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Planaria tissue regeneration

Planaria (Dugesia japonica) were kept in water (Poland Spring; Nestle Waters North America, Stamford, CT, USA) at 18°C. All animals were starved for at least 7 days before the experiments. Tissue regeneration was assessed as described []. In brief, planaria were subjected to head resection postocularly (surgical injury). The posterior portions of the planaria were then placed in spring water containing 0.01% EtOH, or MCTR at indicated concentrations. The extent of tissue regeneration during a 6-day period was determined using captured images of regenerating blastemas at regular intervals (24 h). These images were analyzed using ImageJ software (NIH, Bethesda, MD, USA) to give a tissue regeneration index (TRI) [].

Results

Having matched the physical properties of synthetic, stereochemically defined materials with those of endogenously produced MCTRs from both human and murine systems, we next required assessment of their potent biological actions. Given that MCTRs display tissue regenerative actions, we assessed the ability of each synthetic MCTR to promote tissue regeneration. Planaria were surgically injured by rapidly removing the head portion of the animals and tissue regeneration was followed over time []. Planaria undergo both physiological and restorative regeneration via evolutionarily conserved pathways, making this an ideal system to characterize novel chemical signals involved in tissue regeneration []. When planaria were kept with vehicle alone, maximum tissue regeneration (TRI max) occurred within ~6 days with time to 50% regeneration (T50) occurring by ~2.7 days. Incubation of planaria with MCTR1 led to acceleration in tissue regeneration with a reduction in T50 from ~2.7 days to ~2 days when planaria were incubated with 100nM of MCTR1 and to ~1.9 days when incubated with 1nM (). MCTR2 also accelerated tissue regeneration with planaria, where incubation of surgically injured planaria with MCTR2 reduced T50 to ~1.8 days () and at 1nM reduced T50 to ~1.9 days. The new MCTR3 also demonstrated potent tissue regenerative actions that were dose dependent (). Assessment of their relative potencies at stimulating tissue regeneration demonstrated that MCTR3 and MCTR2 were the most potent of the three, followed by MCTR1. Hence, MCTR3 ≈ MCTR2>MCTR1 ().

E. coli peritonitis

Peritonitis experiments were conducted as in []. Briefly, FVB mice (6-8-weeks old), purchased from Charles River Laboratories (Wilmington, MA, USA) were fed ad libitum Laboratory RodentDiet 20–5058 (LabDiet; Purina Mills, St. Louis, MO, USA) and housed at a maximum of four animals per cage and maintained in 12:12 hour light-dark cycles. Mouse experimental procedures were approved by the Standing Committee on Animals of Harvard Medical School (Protocol 02570) and complied with institutional and U.S. National Institutes of Health (NIH) guidelines. No surgical procedures were conducted and no analgesics were administered. Mice were euthanized by overdose of isoflurane via inhalation in saturated atmosphere, followed by cervical dislocation. If recovery from procedure is compromised as assessed by increase in self grooming, aggressive behavior or rapid movements, self-mutilation, absent activity, lack of eating or drinking, abdominal distention or redness and heat over abdominal area, humane endpoint in Protocol 02570 requires that mice must be euthanized immediately by isoflurane overdose, followed by cervical dislocation. During this procedure, animals did not experience more than slight or momentary pain or distress and no animals died prior to experimental endpoint. E. coli (serotypeO6:K2:H1) was cultured in Luria-Bertani broth and harvested at midlog phase (OD600 nm, ~0.5 absorbance units). In determined experiments mice were administered MCTR1, MCTR2, MCTR3 (50ng/mouse) or vehicle via intraperitoneal (i.p) injection and after 5 minutes inoculated with E. coli (105 CFU/mouse). Exudates were collected at the indicated intervals and leukocyte populations as well as bacterial phagocytosis and macrophage efferocytosis determined using flow cytometry and fluorescent-labeled antibodies []. In select experiments, mice were inoculated with E. coli (105 CFU/mouse), after 12h administered MCTR1, MCTR2, MCTR3 (100ng/mouse) or vehicle i.p. and exudates collected after 24h with leukocyte populations and bacterial phagocytosis determined as above.

Results

Given the increased incidence of bacterial infections [, ] and that MCTR1 and MCTR2 each displays potent actions promoting resolution of infections, we could now test whether each of the stereochemically pure MCTR1, 2 and 3 also carried these actions in murine infections. E. coli (1x105 CFU/mouse) inoculation gave a self-resolving inflammatory response with maximal neutrophil infiltration at ~12 h that subsequently declined, which gave a calculated resolution interval (Ri) of ~12h (). MCTR1 (50ng/mouse) injection immediately prior to E. coli inoculation gave a significant reduction in neutrophil infiltration at the 12 h interval, and shortened the resolution interval from ~12h to ~4h (). Administration of MCTR2 also gave significant reductions in exudate leukocyte counts (~50%), a shortening in the Ri to ~2h and an increase in phagocytosis of E. coli (~50%) by exudate leukocytes. These results with synthetic MCTRs are in accordance with published findings with biologically produced MCTRs [] and confirm the potent actions of these novel mediators in infections. Of note, MCTR3 given immediately prior to E. coli inoculation reduced exudate neutrophil levels by ~54%, shortening the Ri from 12 h to 3 h (). In addition to reducing neutrophil infiltration, each of three MCTR significantly increased leukocyte phagocytosis of E. coli, with MCTR2 and MCTR3 displaying the highest potencies (). MCTR1 was the most potent in stimulating macrophage efferocytosis (), a key defining pro-resolving action.

E. coli peritonitis

Peritonitis experiments were conducted as in []. Briefly, FVB mice (6-8-weeks old), purchased from Charles River Laboratories (Wilmington, MA, USA) were fed ad libitum Laboratory RodentDiet 20–5058 (LabDiet; Purina Mills, St. Louis, MO, USA) and housed at a maximum of four animals per cage and maintained in 12:12 hour light-dark cycles. Mouse experimental procedures were approved by the Standing Committee on Animals of Harvard Medical School (Protocol 02570) and complied with institutional and U.S. National Institutes of Health (NIH) guidelines. No surgical procedures were conducted and no analgesics were administered. Mice were euthanized by overdose of isoflurane via inhalation in saturated atmosphere, followed by cervical dislocation. If recovery from procedure is compromised as assessed by increase in self grooming, aggressive behavior or rapid movements, self-mutilation, absent activity, lack of eating or drinking, abdominal distention or redness and heat over abdominal area, humane endpoint in Protocol 02570 requires that mice must be euthanized immediately by isoflurane overdose, followed by cervical dislocation. During this procedure, animals did not experience more than slight or momentary pain or distress and no animals died prior to experimental endpoint. E. coli (serotypeO6:K2:H1) was cultured in Luria-Bertani broth and harvested at midlog phase (OD600 nm, ~0.5 absorbance units). In determined experiments mice were administered MCTR1, MCTR2, MCTR3 (50ng/mouse) or vehicle via intraperitoneal (i.p) injection and after 5 minutes inoculated with E. coli (105 CFU/mouse). Exudates were collected at the indicated intervals and leukocyte populations as well as bacterial phagocytosis and macrophage efferocytosis determined using flow cytometry and fluorescent-labeled antibodies []. In select experiments, mice were inoculated with E. coli (105 CFU/mouse), after 12h administered MCTR1, MCTR2, MCTR3 (100ng/mouse) or vehicle i.p. and exudates collected after 24h with leukocyte populations and bacterial phagocytosis determined as above.

Results

We also tested whether MCTR carry pro-resolving actions when administered at the peak of inflammation during E. coli infections. Administration of MCTR1 at the peak of inflammation (12h interval) gave reductions in exudate neutrophil counts at 24h and shortened Ri to ~10 h (). In mice given MCTR2 we found reductions in exudate neutrophil counts (~50%) giving an Ri of ~7 h (). At equal doses, MCTR3 also promoted the resolution of E. coli infections, reducing exudate neutrophil counts by ~50% and shortening the Ri to ~9 h (). Together these results demonstrate the potent biological actions of MCTR1 and MCTR2, confirming their original structural assignments (). Moreover, we describe here the unexpected actions of MCTR3 in accelerating the resolution of E. coli infections and establish the rank order potencies in promoting the resolution of infectious-inflammation.

Phagocytosis

Human macrophages and neutrophils were prepared from peripheral blood leukocytes [] and in accordance with the approved Partners Human Research Committee Protocol (1999P001297). Briefly, macrophages were plated in 96-well plates (5x104 cells per well) for 24h, neutrophils were plated in 96-well plates (1x105 cells per well) for 30 min and phagocytosis or efferocytosis was assessed as in []. For efferocytosis, apoptotic PMNs were obtained by culturing neutrophils overnight in PBS-/- (5x106 cells/ml). Apoptotic human PMNs were labeled with bisBenzimide trihydrochloride (Sigma-Aldrich). For efferocytosis, human macrophages were incubated with MCTR1, MCTR2, MCTR3 (1pM-10nM), or vehicle then labeled apoptotic PMNs (2.5x105 cells) and incubated for 60 min (37°C pH 7.45). Fluorescence was measured using a SpectraMax M3 plate reader (Molecular Devices, Sunnyvale, CA, USA), and results were analyzed using SoftMax Pro (Molecular Devices). In select experiments, macrophages were incubated for 15 minutes with vehicle, MCTR1, MCTR2, MCTR3 (1pM-10nM) or a mixture of MCTR1+MCTR2, MCTR2+MCTR3, or MCTR1+MCTR3 (0.1nM each). BacLight Green Bacterial Stain (Life Technologies, Eugene, OR, USA) labeled E. coli (2.5x 106 CFU/well) were added, cells incubated for 60 min (37°C pH 7.45) and fluorescence assessed using the fluorescence plate reader. In separate experiments, human neutrophils were incubated with MCTR1, MCTR2, MCTR3 (1pM-10nM), or vehicle then with BacLight Green Bacterial Stain-labeled E. coli (2.5x 106 CFU/well; 60 min; 37°C pH 7.45) and fluorescence assessed as above.