Endogenous microparticles (MPs) were systematically profiled during the time course of self-limited inflammation. Precursors for specialized proresolving lipid mediators were identified in MPs from inflammatory exudates using liquid chromatography tandem mass spectrometry-based metabolomics. Hence, we postulated that formation of anti-inflammatory and proresolving lipid mediators could underlie beneficial effects attributed to MPs and that this process could serve as a basis for biomimicry. Using human neutrophil-derived MPs, we constructed novel nanoparticles (NPs) containing aspirin-triggered resolvin D1 or a lipoxin A4 analog. Enriched NPs dramatically reduced polymorphonuclear cell influx in murine peritonitis, shortened resolution intervals, and exhibited proresolving actions accelerating keratinocyte healing. The enriched NPs protected against inflammation in the temporomandibular joint. These findings indicate that humanized NPs, termed nano-proresolving medicines, are mimetics of endogenous resolving mechanisms, possess potent beneficial bioactions, can reduce nanotoxicity, and offer new therapeutic approaches.
Uncontrolled inflammation is a fundamental etiology of many pathologies, including cardiovascular diseases, arthritis, and temporomandibular joint (TMJ) disorders (TMDs) (1, 2). Prevalence of TMDs is high, with at least one symptom afflicting a third of United States adults. However, there is an unmet need for effective treatments, as options are limited and often involve behavioral or physical therapies or acute administration of nonsteroidal anti-inflammatories (2). Timely resolution of an inflammatory insult is pertinent for restoration of tissue homeostasis and essential for ongoing health (3). Thus, endogenous control mechanisms of inflammation and its resolution are of considerable interest. Recently, potent specialized chemical mediators derived from essential fatty acids were identified that actively promote inflammation resolution via novel proresolving and anti-inflammatory cascades (4). Originally, microparticles (MPs) were thought to be inert empty vesicles; however, their multiple roles and functional significance during inflammation is now being appreciated. MPs are detected in physiological conditions, with increased numbers disseminated in multiple diseases including rheumatoid arthritis (5). Along these lines, anti-inflammatory properties of MPs shed from polymorphonuclear cells (PMNs) were uncovered (6, 7).
In this study, we investigated the temporal generation and properties of endogenous MPs produced in evolving self-limited inflammatory exudates in vivo and constructed a novel biomimetic system using human PMN-derived MPs to stimulate resolution of inflammation as demonstrated in TMJ disease. This biomimetic construction is highly advantageous as compared with other synthetic nanoparticle (NP) drug delivery systems that have adverse immunotoxic effects (8).
Materials and Methods
Peritonitis was initiated in male FVB mice (6–8 wk; Charles River Laboratories) with zymosan A (1 mg i.p.), and peritoneal lavages were collected at indicated times. Exudate MPs were isolated by removal of leukocytes (4000 × g, 15 min) followed by ultracentrifugation (100,000 × g9).
Preparation of humanized nano-proresolving medicines
Human MPs were prepared (6) and added to thin lipid films in glass flasks (after organic solvent removal by rotary evaporation; 10 min, 25°C) containing fluorescent 1,2-dioleoyl-sn-glycero-3-phoshpo-l-serine-N-7-nitro-2-1,3-benzoxadiazol-4-yl (100 μg; Avanti Polar Lipids) and 7S, 8R, 17R-trihydroxy-docosa-4Z,9E,11E,13Z,15E,19Z-hexaenoic acid (aspirin-triggered resolvin D1 [AT-RvD1], 1 μg; Cayman Chemical) or o-[9,12]-benzo-ω6-epi-lipoxin A4 (LXA4) (1 μg) (10) prepared for these studies by custom synthesis. Intercalation of AT-RvD1 or LXA4 analog and fluorescent phospholipid was performed by aqueous energy dissemination using a sonic dismembrator (output power 15 W, 15 min, 25°C; Fisher Scientific). Humanized NPs were layered on Sephadex G50 columns (Sigma-Aldrich) and fractions collected in 0.2 μm filtered PBS. Incorporation of AT-RvD1 and LXA4 analog was determined using LC-MS/MS and fluorescent phospholipid content confirmed by flow cytometry (BD FACSCanto II; BD Biosciences). NPs and MPs were sized using calibration beads (Corpuscular) by flow cytometry and conventional electron microscopy following negative staining (Tecnai G2 Spirit BioTWIN; Harvard Medical School core facility).
9). Esterified monohydroxy products were assessed in endogenous MPs following methanol/chloroform extraction of phospholipids and overnight saponification with 1 N potassium hydroxide in 90% ethanol. Samples were acidified and extracted with added internal standard d5-17-hydroxy-docosahexaenoic acid (HDHA; 1 ng) for LC-MS/MS–based lipidomics as above.
Peritonitis and wound healing
Human MPs or nano-proresolving medicines (NPRMs) (1 × 104–3 × 105) were given i.v. (or i.p. as indicated) prior to zymosan A (0.1 mg i.p.), and peritoneal leukocytes were assessed at 2, 4, or 12 h. In some experiments, mice were given d5-DHA (1 μg i.v.) prior to zymosan A (1 mg), and peritoneal MPs were collected at 4 h. Wound healing was assessed using an Electric Cell-substrate Impedance Sensing 1600R system (Applied Biophysics) (11). Human epidermal keratinocytes (Lonza) were cultured on 8W1E electrode arrays and, once confluent (∼8000 Ω, using 16 kHz), were wounded (64 kHz, 30 s). Cells were treated with buffer alone, AT-RvD1 NPRMs (10 nM), AT-RvD1 (10 nM), or the equivalent NP number.
Mice were anesthetized (O2: 1 L/min, isoflurane: 2.5%), and CFA was administered (10 μg; 20 μl) into the periarticular space of the left TMJ and 0.9% sterile saline (20 μl) into the right TMJ space (as in Ref. 12). Treatments were then delivered i.v. with buffer (100 μl PBS), AT-RvD1 (10 ng) NPRMs, or o-[9,12]-benzo-ω6-epi-LXA4 (10 ng) NPRMs. After 12 h, PMN infiltration was assessed in the TMJ by myeloperoxidase (MPO) activity by ELISA (Hycult Biotechnology).
Data are mean ± SEM. Multiple group comparisons were made using one-way ANOVA followed by Dunnett’s or Bonferroni post hoc analysis. The p values < 0.05 were considered significant.
Results and Discussion
First, we profiled the time course of MP generation in self-limited acute inflammation (i.e., zymosan-induced peritonitis). The endogenous leukocyte-derived MPs were temporally generated in vivo in inflammatory exudates, with maximum MPs identified within the initiation phase, which gradually declined during the resolution phase corresponding with neutrophilic loss (Fig. 1A). Further characterization of these Annexin A5+ MPs by surface molecule expression demonstrated these were CD11b+, CD45+, and CD54+ (Fig. 1B). To systematically address the LM profiles carried by endogenous MPs, LC-MS/MS–based metabolomics were performed. We discovered that endogenous MPs formed in inflammatory exudates contain esterified biosynthetic precursors of novel specialized proresolving mediators (Fig. 2A). For example, the levels of MP-associated hydroxydocosahexaenoic acids, namely 14-HDHA and 17-HDHA, were high during the initiation phase of acute inflammatory response, decreased during the peak of inflammation, and accumulated in resolution, the phase in which potent anti-inflammatory and proresolving LMs are biosynthesized (13). Fig. 2B shows a representative mass spectrum of 17-HDHA, with the diagnostic ions used for identification criteria denoted, including mass/charge ratio (m/z) 245 and 273. Notably, endogenous MPs were devoid of unesterified monohydroxy fatty acids, suggesting that once liberated from MP membranes, they are made available to leukocytes within the exudate.
Following i.v administration of deuterium-labeled d5-DHA for tracking, circulating d5-DHA is made rapidly available to exudates (14) and was incorporated into endogenous MPs generated during the onset of acute inflammation. Additionally, these MPs also contained enzymatic metabolites of d5-DHA, namely d5-17-HDHA and d5-14-HDHA, deuterated biosynthetic pathway biomarkers for d-series resolvins and maresin biosynthesis, respectively, which reflects activation of these new pathways and their H(p)DHA intermediates (Fig. 2C). These results implicate endogenous MPs as intercellular communicators that can deliver proresolving LM precursors to inflammatory loci. Further experiments were performed to assess whether exogenous DHA was stored within MP phospholipids. When human PMNs were incubated with d5-DHA, MPs were generated containing both d5-DHA and d5-HDHA esterified into phosphatidylcholine (Supplemental Fig. 1A, 1B). Noteworthy, both cytosolic and secretory phospholipase A2 are induced during the resolution phase of inflammation (15). Secretory phospholipase A2 added to resolving MPs liberated esterified precursors from MPs (Supplemental Table I).
Human PMN-derived MPs display anti-inflammatory properties via ALX/formyl peptide receptor 2, the receptor for LXA4, Annexin-A1, as well as RvD1 (6, 16). Additionally, we demonstrated in this study that MPs enhanced efferocytosis (Supplemental Fig. 2) and contain precursors for proresolving LM, thus likely contributing to the beneficial properties attributed to these MPs. Because many biomaterials used for NP drug delivery can activate the circulatory system and cause nanotoxicity (for example, by uptake and activation of dendritic cells) (8), we sought new means to locally administer and activate proresolving cascades in vivo based on the endogenous MP system. Thus, newly constructed nanomedicines should possess multipronged agonist actions in resolution, activating endogenous ALX, releasing precursors for local proresolving mediators, as well as delivering their cargo of uploaded LM.
Harnessing human neutrophil-derived MPs as scaffolds, we constructed NPs containing either AT-RvD1 (4) or a stable analog of LXA4 (10). Following enrichment procedures and energy-induced conversion of MP to NPs, preparations were layered onto size-exclusion chromatography columns to separate free unincorporated lipids from MP-associated lipids, and eluate fractions were collected and taken for analyses including flow cytometry. Characteristically, enriched NPs (Fig. 3A, blue dot plot) were smaller than their MP counterparts (Fig. 3A, red dot plot) and also contained fluorescent phospholipid (Fig. 3B, blue histogram). NP sizing was accomplished by flow cytometry using nanosphere calibration beads (Fig. 3C, 3D). Sizing was further validated using negative stain electron microscopy of MPs (Fig. 3E) and NPRMs (Fig. 3F). Both methods showed smaller, more uniform sizing of NPs following enrichment procedures. Prior to each experiment, incorporation of AT-RvD1 or the LXA4 analog was determined using LC-MS/MS. Representative tandem mass spectrum of NP-associated AT-RvD1 (m/z 375.2) is shown from scheduled MRM of the m/z 375/215 transition pair at 7.4 min (Fig. 3G).
These humanized NPs were inherently anti-inflammatory, even without enriching with LM, counterregulating PMN infiltration in acute zymosan (0.1 mg) peritonitis by ∼25% following i.v. administration of 1 × 105 NPs (Fig. 4A). A similar reduction in PMN influx was observed with 300 ng stable LXA4 analog consistent with earlier findings (17). Remarkably, when the LXA4 analog was incorporated into human NPs, they drastically limited PMN numbers by ∼60%, showing they possess additional anti-inflammatory properties. Importantly, 1 × 105 NPs equated to an incorporation of 5.0–8.8 ng LX analog, effectively demonstrating that much lower amounts were needed locally to reduce PMN infiltration than analog alone. This proved dose dependent, with maximal NPRMs greatly limiting PMN recruitment (Supplemental Fig. 3A). Noteworthy, we found an inhibitory action on PMN recruitment when NPRMs were administered directly into the peritoneum, suggesting a direct action on resident macrophages, dampening their inflammatory response to zymosan (Supplemental Fig. 3B). We also assessed bioactivity of AT-RvD1–enriched NPs in murine peritonitis. Leukocyte recruitment and differential analyses were assessed at three separate intervals to quantify acute inflammatory response using self-limited inflammation (Fig. 4B). Local microbial administration resulted in a rapid increase in PMN infiltration, which peaked at 4 h postchallenge corresponding to the onset of inflammation. By 12 h, PMNs had declined, and the resolution interval (Ri) was calculated (i.e., time taken for maximal PMN to decrease 50%). NPs carrying AT-RvD1 drastically reduced PMN recruitment in microbial peritonitis, reducing maximal PMN infiltration from 9.4 ± 0.7 × 106 to 4.0 ± 0.8 × 106 and also shortened Ri from 7 h to ∼4.5 h (Fig. 4B).
Proresolving bioactions of these humanized nanomedicines were next evaluated in a keratinocyte wound-healing assay using in vitro epidermal abrasion (11). Following focal clearance of keratinocytes with an elevated electrical field pulse, keratinocyte healing from the perimeter into the clearance zone was assessed in real time by impedance sensing. This highly sensitive method indicated that AT-RvD1–containing nanomedicines enhanced wound closure rates as compared with either nonenriched NPs or equimolar amounts of AT-RvD1 (10 nM), demonstrating their proresolving properties (Fig. 4C).
TMDs are a significant clinical problem, causing craniofacial pain (2). Effective treatment of pain arising from inflammation of joints and muscles remains an unmet medical need and is implicated not only in TMDs but also in many other human diseases. To this end, we evaluated whether these nanomedicines would confer protection against CFA-induced inflammation of the TMJ. Indeed, 10 ng AT-RvD1 NPRMs or o-[9,12]-benzo-ω6-epi-LXA4 NPRMs drastically reduced the number of infiltrating PMNs into the inflamed TMJ 12 h after CFA-induced inflammation (Fig. 4D). Importantly, MPO levels in the contralateral TMJ were not elevated, and no significant differences were noted between buffer control and NPRM treatment groups (not shown). Of relevance, both RvD1 and RvE1 also attenuate inflammatory pain (18). Together, these results establish that biomimicking endogenous resolution mechanisms centered on anti-inflammatory and proresolving MPs, enhance the bioactions of potent LMs. NP delivery of AT-RvD1 or a stable analog of LXA4 prevented excessive neutrophilic infiltration to inflamed TMJs, thus circumventing ensuing granuloma formation and further joint destruction associated with overzealous leukocyte recruitment in this complex joint disease.
C.N.S. is an inventor on composition of matter and patents covering lipoxins, resolvins, and related compounds owned by Brigham and Women’s Hospital-Partners Health Care and licensed for clinical development. C.N.S. retains founder stock in Resolvyx Pharmaceuticals.
We thank Maria Ericsson for expert electron microscopy analysis (Harvard Medical School core facility) and Khalil Nasser for technical assistance.
This work was supported by National Institutes of Health Grants DE 019938 (to C.N.S.) and GM38765 (to C.N.S.), Arthritis Research UK Fellowship 18445 (to L.V.N.), and Wellcome Trust Programme Grant 086867/Z/08/Z (to M.P. and R.J.F.).
The online version of this article contains supplemental material.
Abbreviations used in this article:
- aspirin-triggered resolvin D1
- hydroxy-docosahexaenoic acid
- liquid chromatography tandem mass spectrometry
- lipid mediator
- lipoxin A4
- multiple reaction monitoring
- mass/charge ratio
- nano-proresolving medicine
- polymorphonuclear cell
- resolution interval
- temporomandibular joint disorder
- temporomandibular joint.
- Received December 1, 2010.
- Accepted March 14, 2011.
- Copyright © 2011 by The American Association of Immunologists, Inc.