LPS pression of Msx2 and the blastemal marker genes Prrx1 and Sp9 Dpa Treatment Dpa Treatment (Fig. 3D). Macrophage depletion also caused a failure in the acti- vation of Tgf-β1 and TGF target genes Runx2 and fibronectin. Fig. 1. Cytokine regulation during the time course of regeneration. A cross- Down-regulation of TGF-β, fibronectin, and Msx2 was confirmed by species cytokine protein array detects changes in cytokine/chemokine