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G: change in serum free fatty acids from 0 to 30 min of the ITT. Macrophage infiltration into obese adipose tissues suppresses the induction of UCP1 level in mice. Adipose tissue, as well as the liver, was investigated for macrophage infiltration and the presence of inflammatory mediators. These discrepancies may be explained by many factors, including, but not limited to, the type of HFD utilized, the mode of CLD administration (intraperitoneal vs. intravenous), and the dose, timing, and duration of CLD treatment. The effects of operating conditions and local environment, Characterization of Nebulized Liposomal Amikacin (Arikace. Use this unique QR (Quick Response) code with your smart device. Values are meansSE; n = 915 mice per group. Eventually, liposomal encapsulated drugs can specifically be directed towards or attracted by designated pathogenic cells and organs [90] (Fig. On the other hand, IL-6, similar to the finding in epididymal adipose tissue, was downregulated as a result of HFD consumption (P < 0.05). The CD11b + macrophages in lung (A) and colon (B) tissue sections were detected by immunohistochemistry. Reports vary as to whether or not other types of phagocytic cells are killed by clodronate liposomes. 2011 Dec;25(12):4301-11. doi: 10.1096/fj.11-189472. Since this aggregation phenomenon must be a saturable process, multiple injections of clodronate liposomes timed closely together will saturate the binding sites on the neutrophils and/or temporarily deplete serum components which contribute to the aggregation. Hematological findings and complications of COVID19. Conedera F.M., Pousa A.M.Q., Mercader N., Tschopp M., Enzmann V. Retinal microglia signaling affects Mller cell behavior in the zebrafish following laser injury induction. Among them, elevated levels of IL-6 have been known to exacerbate the pathogenic severity of COVID-19 through TNF- mediated activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) signalling [6], [43], [44]. Thus the neutrophil infiltration makes physiological sense, as they are the only primary professional phagocytic cell remaining after macrophage and dendritic cell depletion. Briefly, epididymal and perirenal adipose tissues, as well as a portion of the liver, were homogenized in Mueller buffer containing a protease inhibitor cocktail (Sigma Aldrich) (8). Intranasal instillation of clodronate in liposomes and of diphtheria toxin in CD11c-DTR mice was used to deplete macrophages. In an immunofluorescence based study by Wang et al. Interestingly, while, as expected, the HFD increased adipose tissue macrophage markers, no significant difference was found with respect to gene expression of overall macrophage markers (F4/80 and CD68) or M1 (CD11c) and M2 (CD206) macrophage markers between the HFD groups (Fig. Kyriazopoulou E., Huet T., Cavalli G., Gori A., Kyprianou M., Pickkers P., et al. When the absolute change in serum FFAs over the 30-min period was examined, there was a main effect of CLD treatment to impair the decrease in serum FFAs compared with PBS treatment. ). *Significantly different from start of liposome treatment at 16 wk. Lanthier N, Molendi-Coste O, Cani PD, van Rooijen N, Horsmans Y, Leclercq IA. It has been reported that intravenous administration of 20mg of clodronate encapsulated with unilamellar liposomes significantly reduced macrophages and diminished inflammation in an experimental rat model of arthritis, while treatment of free clodronate had no effect in targeting macrophages [93], [94]. Bar graphs not sharing a common letter (a, b, c, d) are significantly different from one another (P < 0.05). Clodronate treatment significantly depletes macrophages in chickens. Jeong GU, Lyu J, Kim K-D, Chung YC, Yoon GY, Lee S, et al. 3B). Accessibility Rossini M., Braga V., Gatti D., Gerardi D., Zamberlan N., Adami S. Intramuscular clodronate therapy in postmenopausal osteoporosis. SAP models were prepared by injection of sodium taurocholate into the subcapsular space of rat pancreas. A clinical trial by Frediani et al. Besides, the whole animal in vivo experiments described that the use of clodronate-liposome solution leads to efficient depletion of macrophages in the bone marrow, spleen, liver, lungs, brain, gut, peritoneal cavity, lymph nodes and in circulation thereby, suggesting liposome-encapsulated clodronate can spread to various organs in the body [96] (Fig. Santa Cruz A., Mendes-Frias A., Oliveira A.I., Dias L., Matos A.R., Carvalho A., et al. 9). interpreted results of experiments; J.E.B., R.T.E., and M.S.C. Moreover, after controlling for the benefits of weight loss, the accompanying detrimental side effects limit regular CLD treatment as an effective therapeutic strategy. Aladhami AK, Unger CA, Ennis SL, Altomare D, Ji H, Hope MC 3rd, Velzquez KT, Enos RT. Of note, the secretome of activated macrophages, mast cells, endothelial, epithelial cells and microglia appears to be the underlying basis of cytokine storm in COVID-19 [6], [7], [8], [9], [10]. CCR2 / mice were used to assess the contribution of recruited macrophages. A very important result from this study was that DSPC/Chol clodronate liposomes (shown to leak very little in serum) did not inhibit the growth of the RAW 264 cells up to 100 M clodronate (29 g/ml) while the IC 50 (clodronate concentration at which 50% cell growth is inhibited compared to control) of the DSPC/DSPG/Chol formulations were all less than 10 M. The macrophage depleting drug, liposome encapsulated clodronate has gained a greater clinical advantage and use in the treatment of cancer-associated bone diseases and osteoporosis. Driscoll, D.L. PBS-liposome-treated groups were pair-fed to their CLD-treated dietary counterparts. D: intraperitoneal insulin tolerance test (ITT) following a 5-h fast. Besides, liposamal clodronate treatment has been reported to attenuate the zymosan-induced activation of NF-kB, Fos expression and hypothermia [112]. Thus it is imperative that a complete blood count be part of a routine assessment when interventions aimed at modulating macrophage populations are utilized. https://doi.org/10.1128/spectrum.01091-22. Values are meansSE; n = 915 mice per group. However, a time-course study with CLD treatment would be necessary to confirm this. Body composition and metabolic data after 16 wk of dietary treatment [high-fat diet (HFD) or low-fat diet (LFD)] before start of liposome treatment. Pancreatic islets were analyzed for the presence of neutrophils by flow cytometry (A), the production of CXCR2 ligands after culture by ELISA (B), or the expression of CXCL2 by the -cells by flow . However, relative to weight-matched control animals, CLD treatment did not rescue HFD-induced impaired glucose metabolism. injections of either clodronate-loaded liposomes or phosphate-buffered saline (PBS)-loaded liposomes twice during the last week of HFD. drafted manuscript; J.E.B., R.T.E., K.T.V., M.S.C., A.T.S., O.P.M., C.M.R., and E.A.M. Principles of laboratory animal care were followed, and the Institutional Animal Care and Usage Committee of the University of South Carolina approved all experiments. An obese phenotype was successfully achieved, as the HFD-fed mice gained significantly more body weight and fat mass and displayed a significantly greater percentage of body fat (Fig. Initial tests with a single injection of 200 ul iv were well-tolerated by the mice, gave very effective depletion of Kupffer . 3, Fig. Tyurina, O. Gorelik, S. Arepalli, D. Schwegler-Berry, A.F. TaqMan reverse transcription reagents and gene expression assays (Applied Biosystems, Foster City, CA) were used to reverse transcribe and analyze expression of the following genes: F4/80, CD68, CD11c, CD206, MCP-1, TNF, IL-10, IL-6, and IL-1. Neutrophil infiltration into the adipose tissue serves two probable functions: 1) removal of dead immune cells affected by CLD treatment and 2) adoption of the role of macrophages by cleaning up dead adipocytes. Fig. Can emapalumab be life saving for refractory, recurrent, and progressive cytokine storm caused by COVID-19, which is resistant to anakinra, tocilizumab, and Janus kinase inhibitors. The HFD increased all fat pad weights (epididymal, mesenteric, and perirenal) relative to the LFD-fed mice (Fig. Therefore, through the utilization of pair-feeding, our study was designed to determine if macrophage depletion could serve as an effective intervention to reduce obesity-driven adipose tissue inflammation and improve metabolic dysfunction beyond the weight loss seen with reduced energy intake. The duration (2 treatments/wk for 4 wk) of CLD-liposome administration was significantly longer in our study than in these previous studies and may be largely responsible for the discrepancy in outcomes between studies. Opperman K.S., Vandyke K., Clark K.C., Coulter E.A., Hewett D.R., Mrozik K.M., et al. Romagnoli S., Peris A., De Gaudio A.R., Geppetti P. SARS-CoV-2 and COVID-19: from the bench to the bedside. Therefore, we analyzed whole blood to see if CLD treatment impacted hemoglobin levels. As the severity of the disease progresses, COVID-19 escalates kidney injury with obvious signs of hematuria and nephritis due to inflammatory reactions in the glomerular compartments of the kidneys [29]. However, the use of free clodronate molecules in specifically targeting abruptly activated immune cells appears to be a highly challenging approach against COVID-19. After 16 wk on a high-fat diet (HFD) or the AIN-76A control (low-fat) diet (LFD) (n = 30/diet treatment), male C57BL/6J mice were assigned to a CLD- or PBS-liposome treatment (n = 15/group) for 4 wk. Membranes were stained with a Ponceau S solution to verify equal protein loading and transfer efficiency. Boekel L., Wolbink G.J. Once saturation is complete, clodronate liposomes will be available to the phagocytes. (A) To deplete neutrophils . Previous studies revealed that glycyrrhizic derivatives possess antiviral effects against the SARS-CoV-2 and modify the inflammatory signatures in COVID-19 [60], [61]. Moreover, animal experimental studies revealed that treatment of liposamal clodronate leads to depletion of microglia in the brain and retina [110], [111]. Graphical illustration for the clodronate liposomes mediated depletion of activated microglia in COVID-19. Epub 2018 Dec 21. 129, No. The PBS-treated groups were pair-fed the same amount of food as their CLD-treated counterparts. official website and that any information you provide is encrypted See this image and copyright information in PMC. Fluorescent DiI Liposomes: a suspension of artificially prepared lipid vesicles encapsulating an aqueous PBS solution, labelled with the fluorochrome DiI. Southam, M. Dolovich, P.M. OByrne, M.D. Clodronate liposomes was developed by Professor Nico van rooijen of Vrije University in Amsterdam, the Netherlands. 2.Body morphology following dietary [high-fat diet (HFD) or low-fat diet (LFD)] and liposome treatment. Yang M, Chen X, Xu Y. K00 CA234920/CA/NCI NIH HHS/United States, K99 AT009206/AT/NCCIH NIH HHS/United States, R00 AT009206/AT/NCCIH NIH HHS/United States, R21 CA191966/CA/NCI NIH HHS/United States, F99 CA234920/CA/NCI NIH HHS/United States, NCI CPTC Antibody Characterization Program. This finding led us to believe that the tissue PMCs we examined histologically were neutrophils. Moreover, repeated CLD treatment induced neutrophilia and anemia, increased adipose tissue mRNA expression of the proinflammatory cytokines IL-6 and IL-1, and augmented circulating IL-6 and monocyte chemoattractant protein-1 concentrations ( P < 0.05). A method for intratracheal instillation of endotoxin into the lungs of mice. Fig. Microbiol Spectr 2022:e0109122. A.A. Shvedova, E.R. to deplete resident AMs prior to adoptive transfer. Clodronate liposome treatment in a mouse model of colorectal cancer revealed a reduction in the expression of macrophage cell markers and cytokines such as IL-13, TGF-, IL-10 and CCL-17 in the colon leading to arrest in tumor progression [99]. Clodrosome is a multilamellar liposome suspension in which clodronate is encapsulated in the aqueous compartments of the liposomes. Unlike systemic administration, intratracheal administration of free clodronate at higher doses has also been shown to also deplete alveolar macrophages. Autoradiography films were scanned and blots were quantified using scientific imaging software (ImageJ). As expected, the HFD significantly increased the gene expression of the monocyte chemoattractant MCP-1, the proinflammatory cytokine TNF-, and the anti-inflammatory cytokine IL-10 relative to the LFD groups (P < 0.05). al. Additionally, a complete blood count was performed. We hypothesize that the greater degree of toxicity in the LFD CLD group is the result of an immunocompromised state from severe macrophage depletion not reached in the HFD CLD group. This is supported by the observation that Ly6G-positive cells surround adipocytes in the CLD-treated mice. Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro. (However, the earlier Buiting, When the DSPG clodronate liposomes were extruded (avg diameter reduced to 128 nm), they were able to inhibit the growth of L929 fibroblasts, While the Penn-Century Microspray device[21] does generate a larger droplet size, the aerosolization (atomization) occurs within the trachea, so that the entire volume dosed is directly delivered to the lung. B: hepatic lipid accumulation. 4A; P < 0.05). Roles of macrophages in measles virus infection of genetically modified mice. Thus, formulated liposomes can efficiently be attracted and observed by the lungs via pulmonary surfactants. Fig. Moreover, the concept that reduced energy intake is due to CLD-liposome treatment and subsequent macrophage depletion, and not liposome treatment, in general, is supported by the findings of Lee et al., who reported hypothalamic inflammation and reduced energy intake in their macrophage-depleted mice (29). Initially, we used the manufacturers recommended dose of 0.05 mg/g body wt. We employed two reagents to deplete specific effector components in vivo: liposomal clodronate (LC) for depleting macrophages, and cobra venom factor (CVF) for depleting complement. A cohort study by Schurink et al. For this procedure, mice were briefly anesthetized via isoflurane inhalation, and lean mass, fat mass, and percent body fat were assessed by dual-energy X-ray absorptiometry (Lunar PIXImus). Our results suggest that blood monocytes play a central role in regulating neutrophil trafficking in the lung. Front Pharmacol 2021;12. Verschoor C.P., Puchta A., Bowdish D.M.E. In the HFD setting, CLD treatment significantly decreased TNF- gene expression (31%), which corresponded with the similar decrease (30%) in F4/80 mRNA (Fig. The clodronate solution (5 ml) was added to the liposome preparation and mixed thoroughly. Whole blood collected at euthanization (4 wk after liposome treatment) was assessed for mean corpuscular hemoglobin concentration, as well as hemoglobin and %hematocrit. Heath, A. Urtti. Satyanarayanan et al. Notably, in some individuals, COVID-19 vaccination developed destructive macrophage activation and multisystem inflammatory disease similar to the pathogenesis seen in COVID-19 [124], [125]. For negative controls, a serial section was assayed exactly as described for the treated section, but without the primary antibody. As a consequence, the sum of all three fat pads was also increased (Fig. Sakamoto T, Nitta T, Maruno K, Yeh YS, Kuwata H, Tomita K, Goto T, Takahashi N, Kawada T. Am J Physiol Endocrinol Metab. 1 Additionally, a complete blood count was performed. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. 7A; P < 0.05). Inflammatory responses and inflammation-associated diseases in organs. Ali N. Elevated level of C-reactive protein may be an early marker to predict risk for severity of COVID-19. Zheng W, Huang X, Lai Y, Liu X, Jiang Y, Zhan S. Glycyrrhizic acid for COVID-19: findings of targeting pivotal inflammatory pathways triggered by SARS-CoV-2. al. The sample size in the LFD CLD and HFD CLD groups was reduced to 9 and 14 mice, respectively, due to apparent toxicity of CLD treatment. Patel M., Dominguez E., Sacher D., Desai P., Chandar A., Bromberg M., et al. This study was designed to determine if macrophage depletion via clodronate-liposome (CLD) treatment could serve as an effective intervention to reduce obesity-driven inflammatory and metabolic impairments independent of changes in energy intake. Epub 2011 Sep 12. This idea is supported by the significant increase in epididymal mRNA expression of the potent monocyte chemokine MCP-1, as previously shown with acute intraperitoneal CLD-liposome treatment (28), a higher concentration of circulating MCP-1, and tissue neutrophilia in the HFD CLD group. ). However, future experimental evaluations and pharmacodynamicsof liposome encapsulated clodronate in preclinical models of COVID-19 needs to be considered. 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