Recurring Real-Time Monitoring of Inflammations in Living Mice with a Chemiluminescent Probe for Hypochlorous Acid

Open AccessCCS ChemistryCOMMUNICATION6 Jun 2022Recurring Real-Time Monitoring of Inflammations in Living Mice with a Chemiluminescent Probe for Hypochlorous Acid Sen Ye†, Bowei Yang†, Meiling Wu†, Zefeng Chen, Jiangang Shen, Doron Shabat and Dan Yang Sen Ye† Morningside Laboratory for Chemical Biology, Department of Chemistry, TheUniversity of Hong Kong, Hong Kong SAR 999077 †S. Ye, B. Yang, and M. Wu contributed equally to this work.Google Scholar More articles by this author , Bowei Yang† Morningside Laboratory for Chemical Biology, Department of Chemistry, TheUniversity of Hong Kong, Hong Kong SAR 999077 †S. Ye, B. Yang, and M. Wu contributed equally to this work.Google Scholar More articles by this author , Meiling Wu† School of Chinese Medicine, TheUniversity of Hong Kong, Hong Kong SAR 999077 †S. Ye, B. Yang, and M. Wu contributed equally to this work.Google Scholar More articles by this author , Zefeng Chen Morningside Laboratory for Chemical Biology, Department of Chemistry, TheUniversity of Hong Kong, Hong Kong SAR 999077 Google Scholar More articles by this author , Jiangang Shen School of Chinese Medicine, TheUniversity of Hong Kong, Hong Kong SAR 999077 Google Scholar More articles by this author , Doron Shabat *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] School of Chemistry, Faculty of Exact Sciences, Tel Aviv University, Tel Aviv 69978 Google Scholar More articles by this author and Dan Yang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Morningside Laboratory for Chemical Biology, Department of Chemistry, TheUniversity of Hong Kong, Hong Kong SAR 999077 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.021.202101108 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Probes for in vivo imaging of hypochlorous acid (HOCl), one of the most important reactive oxygen species in innate immunity, are urgently needed to understand the pathogenesis of autoimmune and neuroinflammatory disorders. As a strong oxidant, HOCl could bleach near-infrared sensors and inactivate luciferase readily, making in vivo imaging overwhelmingly challenging. Via fine-tuning of a selective HOCl sensing moiety, HOCl stable spacer, and bright chemiluminescent scaffold, we have developed HOCl-CL-510 as a highly selective and sensitive probe for HOCl detection both in vitro and in vivo. In particular, we achieved recurring real-time monitoring of HOCl in both acute and chronic inflammation models in living mice, providing a new chemical tool for dynamic monitoring of disease development with reduced usage of experimental animals. Download figure Download PowerPoint Introduction Hypochlorous acid (HOCl) is probably the most important disinfection reagent, widely used by humans in daily life and by neutrophils in innate immunity.1,2 As a strong oxidant and chlorination reagent, HOCl readily reacts with proteins, amino acids, lipids, and DNA, which could inactivate Escherichia coli within 0.1 s by destroying its vital systems.3 Neutrophil HOCl production, however, is a highly regulated event to precisely kill pathogens near inflammatory sites without broad damage, whereby signal transduction, neutrophil recruitment and activation, and HOCl burst are delicately orchestrated.4,5 Cellular HOCl is primarily produced from a reaction between H2O2 and chloride ion catalyzed by peroxidases (e.g., myeloperoxidase, MPO). If the delicate balance of the inflammatory cascade becomes deregulated, an autoimmune disorder could occur with aberrant HOCl production.6 In particular, rheumatoid arthritis is associated with higher plasma MPO levels and elevated HOCl production, which could further chlorinate tyrosine residues, leading to collagen damage.7 On the other hand, manually increasing HOCl in a precise location could contribute to enhanced immunity and improve the therapeutic efficacy.8 For example, MPO-derived nanomedicine was developed to promote platinum drug chemotherapy via in situ HOCl generation in a tumor.9 In this regard, selective and sensitive in vivo detection of HOCl is critical to understand its essential roles in pathogenesis and therapeutics.1,10 During the past decade, numerous HOCl fluorescent probes have been developed with high selectivity, sensitivity, and spatiotemporal precision to study its dynamic formation in living cells, tissue slices, and transparent zebrafishes.11–14 Recently, remarkable progress has been made to utilize near-infrared (NIR) HOCl fluorescent probes for mice imaging.15–19 However, since HOCl is a strong oxidant that could destroy the conjugating system readily (Figure 1a), a lack of HOCl-stable NIR fluorophore has limited further advancement in this field.1,2 An alternative for mice imaging is the luciferin/luciferase system, which requires luciferase transfection, luciferin, oxygen, and adenosine 5′-triphosphate (ATP) to release bioluminescence.20,21 However, luciferase could be inactivated by HOCl. Figure 1 | (a–c) Strategies for HOCl in vivo imaging. Download figure Download PowerPoint Recently, chemiluminescence has become an emerging platform for bioimaging without any laser excitation and luciferase transfection.22–25 Specifically, 1,2-dioxetane has been extensively used in activity-based sensing: a carefully designed masking group could be deprotected selectively by its analyte or enzymatic activity to release the energy stored in the chemical scaffold in terms of light emission.26–30 Chemiluminescent probes have demonstrated advantages of high sensitivity, low background, tunable kinetics, and operational simplicity.31–35 Very recently, a chemiluminescent probe derived from traditional aminophenyl fluorescein (APF) was developed for in vivo imaging of HOCl.36 Herein, we report the design, synthesis, characterization, and bioimaging applications of HOCl-CL-510, our uniquely designed HOCl chemiluminescent probe. HOCl-CL-510 is highly selective and sensitive via activity-based sensing of 2,6-dichlorophonel toward HOCl, which could be extensively applied to in vitro biochemical systems, cellular assays, and living mice. Endogenous HOCl production of macrophages in response to nutrient depletion was robustly visualized. Moreover, recurring real-time HOCl monitoring was successfully performed in the lipopolysaccharide (LPS)-stimulation mice model and an arthritis mice model with HOCl-CL-510, thereby providing a versatile approach for in vivo dynamic sensing. All experimental animal protocols were conducted in accordance with the National and Institutional Guidelines on Ethics and Biosafety, with approval by the Committee on the Use of Live Animal in Teaching and Research (CULATR) at The University of Hong Kong (Approval No. CULATR 5069-19). Results and Discussion Design and synthesis of Probe 1, Probe 2, and HOCl-CL-510 Our design relied on activity-based sensing,37 during which the masked phenoxy-dioxetane could be selectively deprotected by HOCl to release chemiluminescence. Since HOCl is a highly reactive bleaching reagent that could be scavenged via oxidation and chlorination, the HOCl responsive moiety should meet the stringent requirements of high sensitivity, selectivity, and rapid response to enable HOCl chemiluminescent imaging. By introducing two ortho chlorine substituents into phenol (2,6-dichlorophonel moiety), the electron-rich phenoxide could be the predominant species under physiological conditions, showing markedly enhanced reactivity toward HOCl.13 The designed probe Probe 1 (Figure 2a) was synthesized successfully and characterized (see Supporting Information). Figure 2 | (a) Chemical structures of Probe 1, Probe 2, and HOCl-CL-510. (b) Time course of HOCl-CL-510 (10 μM) reacted with various ROS. [ROS] = 100 μM, except 10 μM for •OH, ONOO–, and HOCl. Measurements were conducted using potassium phosphate buffer, pH 7.4, at 37 °C. (c) Luminescence response of HOCl-CL-510 reacted with various ROS; bars represent emission intensities at 0 (light grey), 15 (grey), 30 (dark grey), and 45 (red) s. (d) Luminescent image and profile of HOCl-CL-510 (5 μM) treated with increasing amounts of HOCl (0–10 μM). The image was acquired within 2 min after HOCl addition. (e) The luminescence intensity of HOCl-CL-510 as a function of HOCl concentrations (data are mean ± S.D., n = 3). Download figure Download PowerPoint First, we evaluated the chemiluminescent responses of Probe 1 toward HOCl by adding physiological levels (μM range) of HOCl and measuring the light emission without any laser excitation. As shown in Supporting Information Figure S1, Probe 1 (10 μM) only gave a moderate response upon 10 μM HOCl treatment. Surprisingly, the chemiluminescent response started to show a dose-dependent increase between 10 and 20 μM of HOCl. However, the sparse photon released and insensitivity toward HOCl made Probe 1 unsuitable for HOCl in vivo imaging. To increase the photon budget for bioimaging, we sought to replace the carboxylic ester group in Probe 1 with a carboxylic acid group in Probe 2, which yielded better solubility in aqueous solution and improved the chemiluminescent kinetics.24 Probe 2 indeed showed higher radiance, but it was still not sensitive toward HOCl ( Supporting Information Figure S2). We reasoned that the middle aromatic spacer in Probe 1 and Probe 2 might scavenge HOCl, as HOCl is also a potent chlorination reagent. By introducing an electron-withdrawing substituent (i.e., chlorine atom) on the electron-rich spacer, we finally designed HOCl-CL-510, presuming that it might prevent undesirable HOCl consumption to improve the sensitivity. Reactivity and selectivity of HOCl-CL-510 toward HOCl Probe HOCl-CL-510 was synthesized following a scheme similar to that of Probe 1. To our delight, HOCl-CL-510 showed markedly improved performance. Upon treatment with 10 μM HOCl, HOCl-CL-510 (10 μM) produced a 216-fold enhancement of chemiluminescent signal within 30 s (Figure 2b and Supporting Information Figure S2); the radiance of HOCl-triggered chemiluminescence is also increased 1000 times (from 105 to 108 p/s/cm2/sr), compared with Probe 1. More importantly, HOCl-CL-510 was highly selective toward HOCl; other potentially competing reactive oxygen species (ROS) tested including 1O2, ROO•, TBHP, NO, O2•–, H2O2, •OH, and ONOO–, showed only negligible responses (Figures 2b and 2c). To evaluate its real performance for HOCl imaging, 5 μM HOCl-CL-510 was treated with 0, 2, 4, 6, 8, and 10 μM HOCl in a 96-well plate, and the photon released was acquired by an IVIS® Spectrum Imaging System. We observed a dose-dependent increase and a linear relationship between the luminescence intensities and HOCl concentrations (Figures 2d and 2e), indicating that our probe could robustly visualize micromolar HOCl. Morever, the cleaved benzoate product, after HOCl-triggered chemiexcitation, was detected successfully using ultra-performance liquid chromatography coupled with mass spectroscopy (UPLC-MS) analysis, further confirming our design principle (Figure 1c and Supporting Information). Our data supported that HOCl-CL-510 is a selective and sensitive chemiluminescent probe for HOCl. Molecular imaging of endogenous HOCl production in living cells Cytotoxicity test (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT cell viability assay) also confirmed that HOCl-CL-510 has low cellular toxicity ( Supporting Information Figure S3). We then applied HOCl-CL-510 in live-cell imaging. Previous work by other investigators revealed that elevated superoxide and hydrogen peroxide could be detected during the starvation challenge.38–40 Also, our study confirmed that H2O2 was generated rapidly during amino acid restriction by Hank’s balanced salt solution (HBSS).41,42 The endogenous HOCl production, however, is far less investigated. Thus, we considered examining the production of HOCl in a 96-well plate using HOCl-CL-510. In this experiment, RAW264.7 macrophages were treated with HBSS at varying times (0–24 h) before imaging. N-Acetylcysteine (NAC; 5 mM) was added to intervention groups and coincubated for 10 min to remove HOCl before imaging. Then HOCl-CL-510 (5 μM) was added to image the endogenous HOCl generated from starvation. As shown in Figure 3, starvation-induced HOCl and basal HOCl could trigger our chemiluminescent probe to release photon fluxes effectively. Two-hour starvation already induced a noticeable increase of luminescent signal, while 24 h starvation led to about onefold enhancement of photon fluxes, compared with untreated groups. Those photon fluxes could be abolished by NAC, a nonselective yet effective HOCl scavenger, which confirmed that HOCl-CL-510 could detect cellular HOCl selectively. As 1,2-dioxetane might slowly react with alkyl thiols at higher concentrations,43 taurine was also used to attenuate the starvation-induced chemiluminescence with HOCl-CL-510 ( Supporting Information Figure S4). Collectively, the robust performance of cell imaging in the 96-well plates could be explored further to develop high-throughput cellular assays for HOCl stimulating reagents or scavengers.14 Since a laser is not needed for chemiluminescent imaging, those autofluorescent drugs and compounds in a high-throughput screening library could, in principle, be screened by using HOCl-CL-510 without worrying about false positive hits due to autofluorescence. Figure 3 | Chemiluminescent imaging of starvation challenged RAW264.7 macrophages. Cells were treated with HBSS for 0–24 h, then HOCl-CL-510 (5 μM) was added. The chemiluminescence images were acquired with an acquisition time of 0.1 min. NAC (5 mM) was added 10 min before imaging in intervention groups (data are mean ± S.D., n = 3). Download figure Download PowerPoint Recurring in vivo imaging of endogenous HOCl in stimulated mice After confirming that HOCl-CL-510 could detect HOCl in both chemical systems and cellular assays, we further applied the probe to detect HOCl in living mice. First, we attempted the subcutaneous injection of HOCl in living mice and employed HOCl-CL-510 for HOCl detection. As shown in Supporting Information Figure S5, the probe detected the exogenously injected HOCl in the mice with high sensitivity and excellent signal-to-noise ratio, owing to the bright chemiluminescent nature and rapid response of the probe. LPS, composed of a lipid (e.g., Lipid A) and O-antigens, could induce an acute immune response, including toll-like receptor 4 (TLR-4) signaling, transcription factor (e.g., NF-κB) activation, cytokine production, and finally, macrophage stimulation.5,44,45 Endogenous HOCl burst is expected during an LPS-challenged inflammatory cascade in living mice. Thus, we performed an experiment, giving the mice in treatment group intraperitoneal (i.p.) injection of LPS, while the mice in the control group received i.p. injection of the same amount of phosphate-buffered saline (PBS) as the vehicle. Taurine (a selective HOCl scavenger) or NAC (a broad-spectrum antioxidant) was injected into the intervention group. Then the mice were injected with HOCl-CL-510 (0.25 mM in 100 μL PBS) probe 3 h after LPS stimulation. As shown in Figure 4a, the unstained mice (no probe injection) did not show any background photon fluxes. LPS challenged HOCl burst could be robustly visualized as a onefold increase in photon fluxes in the LPS treatment group using HOCl-CL-510, compared with the control group. Moreover, this increased chemiluminescent signal could be attenuated effectively by taurine or NAC treatment, with taurine being even more potent than NAC in removing HOCl in vivo. The HOCl-triggered photon fluxes lasted for ∼20 min in the living mice, providing an adequate time window for imaging. Figure 4 | Recurring in vivo chemiluminescent imaging of HOCl-CL-510 in response to LPS stimulated HOCl production in mice. HOCl-CL-510 (100 μL 0.25 mM in PBS) was i.p. injected, and the chemiluminescence images were acquired over time with an acquisition time of 2 min (red 3 min; black 9 min; grey 15 min; light grey 21 min). (a) Representative images of mice and photon flux quantification of 3 h LPS (0.3 mg) stimulation. NAC (1.6 mg) and taurine (1.5 mg) were i.p. injected into the mice in the intervention groups. (b) The chemiluminescent signal is completely gone after 3 h of probe injection. (c) Recurring in vivo chemiluminescent imaging of mice and photon flux quantification of 6 h LPS stimulation. Download figure Download PowerPoint The self-decaying property of HOCl-CL-510 was explored further for recurring real-time in vivo imaging. The fluorophores of traditional fluorescent probes could only be degraded slowly; thus, repetitive imaging might be troublesome due to residual signal (the more stable, the worse). In our case, the chemiluminescent signal disappeared after 3 h in the living mice (Figure 4b). Upon a second round of probe injection in the same mice after 6-h LPS stimulation (Figure 4c), HOCl could still be visualized selectively. Finally, we employed the probe in a 14-day chronic inflammation model of arthritic mice induced by complete Freund’s adjuvant (CFA, inactivated mycobacterium). Upon CFA stimulation, subsequent macrophage recruitment and activation results in a HOCl burst in the tibiotarsal joint,46,47 believed to play a critical role in the initiation and progression of arthritis.7,16 CFA was injected into the right ankle of mice, and then apparent inflammation and swelling could be observed on the next day, indicating successful recruitment of macrophages for developing arthritis. HOCl-CL-510 was injected into the lower right or left ankle to detect in situ generated HOCl. As shown in Figure 5d, CFA induced a significantly higher level of HOCl readily near the inflammatory site on day 1 (see Supporting Information Figure S6 for full mice images). After 7 days, the swelling was still observable on the right ankle, and HOCl burst persisted (Figures 5b and 5c), visualized successfully by in vivo recurring real-time imaging with HOCl-CL-510. On day 14 post CFA injection, the stimulated chronic inflammation on the right ankle could still be detected robustly, and no translocation of inflammation to the left ankle was observable (Figure 5d). Figure 5 | Recurring in vivo chemiluminescent imaging of HOCl-CL-510 in an arthritic mice model. (a) 14-day workflow for recurring imaging of arthritis mice model. (b) HOCl-CL-510 (30 μL 0.4 mM in PBS) was injected into the tibiotarsal joints of model mice and control mice, and the chemiluminescence images were acquired three times with an auto acquisition setting (typically 0.5 min). The arthritis model was induced by injecting CFA into the right ankle of mice, and the left ankle was regarded as a control. (c) Quantification of total fluxes in Figure 5b. (d) Representative chemiluminescent images were taken on the 1, 7, and 14 days post CFA injection. Download figure Download PowerPoint As a proof of concept, our self-decaying probe HOCl-CL-510 enabled the first in vivo recurring real-time imaging of HOCl in both acute and chronic inflammation models, providing a starting point for time-lapse monitoring of disease development using the same mice. However, increasing the sample size would enable a more robust evaluation to reduce biological differences between individual animals. A good balance between recurring imaging of the same mice and biological replicates is needed, which also aligned with the principles of 3Rs (Replacement, Reduction, and Refinement) for animal research. Conclusion Through fine-tuning of the HOCl-responsive moiety, spacer, and phenoxy-dioxetane luminophore, we have successfully developed a novel HOCl probe, HOCl-CL-510, for recurring real-time monitoring of HOCl. The rapid HOCl sensing, HOCl-stable spacer, and unique chemiexcitation emission are critical for its high sensitivity in bioimaging. Moreover, its self-decaying property implies a rapid signal clearance without interferences with optical residuals, enabling dynamic monitoring of HOCl during pathogenesis. We believe that this work would provide a valuable molecular probe for studying HOCl-related disease models and screening of new therapeutics in cells and animals, thereby revealing important design principles for HOCl sensing and defining new directions for bioimaging to enhance animal welfare. Supporting Information Supporting Information is available and includes detailed experimental procedures, probe synthesis, characterization data, imaging protocols, and six supporting figures. Conflict of Interest The authors declare no conflict of interest. Funding Information This work was supported by The University of Hong Kong, Morningside Foundation, Hong Kong Research Grants Council Area of Excellence Scheme (grant no. AoE/P-705/16 to D.Y.) and National Natural Science Foundation of China (grant no. 21961142011 to D.Y.). D.S. is thankful to the Israel Science Foundation-China Joint Funding Program. 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