Identification of 14-series sulfido-conjugated PNAS PLUS mediators that promote resolution of infection and organ protection Jesmond Dalli, Nan Chiang, and Charles N. Serhan1 Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Harvard Institutes of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115 Edited by Carl F. Nathan, Weill Cornell Medical College, New York, NY, and approved September 25, 2014 (received for review August 5, 2014) Upon infection and inflammation, tissue repair and regeneration up-regulation of select SPMs [including Resolvin (Rv) D1, RvD5, are essential in reestablishing function. Here we identified potent and Protectin D1]. These mediators enhance bacterial killing and molecules present in self-limited infectious murine exudates, clearance along with regulating phagocyte recruitment (11). regenerating planaria, and human milk as well as macrophages The levels of these potent leukocyte agonists decline during that stimulate tissue regeneration in planaria and are proresolv- the later phase of the self-limited inflammatory response (11), ing. Characterization of their physical properties and isotope allowing the possibility that other signals may be produced that tracking indicated that the bioactive structures contained docosa- regulate leukocyte responses to promote tissue repair and re- hexaenoic acid and sulfido-conjugate (SC) of triene double bonds generation. Given the pivotal roles of chemical signals in infec- that proved to be 13-glutathionyl, 14-hydroxy-docosahexaenoic tions, we investigated whether mediators within self-resolving acid (SCI) and 13-cysteinylglycinyl, 14-hydroxy-docosahexaenoic infections could regulate tissue repair and regeneration without acid (SCII). These molecules rescued Escherichia coli infection- immunosuppression. Because maresin 1 (7R,14S-dihydroxy- mediated delay in tissue regeneration in planaria, improving re- docosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid; MaR1) displays generation intervals from ∼4.2 to ∼3.7 d. Administration of SCs potent proresolving and tissue-regenerative actions (12, 13), we protected mice from second-organ reflow injury, promoting repair investigated whether previously undescribed signals are produced via limiting neutrophil infiltration, up-regulating Ki67, and Roof during self-limited infections that regulate tissue regeneration. In plate-specific spondin 3. At nanomolar potencies these conjugates this report, we identify a new pathway and mediators in planaria, also resolved E. coli infections by limiting neutrophil infiltration mouse, and human tissues that promote repair and regeneration and stimulating bacterial phagocytosis and clearance as well as during infection. Identification of two new sulfido-conjugate (SC) efferocytosis of apoptotic cells. Together, these findings identify mediators provides the first evidence, to our knowledge, for previously undescribed conserved chemical signals and pathways autacoids produced during the resolution of infections that signal in planaria, mouse, and human tissues that enhance host responses innate host responses and accelerate repair. to contain infections, stimulate resolution of inflammation, and pro- mote the restoration of function. Results and Discussion To obtain self-resolving infectious exudates and assess tissue regeneration | omega 3 | leukocytes | inflammation | eicosanoids regeneration, we used murine Escherichia coli peritonitis rele- vant to human infections and mapped leukocyte trafficking. iven the rise in antibiotic-resistant infections and the critical E. coli inoculation at 105 colony-forming units (CFUs) per mouse Grole that barrier breach plays in microbial invasion, identifi- i.p. gave a self-limited host response that reached maximal cation of new endogenous signals that promote pathogen clear- neutrophil infiltration at 12 h and subsequently declined (Fig. 1A). ance and tissue repair/regeneration is of wide interest (1). When self-limited, acute inflammation is a host-protective response Significance (2, 3) that is orchestrated by chemical mediators and is an active process generated by evolutionarily conserved biosynthetic path- Inability of the body to contain infections may lead to collat- ways (3, 4). During initiation of inflammation, potent mediators eral organ damage resulting from unchecked innate immune are locally produced that promote vascular leakage, leukocyte responses. Here we investigated the chemical signals produced recruitment, and pain (5, 6). In disease, these pathways can be by immune cells to expedite clearance of bacteria and promote dysregulated, leading to heightened inflammatory responses and organ repair and tissue regeneration. We identified molecules perpetuation of the disease state (6–9). One approach to regu- produced during self-limited infections and in human milk that lating exuberant inflammatory responses is inhibition of initiating promote clearance of bacteria as well as accelerate tissue re- mediators (e.g., eicosanoids, including prostaglandins) via bio- generation. In addition, these molecules also protected organs synthetic enzyme inhibitors (6, 10) and receptor antagonists (5). In from exuberant inflammatory responses by limiting select the context of infection, this approach may be of limited clinical white blood cell recruitment and up-regulating the expression utility and can have potential drawbacks, including immune sup- of proteins involved in tissue repair. Therefore, these results pression (7, 8). identify new resolution moduli that regulate phagocytes to In self-limited inflammation, endogenous programs are acti- clear bacteria and activate the regeneration milieu. INFLAMMATION vated at the onset that regulate the amplitude of the inflammatory IMMUNOLOGY AND responses and stimulate resolution (3, 9). Central to these host- Author contributions: C.N.S. conceived the overall research plan and experimental design; protective responses are novel families of endogenous chemical J.D., N.C., and C.N.S. designed research; J.D. and N.C. performed research; J.D. and N.C. mediators termed specialized proresolving mediators (SPMs) (9). analyzed data; and J.D., N.C., and C.N.S. wrote the paper. In sterile inflammation and injury, these mediators actively limit The authors declare no conflict of interest. further neutrophil recruitment and promote macrophage clear- This article is a PNAS Direct Submission. ance of apoptotic cells and tissue debris (7–9). In self-resolving 1To whom correspondence should be addressed. Email: [email protected]. infections, endogenous resolution programs are also activated This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. during the early stages of inflammatory responses, with the 1073/pnas.1415006111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1415006111 PNAS | Published online October 16, 2014 | E4753–E4761 Downloaded by guest on September 25, 2021 Monocyte/macrophage numbers increased between 4 and dose-dependently accelerated head regeneration (r2 = 0.91) as 24 h, demarking onset of the resolution phase. We next isolated early as 2 d after surgery, shortening T50 to ∼3.3 d. For direct products from resolving infectious exudates (i.e., 24 h) and comparison, maresin 1 (Fig. 1B and Fig. S1A), the known dihy- assessed their ability, with planaria, to stimulate tissue regen- droxy-containing potent macrophage-derived proresolving me- eration. Because MaR1 stimulates tissue regeneration (12) and diator (12), accelerated this process to essentially the same elutes within methyl formate fractions from C18 solid-phase extent. Toward human translation, and because milk carries extraction [referred to as solid-phase extraction chromatographic nutrients and is appreciated to have products relevant to infant (SPE-C) isolate fraction 1], we sought evidence for signals in development and immune status (15), we also assessed tissue- distinct chromatographic fractions. Here we assessed eluates in regenerative properties of human milk isolates using the same methanol fractions (SPE-C isolate fraction 2) for previously chromatographic fractions (Fig. 1B). Incubation of planaria with undescribed signals that displayed tissue-regenerative properties. human milk isolates dose-dependently accelerated regeneration ∼ ∼ Planaria undergo both restorative and physiological regeneration and reduced T50 from 4.3 to 3.5 d (Fig. 1B and Fig. S1B). via evolutionarily conserved pathways, making this an ideal sys- These results demonstrate that both mouse resolving exudates tem to identify chemical signals involved in tissue regeneration and human milk possess tissue-regenerative properties that elute (14). To this end, planaria (Dugesia japonica) were injured on within the methanol fractions from solid-phase C18 extractions. day 0 and time-dependent head regeneration was monitored. To Next, we investigated whether these bioactive molecules reg- quantitate regeneration, we calculated tissue-regeneration in- ulated signaling pathways that trigger head-to-tail differentiation dices (TRIs) (12). Following head resection, regeneration en- in D. japonica (16). Two days postinjury, in regenerating blaste- mas, isolates from both mouse resolving exudates and human sued, giving a TRImax (maximum tissue regeneration) at 6 d and milk significantly increased expression of the fibroblast growth T50 (the interval at which 50% regeneration, TRI50, occurred) at ∼4.3 d (Fig. 1B).