Organic and inorganic nanoparticles (NPs) have shown promising outcomes in transdermal drug delivery. NPs can not only enhance the skin penetration of small/biomacromolecule therapeutic agents but can also impart control over drug release or target impaired tissue. Thanks to their unique optical, photothermal, and superparamagnetic features, NPs have been also utilized for the treatment of skin disorders, imaging, and biosensing applications. Despite the widespread transdermal applications of NPs, their delivery across the stratum corneum, which is the main skin barrier, has remained challenging. Microneedle array (MN) technology has recently revealed promising outcomes in the delivery of various formulations, especially NPs to deliver both hydrophilic and hydrophobic therapeutic agents. The present work reviews the advancements in the application of MNs and NPs for an effective transdermal delivery of a wide range of therapeutics in cancer chemotherapy and immunotherapy, photothermal and photodynamic therapy, peptide/protein vaccination, and the gene therapy of various diseases. In addition, this paper provides an overall insight on MNs’ challenges and summarizes the recent achievements in clinical trials with future outlooks on the transdermal delivery of a wide range of nanomedicines. Skin is responsible for enclosing and protecting the human body against the invasion of microorganisms, allergens, toxins, and UV irradiation . Meanwhi®, Transfersomes®, Invasomes®, Vesosomes®, Proliposomes, and Pharmacosomes® have been introduced as reviewed before . In. In98]. Despite the efforts made to develop new cancer treatment strategies, chemotherapy has remained an important therapeutic modality. Numerous nanomaterials have been investigated as drug carriers to enhance the therapeutic efficacy and bioavailability of anti-cancer agents. Nonetheless, the systemic administration of anti-cancer therapeutics can trigger serious side effects . Using tAs an immune reactive component, skin tissue could be a site for an efficient immune response against a wide range of antigens. Different immune cells including the dendritic cells (DCs), Langerhans cells (LCs), and antigen-presenting cells (APCs) are involved in an efficient immune response. A high density of these cells, as well as their accessibility, has highlighted skin as one of the most important vaccination sites . In receConsidering the potential of MNs to improve percutaneous delivery, MN-mediated transcutaneous immunization can be an effective method for the stimulation of immune responses. For instance, dMNs consisting of poly (methyl vinyl ether) and maleic anhydride (PMVE/MA) laden with PLGA NPs-containing model antigen  was utilized to target LCs. This approach has shown proper in vivo protection against melanoma (expressing B16 antigens) and para-influenza in a murine model by activating antigen-specific CD8+ T lymphocytes in tumors and viruses, respectively. Moreover, this study showed that antigen encapsulation in NPs can prolong the antigen’s retention time in the skin and enhance the antigen stability in MNs. Therefore, this approach could provide proper antigen protection in the case of OVA-expressed melanoma cells . A few sPhotothermal therapy (PTT) with near-infrared (NIR) irradiation is an interesting alternative technique for chemotherapy that can be combined with conventional chemotherapy to promote a synergistic effect. In PTT, the photothermal agents convert light to heat, which can induce thermal cell injury, membrane damage, and protein denaturation . FurtherTo achieve combined synergistic effects, chemotherapeutic drugs and photothermal agents should be simultaneously delivered precisely to the tumor site. However, finding an administration route for co-delivery has remained a big challenge that can be addressed by MNs. For this purpose, DOX-encapsulated dissolvable hyaluronic acid MNs (DOX-loaded HA MNs) containing a PEGylated gold nanorod (PEGylated-GNR) as a NIR light-responsive agent was developed for human epidermoid cancer therapy . In thisAfter inserting MNs into the tumor site, the MNs dissolved and released ICG NPs and 1-methyl-tryptophan (1-MT) as an indoleamine 2, 3-dioxygenase (IDO) blockade. Following NIR irradiation, the PTT effect of ICG-NPs can ablate tumor cells, warn the immune system, and promote interferon-γ (IFN-γ) secretion. Furthermore, the pre-apoptotic calreticulin (CRT) translocation starts generating an “eat me” signal to the cell surface that causes the uptake of death cancer cells by APCs. Other damage-mediated molecular patterns including some heat shock proteins (HSP70) can also help in the APCs’ maturation. Then, the released 1-MT can prevent the IDO activity to catalyze the tryptophan (Trp) degradation into kynurenine (Kyn). The local co-delivery of ICG-NPs with IDO blockade not only can destroy the primary tumor cells but also inhibit the growth of distant tumor cells and lung metastases. Reprinted with permission from .Photodynamic therapy (PDT) is a non-invasive approach to treat various tumors and non-neoplastic diseases . PDT inv®S-97 MNs) was recently explored for the skin delivery of the NIR photosensitizer, Redaporfin™ [MN-mediated PDT has been investigated for its possible applications in tumor treatment. Donnelly et al. explored the ability of solid silicon MNs for tumor therapy utilizing the poke-and-patch approach . They shaporfin™ . In vitraporfin™ . NotablyAs mentioned before, the delivery of highly potent photosensitizers to the tumor site is vital for the excellent therapeutic efficacy of the PDT approach. Some recent studies reported photosensitizer-loaded NP formulations for not only controlled delivery but also for combinational therapy incorporating two or more drugs ,145. ForWhen used alone, the PDT approach suffers from several challenges, such as low efficacy and systemic phototoxicity ,148. To 3 microparticles showed notable mechanical strength and sustained release in comparison with pure PVP MNs [MN technology has offered a revolutionary platform for protein delivery. There are generally serious challenges for the TDD of therapeutic proteins such as their susceptibility to degradation and relatively large molecule size . Concern PVP MNs . This st PVP MNs . For thi PVP MNs . Glucose PVP MNs . Altoget2O2-responsive NPs were designed to obtain fast and painless administration [x was co-loaded into the NPs to catalyze blood glucose to gluconic acid and generate H2O2 as a reaction byproduct resulting in NPs disintegration and insulin release. Therefore, the NPs act as both the glucose-sensing agent moieties and the insulin release actuator. NPs can provide basal insulin release in a controllable manner. Moreover, they facilitate insulin release in response to a hyperglycemic condition [The ongoing development of smart insulin delivery based on MN technology in combination with NPs has shown the potential to improve the quality of life of diabetic patients. In recent works, the MNs integrated with insulin-loaded, Hstration ,162. In ondition . These rMNs have been utilized for transdermal vaccination and the immunotherapy of skin tumors. Zhou et al. developed MNs containing transfersomes co-encapsulated anti-PD1 (antigen) and polyinosinic:polycytidylic acid (adjuvant) which were functionalized with αCD40 (DCs targeting ligand). The accumulation of the transferosomes in tumor-draining lymph nodes enhanced the maturation of DCs and improved Th1 immune responses. An improved T cell activation and infiltration was reported in a mouse melanoma model. Moreover, the activity of regulatory T cells decreased in the tumor site, reverting the immunosuppressive tumor microenvironment . This stMost vaccines are currently administered using hypodermic needles, necessitating expert administration, cold chain storage, and the transportation of liquid formulations. In addition, most vaccines are formulated in liquid necessitating close temperature control during transport, storage, and distribution  . The iss+ and CD8+ T cell activation [As mentioned earlier, MNs have contributed to the delivery of many NPs into the skin that can be extended to vaccine delivery. The main reason behind the use of NPs in combination with MNs to deliver the antigen is the improved stability and controlled release of antigen for inducing higher immunogenicity. For example, chicken OVA was encapsulated into PLGA-NPs, which was then incorporated in dMNs. Using this approach, MNs slowly released the antigen to lymph nodes occupied with DCs. This strategy led to the successful in vivo immune system activation against influenza and melanoma tumors . In anottivation . Table 3Gene therapy, or replacing a malfunctioning gene with therapeutic nucleic acids , can be N, N, N-trimethyl chitosan/DNA polyplexes. MNs assisted in the co-delivery of the DNA vaccine and PTX to DCs, which resulted in synergistic effects on the DCs maturation and antigen-presenting function, consequently enhancing the immune stimulation and decreasing the immune escape [MNs are especially considered in the transcutaneous delivery of DNA vaccines. For example, the double conjugate of PEI to mannose (Man) and cell-penetrating peptide (CPP: a sequence of TAT: RRRQRRKKRC-SH) were used to condense the DNA vaccine, which was then applied in transcutaneous immunization using solid MNs. This system effectively activated the Trp2-specific response, leading to effective immune system activation against B16 melanoma in BALB/c mice . In thise escape .As mentioned earlier, the applicability of MNs in TDD has been investigated since 1998 . To dateGiven the thorough research performed in recent years on various types of MNs, there are several critical issues to address for their widespread applications in clinical settings. MN applications may lead to short- and long-term safety concerns in patients. It is generally believed that there is no major complication with the short-term use of MNs; nevertheless, the long-term application of MNs can cause erythema (skin redness) as well as pain based on their size and the number of needles. In addition, the effectiveness of MNs as a pore-forming pretreatment depends on the availability of their open micropores. The skin type can also affect the micropore closure time following the MN’s application. Recently, it was reported that the micropore closure time can vary in human subjects of different ethnic/racial backgrounds, with a longer micropore closure time in colored skin. These findings indicate that further research is warranted to verify the safety and efficacy of MNs in a variety of human populations . AnotherDue to the emerging features of NPs and their promising clinical outcomes, rapid developments in NP-laden MNs are expected, but further research is required to exploit their therapeutic and diagnostic potentials. Apart from the efficacy of NPs, the safety of their use via the transdermal route should be addressed. Furthermore, incorporating NPs into the MN matrix can change their mechanical strength. There are a variety of modes resulting in the MNs’ mechanical failure, such as base-plate fracturing, MNs folding, and warping. Consequently, the mechanical properties of MNs should be characterized using different mechanical tests including axial and transverse force, base-plate strength, and shear strength analysis . Moreove.Minimally invasive TDD has remained a challenge requiring more advanced delivery systems to address the limitations associated with conventional therapies. Among numerous TDD systems, NPs have attracted significant attention in recent years due to their potential features such as effective delivery carriers, not only to reduce the side effects associated with conventional formulations, but also to enhance skin permeation, depot drug action, and targeted delivery to impaired skin cells. Moreover, due to the interesting characteristics of certain NPs, including optical, photothermal, and superparamagnetic properties, novel therapeutic, imaging, and biosensing applications are emerging. MNs-based delivery systems have attracted much attention in improving the function of NPs in TDD due to their non-invasive and pain-free delivery characteristics, without a first-pass effect as well as infection and safety problems. Interestingly, the MNs-array technology can shuttle NPs directly into the skin layers. In recent years, MNs, in combination with NPs containing therapeutic agents, have advanced TDD to a new level to effectively treat skin disorders. A combination of MNs and NPs has shown numerous benefits over conventional systems, such as the improved skin penetration of NPs, prolonged or controlled drug release, and the possibility of new add-on therapeutics . Although not fully explored, NPs might play a vital role in cancer chemotherapy using MNs. Moreover, these combined systems have been especially explored for vaccination, immunotherapy, and gene delivery. For a successful delivery of NPs in combination with MNs, several features are warranted, such as the type, physicochemical, pharmacokinetic, and biologic features of NPs as well as the strategy used for delivery by MNs. Overall, MNs can be applied in the TDD of NPs, either drug-loaded or alone, by properly developing and reliably evaluating their practical aspects in clinical trials. These combined systems would have a significant impact on the future of nanomedicine to effectively treat skin disorders as well as systemic applications


We observed a significant increase in CX3CR1 mRNA levels at 2 h and CX3CR1 protein expression at 4 h. CX3CR1-induced VCAM-1 expression in response to low shear stress by activating NF-κB signaling pathway in VECs. Our findings demonstrate that low shear stress increases CX3CR1 expression, which increases VCAM-1 expression due to elevated NF-κB activation. The current study provides evidence of the correlation between shear stress and atherosclerosis mediated by CX3CR1.Atherosclerosis is a significant cause of mortality and morbidity. Studies suggest that the chemokine receptor CX3CR1 plays a critical role in atherogenesis. Shear stress is an important mechanical force that affects blood vessel function. In this study, we investigated the effect of shear stress on CX3CR1 expression in vascular endothelial cells (VECs). First, cells were exposed to different shear stress and then CX3CR1 mRNA and protein were measured by quantitative RT-PCR and western blot analysis, respectively. CX3CR1 gene silencing was used to analyze the molecular mechanisms underlying shear stress-mediated effects on CX3CR1 expression. CX3CR1 mRNA and protein expression were significantly increased with 4.14 dyne/cm Although risk factors, such as hypertension and diabetes, lead to vascular endothelial cell (VEC) dysfunction throughout the vasculature , 2, athe2 . Normal 2 , but lowfunction , 8, and function .Inflammation plays a primary role in atherogenesis, and numerous studies have shown that the inflammatory response is a key factor in the progression of atherosclerosis in vascular disease . CX3CL1 NF-κB is a critical regulator of many cell processes, including cytokine production that influences inflammation and stress responses . VCAM-1 2 air incubator at 37 °C. Pancreatin (1 mL) was added per culture dish to detach the cells from the plates. Cell suspensions were seeded onto sterile glass slides (75 × 20 mm) coated with 1 mL fibronectin , and were further incubated in empty culture dishes.HUVECs derived from EA.hy926 cell lines were purchased from the Shanghai Cell Bank  and maintained in HyClone™ Medium 199  supplemented with 10% fetal calf serum and 1% penicillin and streptomycin. Cell cultures were grown in a humidified 5% COτ is shear stress, µ is medium viscosity (0.72 mPa·s), Q is volumetric flow rate, h is chamber height (0.8 mm), and W is chamber width (95 mm). Shear stress of varying levels  was applied to EA.hy926 cells grown on glass slides for 2 h. The cells were then subjected to a fixed shear stress (4.14 dyne/cm2) for different durations .A parallel-plate flow chamber system (185 × 95 × 0.8 mm) was incorporated into a closed-loop perfusion system containing serum-free M199 medium driven by a roller pump. Shear stress was calculated according to the equation −ΔΔCT method.Cellular RNA was extracted from each individual glass slide using TRIzol Reagent  according to the manufacturer’s protocol. RNA quantity and quality were determined by the A260/A280 ratio using a Nano Drop 1000 spectrophotometer. Two-step reverse transcription (RT) was performed using a RevertAid First Strand cDNA Synthesis Kit  following the manufacturer’s instructions. qRT-PCR was performed using a QuantiFast SYBR Green PCR Kit . The following primers were used : CX3CR1: forward 5’-catcaccgtcatcagcattga-3’, reverse 5’-ggtagtcaccaaggcattcatt -3’; β-actin: forward 5’-ggtagtcaccaaggcattcatt-3’, reverse 5’-ctccttaatgtcacgcacgat-3’. The reaction mix consisted of 2 × QuantiFast SYBR Green PCR Master Mix (10 µL), forward primer (1 µL), reverse primer (1 µL), RNase-free water (6 µL), template cDNA (2 µL), and RNase-free water for a total volume of 20 µL. The CFX96 Real-Time System  was employed for this reaction. Relative gene expression was calculated using the 2Total cellular protein on the glass slides was extracted using RIPA lysis buffer containing protease and phosphorylase inhibitors . Protein concentration was measured using the Pierce BCA Protein Assay Kit according to the manufacturer’s instructions. Proteins were separated using 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes  by electroblotting. Membranes were incubated in 5% skimmed milk in Tris buffered saline with Tween 20 (TBST)  for 1 h to block nonspecific binding. Subsequently, the membranes were incubated overnight with either rabbit anti-CX3CR1 , NF-κB P65 , VCAM-1 , or rabbit anti-GAPDH  primary antibodies at 4 °C. The membranes were washed three times with TBST and incubated with secondary antibody conjugated to HRP  for 2 h. Protein bands were visualized using a color development solution (1:1) for 3 min. Images were acquired using a chemiluminescence imager (Bio-Rad).All incubations were performed at standard laboratory room temperature (22 ± 1 °C) unless otherwise stated. For cell immunofluorescence, the ECs were fixed on glass slides with 4% paraformaldehyde for 1 h and then washed three times using PBS. The cells were blocked with 5% BSA-PBS  for 30 min, followed by overnight incubation with an appropriate primary rabbit anti-CX3CR1 antibody  at 4 °C. The slides were then washed and incubated for 2 h with secondary biotinylated Alexa Fluor 594 anti-rabbit IgG antibody . Studies using these antibodies have been previously published, and related details are available from the manufacturers’ websites.The culture medium was supplemented with 10% fetal bovine serum (FBS) and 100 units/mL of penicillin and streptomycin. Construct lentiviral vectors and lentivirus were produced by GENECHEM . The lentiviruses were constructed encoding sequences targeting CX3CR1 mRNA (siCX3CR1 target-CTTGTCTGATCTGCTGTTT); empty vector was used as the negative control. Stable cell lines were generated by lentivirus infection, which was carried out in a six-well plate with serum-free M199 medium. HUVECs were transduced with lenti-si-CX3CR1 at an infection multiplicity of infection = 10 at 37 °C with 5 µg/mL polybrene and enhanced for 72 h according to the manufacturer’s guidelines. The culture medium was replaced with fresh medium containing 10% FBS, and the cells were continuously cultured for 6–8 days followed by selection with flow cytometry.3/well) were plated and treated in 96-well plates with various concentrations of PDTC  for 0, 12, 24, 48, and 72 h. Then, 200 µL of MTT  was added to the cells for 4 h, the MTT fluid was removed, and 150 µL of DMSO  was added to the wells. After shaking at a low speed for 10 min, the optical density values of the cells were measured according to the manufacturer’s instructions.HUVECs , the least-significant difference test, or multiple ANOVA with SPSS software version 17.0. P values < 0.05 were considered significant.All results are expressed as mean ± standard error of the mean. Statistical comparisons between two groups were performed using a Student’s 2) for 2 h. We found CX3CR1 expressed in HUVECs. After each experiment, the cells were collected to measure CX3CR1 mRNA levels and protein expression. Both CX3CR1 mRNA levels . CX3CR1 mRNA levels in the cells peaked at 2 h , including laminar shear stress and disturbed flow, which can affect endothelial cell function , 23. Thi2 is essential to maintain the structural and functional integrity of VECs [2) can also increase CX3CR1 expression that is dependent on NF-κB activation in EA.hy926 cells. CX3CR1 belongs to a family of G protein-coupled receptors, at the same time, we also subjected CX3CR1 protein were expression remained in the cell membrane after exposure to shear stress.Shear stress of 5–12 dyne/cm of VECs . Blood f of VECs , 26. Num of VECs . Cheng e of VECs , 29. Our2) [Our current findings support our previously published study demonstrating that CX3CL1 expression increases under low shear stress (4.14 dyne/cm2) . However2) . We prev2) . InhibitSubsequently, we investigated the mechanism by which low shear stress-induced CX3CR1 expression. We found that VCAM-1 increased in response to low shear stress after CX3CR1 silencing. Some findings indicated that atherosclerosis promoted low shear stress by activating the soluble form of VCAM-1 . A previIn conclusion, Our study demonstrates that low shear stress increases CX3CR1 expression and that CX3CR1-induced VCAM-1 expression is mediated by the NF-κB signaling pathway in response to low shear stress. The current data provide evidence of the correlation between shear stress and atherosclerosis that is regulated by CX3CR1.Suggested reviewers: Zuzanna Rowinska: z.rowinska@klinikum-bochum.de. Rachana Shah: shahr@email.chop.edu. Andreas Ludwig: aludwig@ukaachen.de.


Cx3cr1. Here, we investigated the contribution of mouse Cx3cr1+ and Cx3cr1neg i-OCLs to bone loss. We showed that Cx3cr1+ and Cx3cr1neg i-OCLs differ considerably in transcriptional and functional aspects. Cx3cr1neg i-OCLs have a high ability to resorb bone and activate inflammatory CD4+ T cells. Although Cx3cr1+ i-OCLs are associated with inflammation, they resorb less and have in vitro an immune-suppressive effect on Cx3cr1neg i-OCLs, mediated by PD-L1. Our results provide new insights into i-OCL heterogeneity. They also reveal that different i-OCL subsets may interact to regulate inflammation. This contributes to a better understanding and prevention of inflammatory bone destruction.Bone destruction relies on interactions between bone and immune cells. Bone-resorbing osteoclasts (OCLs) were recently identified as innate immune cells activating T cells toward tolerance or inflammation. Thus, pathological bone destruction not only relies on increased osteoclast differentiation, but also on the presence of inflammatory OCLs (i-OCLs), part of which express  Chronic inflammation and bone destruction are frequently associated due to complex interactions between activated immune cells and progenitors of bone-resorbing osteoclasts (OCLs). While they have long been attributed to the sole effect of immune cells on osteoclastogenesis, recent evidences demonstrated that these interactions are reciprocal . Indeed,hi monocytes (MNs) (+ and CD8+ T cells (tolerogenic-OCLs/t OCLs) (+ T cells (inflammatory-OCLs/i-OCLs) (OCLs are long-lived multinucleated cells arising from bone marrow (BM) progenitors . Howeveres (MNs)  and dendes (MNs) . Furtheres (MNs) . We and /t OCLs) , whereas/i-OCLs) . Thus, aCx3cr1 decreases and is very low in t-OCLs  mice, a model where osteoclastogenesis is driven by TNF-α and RANK-L-producing CD4+ T cells (+ OCLs previously described in inflammatory colitis (+ and Cx3cr1neg i-OCLs as well as Cx3cr1-deficient i-OCLs by RNA-sequencing (RNA-seq) analysis and in vitro functional assays. We showed that Cx3cr1 deficiency does not change the bone resorption and T cell activation capacity of mature Cx3cr1+ i-OCLs demonstrating that the Cx3cr1 protein per se does not play a major role in these functions. Furthermore, we showed that Cx3cr1 is a marker that identifies two distinct subsets of i-OCLs (Cx3cr1neg and Cx3cr1+) having different immune functions. Our findings unveil the heterogeneity of i-OCLs and their contribution to inflammation and bone loss. These new insights into osteoimmunology and OCL heterogeneity contribute to a better understanding of the bone microenvironment regulation and the underlying molecular processes during inflammatory bone destruction.We addressed these questions using cr1 gene . Consequng cells . We eval T cells . These c colitis . We also+ i-OCLs increased in inflammatory colitis associated with bone destruction  mice. We previously showed that Cx3cr1truction  and we ctruction . To evalP/+ mice . They alP/+ mice . Thus, CCx3cr1GFP/GFP mice and WT Cx3cr1GFP/+ mice. While both DCs and Ly6Chi MNs have been involved in the formation of OCLs in pathological conditions, only DCs were shown to give rise to Cx3cr1+ i-OCLs  specific for the immunodominant ovalbumin (OVA) peptide  between the 2 populations. Principal Component Analysis (PCA) of all detectable genes revealed that Cx3cr1+ (GFP+) and Cx3cr1neg (GFPneg) i-OCLs were clustered into 2 distinct populations (2FC ≥ 1)  . In addixpressed . In partf Cx3cr1  was redun by LPS  was incrg i-OCLs . These dived DCs . Thus, tg i-OCLs  stronglyneg i-OCLs expressed higher levels of genes responsible for bone resorption  cells  cells , in accog) cells .+ T cells (+ i-OCLs had significant higher expression of immunosuppressive genes (CD274 (PD-L1), Lgals9  and Tnfrsf14 (HEVM) confirmed these findings  were added to the culture  highly stimulated CD4+ T cell proliferation, which was dramatically decreased by the addition of Cx3cr1+ i-OCLs  and a reduced osteoclastogenesis. Although Cx3cr1-deficient mice were reported to have slightly reduced bone density . Bone destruction in MM is supported by malignant plasma cells that favor OCL differentiation, including from DCs, through high RANK-L, IL-17 and TNFα production . In thes+ i-OCLs . Thus, o+ and Cx3cr1neg i-OCLs both induce TNFα-producing CD4+ T cells and are resorbing cells, we showed that mature Cx3cr1neg i-OCLs are more efficient to induce CD4+ T cell proliferation and have a higher resorption capacity in vitro than Cx3cr1+ i-OCLs. This suggests that Cx3cr1neg i-OCLs play a major role in inflammatory bone destruction such as in osteoporosis and chronic inflammatory diseases. Interestingly, when comparing Cx3cr1+ i-OCLs from Cx3cr1-deficient Cx3cr1GFP/GFP and WT Cx3cr1GFP/+ mice no differences were observed. Thus, the Cx3cr1 protein as such is not essential for the immunomodulatory and bone resorbing function of Cx3cr1+ i-OCLs. However, it remains a membrane marker allowing to distinguish between 2 i-OCLs subsets (namely Cx3cr1+ and Cx3cr1neg i-OCLs) exerting different immunomodulatory roles. Apart from the lack of Cx3cr1 expression and their immunogenic effect, there is currently no specific marker for the identification of Cx3cr1neg i-OCLs. Thus, an in-depth characterization of these cells is necessary to fully address their specific role. Interestingly, Cx3cr1neg i-OCLs induce high proportions of TNFα and IFNγ-producing CD4+ T cells in vitro. These cytokines are potent inducers of Cx3cr1 and PD-L1 . Animals were maintained under a 12 hr light/12 hr dark cycle with free access to water and standard mouse diet. For ovariectomy, female mice were randomly divided into 2 groups, which were then sham-operated (SHAM) or ovariectomized (OVX). Ovariectomy and sham surgery were performed on 6 weeks old Cx3cr1GFP/+ and Cx3cr1GFP/GFP female mice after anesthesia with isoflurane. The animals were intensively monitored for 72 hr after the procedure. Animals were weighed at the beginning of the study and twice a week thereafter. Six weeks after ovariectomy, mice were sacrificed. Uteri were weighed to control the quality of ovariectomy (not shown). Bones were used either for bone marrow cell isolation and further flow cytometry analysis or for subsequent µCT and histological analysis. Approval for animal experiments was obtained from the Institutional Ethics Committee on Laboratory Animals  and experiments were conducted in compliance with ethical regulations for animal testing and research.OT-II mice were purchased from Charles River Laboratory and C57BL/6 Femora were fixed in 4% paraformaldehyde (PFA) overnight. Subsequently, microcomputed tomography (µCT) was performed using the Skyscan 1176 µCT system  at the preclinical platform ECELLFRANCE . Scans were performed using isotropic voxels size of 18 µm, voltage of 50 kV, current of 500 mA, 0.5 mm aluminum filter, 180 degrees with a 0.7-degree rotation step and 210 ms exposure time. Data 3D reconstructions were generated for visual representation using NRecon software .For histological analysis, fixed femora were decalcified in 10% EDTA for 10 days, embedded in paraffin and tartrate-resistant acid phosphatase (TRAcP) staining was performed on 7 µm sections following manufacturer’s recommendations (Sigma).+ DCs or from blood CD11b+ Ly6Chi MNs of 6 week old WT Cx3cr1GFP/+ and deficient Cx3cr1GFP/GFP mice as previously described (6 cells/well in RPMI medium (ThermoFisher Scientific) supplemented with 5% serum , 1% penicillin-streptomycin, 50 µM β-mercaptoethanol (both from ThermoFisher Scientific), 10 ng/ml IL-4 and 10 ng/ml GM-CSF (both from PeproTech). CD11c+ DCs were sorted using anti-CD11c antibody  and anti-biotin microbeads (Miltenyi Biotec). Alternatively, when indicated, i-OCLs were obtained from blood SSClo CD11b+ Ly6Chi MNs of Cx3cr1GFP/+ OVX mice sorted on a FACS-Aria II (BD Biosciences) using anti-CD11b and Ly6C antibodies. For i-OCLs culture, 2 × 104 CD11c+ DCs or CD11b+ Ly6Chi MNs were seeded/well on 24-well plates in MEM-alpha (ThermoFisher Scientific) supplemented with 5% serum , 1% penicillin-streptomycin, 50 µM β-mercaptoethanol, 25 ng/ml M-CSF and 30 ng/ml RANK-L (both R and D). Cells were cultured at 37°C and 5% CO2. Medium was changed every 3–4 days. Fully differentiated i-OCLs were stained with TRAcP (Sigma-Aldrich) according to manufacturer’s instructions or collected from the culture plates using Accutase (Sigma-Aldrich) and used for subsequent FACS analysis or FACS sorting based on their nuclei number using H33342 (Sigma-Aldrich) as described previously (6 BM cells/well in 24-well plates) in MEM-alpha supplemented with 5% serum , 1% penicillin-streptomycin, 50 µM β-mercaptoethanol and 25 ng/ml M-CSF for a total of 6 days.As OCLs cannot be isolated directly ex vivo in sufficient number for subsequent functional analysis, inflammatory OCLs (i-OCLs) were differentiated in vitro from BM-derived CD11cescribed . Brieflyeviously . When inCx3cr1GFP mutant mice were labeled with antibodies specific for murine anti-CD80, CD86 and IAb  antibodies with a PercP-conjugated streptavidin in PSE for 15 min on ice. For antigen-uptake, mature OCLs were collected and incubated in the presence of 25 µg/ml ovalbumin (ThermoFisher Scientific) for 2.5 hr at 37°C before adding 5 µg/ml H33342 staining for another 30 min and subsequent FACS acquisition. Gating strategies for Cx3cr1+ and Cx3cr1neg i-OCLs are shown in Mature in vitro differentiated i-OCLs were analyzed and sorted based on their multinucleation and GFP expression as described previously  using flFor ex vivo FACS analysis of OCL progenitors, after red blood cell lysis (Sigma-Aldrich) BM cells were labelled with anti-CD11c, CD11b and Ly6C antibodies. For FACS analysis of macrophages, BM-derived macrophages were labeled with anti-CD68 and CD169 antibodies.+ T cells were stimulated with PMA, ionomycin and Brefeldin A, labelled with an anti-CD4 antibody and fixed with 4% formaldehyde overnight as described  in MEM-alpha containing 10% FBS  and 30 ng/ml RANK-L (R and D). After 48 hr, matrix dissolution activity was evaluated by removing the cells using distilled sterile water and staining with 2% Alizarin Red (Sigma-Aldrich) for 1 min. Imaging was performed using an Axio Observer D1 microscope (Zeiss) and images were taken using AxioVision Rel. 4.8 software (Zeiss). Resorbed areas were quantified using Fiji/ImageJ software (Mature in vitro differentiated i-OCLs were sorted based on their multinucleation and GFP (Cx3cr1) expression as described above . A totalsoftware .+ and Cx3cr1neg i-OCLs were sorted based on their multinucleation and GFP expression as mentioned above  peptide (ThermoFisher Scientific). Spleens of 8 weeks old OT-II mice were used to isolate CD4+ T cells that express transgenic OVA-specific αβ-TCRs by using the Dynabeads Untouched Mouse CD4 Cell Isolation Kit (ThermoFisher Scientific) according to manufacturer’s specifications. To analyze T cell proliferation, CD4+ T cells were labelled with 250 µM CFSE for 10 min at 37°C. OVA-loaded i-OCLs and CD4+ T cells were seeded in a ratio 1:20 in IMDM medium supplemented with 5% FCS , 1% penicillin-streptomycin (Gibco) and 50 µM β-mercaptoethanol (Gibco). T cell proliferation was assessed by FACS after 3–5 days.Mature in vitro differentiated Cx3cr1ed above  and seed+ i-OCLs, sorted Cx3cr1neg i-OCLs were stimulated with 600 nM OVA for 3 hr at 37°C, washed 3 times with IMDM medium and cultured together with CFSE-labelled CD4+ T cells from transgenic OT-II mice on 96-well plates as described above. Cx3cr1+ i-OCLs were sorted and added to the coculture without ovalbumin challenge in different ratios between Cx3cr1neg vs Cx3cr1+ i-OCLs . T cell proliferation was assessed using FACS after 3–5 days of coculture.Implication of the PD-1/PD-L1 axis was evaluated by adding 5 µg/ml isotype control or anti-PD-1 ) antibodies at the beginning to the coculture. To validate the immune suppressive effect of Cx3cr1Total RNA (100 ng) from 4 biological replicates (from 4 different mice) in each group was extracted from in vitro differentiated i-OCLs  as described previously  was used. Statistical significance was considered at p<0.05. Experimental values are presented as mean ± standard deviation (SD) of at least three biological replicas. Error bars for gene expression analysis of humans and mice using RT-qPCR show the mean value with 95% confidence interval. All experiments were repeated with a minimum of three biological replicates and at least two technical replicates.All data were analyzed using Graph Pad Prism 7.0 software using an appropriate two tailed student’s  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The authors studied the heterogeneity of inflammatory osteoclasts (i-OCL) using Cx3cr1 as a marker of osteoclast heterogeneity in a mouse model of OVX-induced bone resorption. Cx3cr1-neg OCLs showed higher capacity for bone matrix dissolution, phagocytosed less OVA antigen, and had higher capacity to induce T cell proliferation in vitro, compared to Cx3cr1+ OCLs.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kathryn Cheah as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Dissecting the phenotypic and functional heterogeneity of inflammatory osteoclasts by the expression of CX3CR1" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Essential revisions:1) The conclusions rely on (1) gene expression profiling of sorted CX3CR1+ and CX3CR1- cells, and (2) in vitro culture followed by functional analysis of said sorted cells. However, for these in vitro experiments, the culture conditions employed prior to analysis are not clear. The Materials and methods section suggests that there was extensive culture and stimulation prior to analysis following sorting, but these conditions are not explicitly stated in the main text. If cells were extensively cultured with stimulating cytokines prior to functional analysis, then this would be expected to drastically change their functional profile compared to cells sorted directly from the in vivo environment. This would affect the results in Figures 2, 4, 5, and 6.2) While Cx3cr1+ and Cx3cr1- cells have different effects on T cell proliferation, that doesn't necessarily mean that one regulates the activity of the other in vivo. To make this conclusion it would be necessary to deplete selectively one population in vivo and observe the effects on the other population.3) Given that macrophages can fuse into multinucleated cells and that Cx3cr1 is a marker of macrophage heterogeneity, it has not been demonstrated that the cells were indeed OCLs and not macrophages. Evidence of the cell identity, OCL or macrophage should be provided.4) The absence of Cx3cr1 in Cx3cr1 gfp/gfp mice resulted in reduced recruitment of dendritic cells and Ly6C-hi monocytes to the sites of bone resorption in OVX animals. This result would be expected because Cx3cr1 is well known to be involved in leukocyte trafficking. The authors concluded that this result shows that Ly6C-hi monocytes are progenitors of inflammatory OCLs. This conclusion is not supported by the results.5) The authors conclude that Cx3cr1+ cells express higher levels of immunosuppressive genes compared to Cx3cr1- cells. Because the list of genes clearly includes many genes that are immunostimulatory , the conclusions about immunosuppressive gene expression profile in Cx3cr1- cells are not clearly supported.6) In Figure 1 the authors claim that CXC3R1 positive osteoclasts are needed for the full bone resorbing activity of osteoclasts after ovariectomy. The claim is based on only minor differences and whether CXC3R1 positive osteoclasts were preset or not, the osteoporosis phenotype develops in an equally severe manner. Moreover, these data contradict a statement made later in the paper that CXC3R1 positive osteoclasts are not needed for bone resorption.7) Data shown in Figure 2 (no difference in the bone resorption capacities of CX3CR1+ cells isolated from GFP/+ mice and CX3CR1- cells isolated from GFP/GFP mice) and those shown in Figure 4 (large differences in bone resorptive capacities of CX3CR1+ and CX3CR1-negative cells derived from dendricytes) are contradictory. This inconsistency calls into question the validity of using cells derived from the dendricytes in culture if they behave differently from the mature cells isolated from the genetically modified mice. Essential revisions:1) The conclusions rely on (1) gene expression profiling of sorted CX3CR1+ and CX3CR1- cells, and (2) in vitro culture followed by functional analysis of said sorted cells. However, for these in vitro experiments, the culture conditions employed prior to analysis are not clear. The Materials and methods section suggests that there was extensive culture and stimulation prior to analysis following sorting, but these conditions are not explicitly stated in the main text. If cells were extensively cultured with stimulating cytokines prior to functional analysis, then this would be expected to drastically change their functional profile compared to cells sorted directly from the in vivo environment. This would affect the results in Figures 2, 4, 5, and 6.ex-vivo isolated osteoclasts would have been the best approach. However, up to now, there are no publications investigating osteoclast function on murine ex vivo-isolated osteoclasts. These cells are rare and firmly attached to the bone and to date there's no procedure in the literature to isolate them in sufficient number to perform subsequent analysis. Consequently, functional assays on osteoclasts always use in vitro-generated cells and much of osteoclast biology described so far in the literature has been investigated in such conditions . Moreover, at the moment there is no marker to distinguish between inflammatory and tolerogenic Cx3cr1neg osteoclasts, which make it impossible to sort them ex vivo. Up to know, the only way to obtain these cells is to differentiate them from their respective known progenitors. Of course, we agree with the reviewers that this may affect their function, but to the same extend as in all studies investigating osteoclast activity and published so far and we don't have any other option.The reviewers raised an important point. We generated in vitro inflammatory OCLs in the presence of RANK-L and M-CSF, the 2 main osteoclastogenic factors, according to the gold standard protocol for in vitro osteoclastogenesis and starting from their known progenitors, BM-derived dendritic cells . Of course, we agree that investigating osteoclast function on To make the procedure of osteoclast preparation clearer as requested, we revised the main text and included more details in the Results and Materials and methods sections as well as in the figure legends. We also added in Figure 2 and Figure 3 a schematic representation of osteoclast preparation.2) While Cx3cr1+ and Cx3cr1- cells have different effects on T cell proliferation, that doesn't necessarily mean that one regulates the activity of the other in vivo. To make this conclusion it would be necessary to deplete selectively one population in vivo and observe the effects on the other population.The reviewers are perfectly right and demonstrating the interaction between the 2 i-OCLs subsets in vivo would require specific depletion in vivo of one of these subsets. We are currently investigating in more details the heterogeneity of the different OCL subsets in order to identify suitable factors that would allow to specifically target one or the other OCL subset. This study, and in particular the validation of markers, requires time and the data will be published in a future manuscript.However, to address the reviewer's concern and to avoid any over-interpretation of our results, in the revised manuscript we softened our conclusion and clearly stated that the mechanisms we described are only shown in vitro and remain to be demonstrated in vivo.3) Given that macrophages can fuse into multinucleated cells and that Cx3cr1 is a marker of macrophage heterogeneity, it has not been demonstrated that the cells were indeed OCLs and not macrophages. Evidence of the cell identity, OCL or macrophage should be provided.+ and Cx3cr1neg subsets of OCLs do not express these macrophage markers but share osteoclast hallmarks, such as TRAcP expression and resorption capacity . These data were included in the revised manuscript and confirm that the cells we are analyzing are bona fide OCLs, whether they express or not Cx3cr1.To prove the identity of the cells we are analyzing, we performed additional experiments as requested. In particular, because in our protocol osteoclasts are generated in the presence of RANK-L but also M-CSF, a potent inducer of macrophages, we compared these osteoclasts to macrophages generated from bone marrow cells in the presence of M-CSF. In these conditions, we didn't observed cell fusion in macrophages. Moreover, our results showed that while BM-derived macrophages express the 2 BM macrophage markers CD68 and CD169 but are negative for TRAcP expression , both Cx3cr14) The absence of Cx3cr1 in Cx3cr1 gfp/gfp mice resulted in reduced recruitment of dendritic cells and Ly6C-hi monocytes to the sites of bone resorption in OVX animals. This result would be expected because Cx3cr1 is well known to be involved in leukocyte trafficking. The authors concluded that this result shows that Ly6C-hi monocytes are progenitors of inflammatory OCLs. This conclusion is not supported by the results.hi monocytes are involved in osteoclastogenesis in particular in conditions related to inflammatory bone destruction , which suggest that they could be progenitors of Cx3cr1+ osteoclasts that are also associated to the same conditions . To address the capacity of Ly6Chi monocytes to differentiate into Cx3cr1+ osteoclasts, we sorted Ly6Chi monocytes from the blood of WT Cx3cr1GFP/+ mice and cultured them with M-CSF and RANKL for 6 days as shown in Figure 1—figure supplement 1A. We analyzed the resulting osteoclasts by FACS and found that about 35% of these osteoclasts are GFP/Cx3cr1+  demonstrating that Ly6Chi monocytes are indeed progenitors of Cx3cr1+ osteoclasts, which support our conclusion. To make this point more visible and clearer in the revised version of the manuscript, we modified the text in the Results section and added a schematic representation of the experiment .Ly6C5) The authors conclude that Cx3cr1+ cells express higher levels of immunosuppressive genes compared to Cx3cr1- cells. Because the list of genes clearly includes many genes that are immunostimulatory , the conclusions about immunosuppressive gene expression profile in Cx3cr1- cells are not clearly supported.+ cells, not of Cx3cr1neg cells.There might be a typo error (Cx3cr1- instead of Cx3cr1+) in the last sentence of the reviewer's comment because we only discussed the immunosuppressive capacity of Cx3cr1+ osteoclasts do not only express high levels of immunosuppressive genes but also of genes involved in inflammation , which is in line with their capacity to stimulate to some extend the proliferation of inflammatory CD4+ T cell . Thus, we agree that just the RNA expression profile of these OCLs is not fully supporting their immunosuppressive role. In the revised manuscript, the comment on the RNAseq analysis for this figure in the Results section has been rephrased accordingly. However, our conclusions do not only rely on the expression profile of osteoclasts, but also on in vitro functional analysis that clearly show:The reviewers are perfectly right and Cx3cr1+ OCLs is increased when blocking the immunosuppressive checkpoint protein PD1 in vitro , 2) that in an immunosuppressive assay, Cx3cr1+ OCLs reduce the capacity of Cx3cr1neg OCLs to stimulate T cell proliferation  which is a clear functional demonstration of their immunosuppressive effect, at least in vitro as mentioned in the revised version.1) that the T cell activation capacity of Cx3cr16) In Figure 1 the authors claim that CXC3R1 positive osteoclasts are needed for the full bone resorbing activity of osteoclasts after ovariectomy. The claim is based on only minor differences and whether CXC3R1 positive osteoclasts were preset or not, the osteoporosis phenotype develops in an equally severe manner. Moreover, these data contradict a statement made later in the paper that CXC3R1 positive osteoclasts are not needed for bone resorption.We agree that the differences observed when comparing OVX WT mice and OVX Cx3cr1-deficient mice  for the microscanner analysis are minor but they are significant and are further supported by a reduced osteoclast number . But to follow the reviewer concern, we rephrased the comment of this figure in the Results section to indicate this low degree of variation.+ OCLs are required for bone loss in OVX mice. Figure 1 is not addressing the role of Cx3cr1+ osteoclasts but the implication of the Cx3cr1 protein in bone destruction associated to ovariectomy, which are 2 different questions. In Figure 1, we showed that Cx3cr1, as a protein, is important for the recruitment of OCL progenitors in the bone marrow of OVX mice . Moreover, we showed that i-OCLs from Cx3cr1-deficient and from WT mice have the same resorption activity , which demonstrates that the protein Cx3cr1 is not important for this activity.However, there might be a misunderstanding as we didn't claim that Cx3cr1+ OCLs are not needed for bone resorption, we only showed that they resorb less than the Cx3cr1neg OCLs .Moreover, we didn't claim that Cx3cr1But to avoid any misunderstanding, we modified the text to make it clearer and to describe in more details the mouse model we used. We also added in Figure 2 and 3 a schematic representation of the preparation of the cells to avoid any confusion.7) Data shown in Figure 2 (no difference in the bone resorption capacities of CX3CR1+ cells isolated from GFP/+ mice and CX3CR1- cells isolated from GFP/GFP mice) and those shown in Figure 4 (large differences in bone resorptive capacities of CX3CR1+ and CX3CR1-negative cells derived from dendricytes) are contradictory. This inconsistency calls into question the validity of using cells derived from the dendricytes in culture if they behave differently from the mature cells isolated from the genetically modified mice.As explained in the first point above, due to methodological issues, in particular the absence of a protocol to isolate sufficient osteoclasts or to sort ex vivo specific subsets of OCLs, there is no other possibility to investigate the function of these OCLs than the use of in vitro differentiated cells. In particular in our study, all inflammatory osteoclasts used in functional assays were derived from dendritic cells in exactly the same culture conditions, as already described . Thus, even if we cannot exclude that the culture conditions could affect osteoclast function compared to in vivo, it would be to the same extend in all our analysis. It would also be to the same extend as in all studies investigating osteoclast activity published so far.+ osteoclasts coming either from WT (Cx3cr1GFP/+) mice or from mice baring a non-functional Cx3cr1 gene (Cx3cr1GFP/GFP). There are no data on Cx3cr1neg cells in Figure 2. And indeed, we showed that Cx3cr1+ cells have equivalent resorption capacity whether Cx3cr1 is functional or not , which demonstrates that the protein Cx3cr1 is not important for this activity.Regarding the reviewer's concern on potential inconsistency in our results, there might be a misunderstanding. In Figure 2, we addressed the role of Cx3cr1, as a protein, in bone resorption and T cell activation by osteoclasts. This was achieved comparing Cx3cr1+ and Cx3cr1neg i-OCLs from WT mice differ in their resorption and T cell activation capacity .In contrast, in Figure 4, we addressed, in WT mice, the specific function of the 2 distinct subsets of osteoclasts that are identified using Cx3cr1 as a membrane marker. We showed that Cx3cr1Thus, Figures 1-2 and Figures 3-6 explore 2 completely distinct aspects that could not be compared.+ OCLs having a non-functional Cx3cr1 gene could not be considered equivalent to Cx3cr1neg OCLs from WT mice. Indeed, as shown in + i-OCLs from WT and mutant mice are closely related, both differ considerably from Cx3cr1neg cells from WT mice as they cluster in 2 completely distinct groups (panel A). This was further confirmed by matrix similarity analysis of our transcriptomic data that showed again that the 2 populations are distinct (panel B). Therefore none of the conclusions made for one of these population can be applied to the other.Importantly, Cx3cr1+ and Cx3cr1neg i-OCL could be confusing. Thus, in the revised manuscript we modified the text to make it clearer and we added schematic representations of the preparation of OCLs in the figures.However, we recognize that switching from the analysis of the implication of the Cx3cr1 protein in bone loss to the analysis of the respective role of Cx3cr1


APC or activating mutations in β-catenin (CTNNB1) lead to signaling overactivation and subsequent intestinal hyperplasia. Numerous classes of medicines derived from synthetic or natural small molecules, including alkaloids, have benefited the treatment of different diseases, including cancer, Piperine is a true alkaloid, derived from lysine, responsible for the spicy taste of black pepper (Piper nigrum) and long pepper (Piper longum). Studies have shown that piperine has a wide range of pharmacological properties; however, piperine molecular mechanisms of action are still not fully understood. By using Wnt/β-catenin pathway epistasis experiment we show that piperine inhibits the canonical Wnt pathway induced by overexpression of β-catenin, β-catenin S33A or dnTCF4 VP16, while also suppressing β-catenin nuclear localization in HCT116 cell line. Additionally, piperine impairs cell proliferation and migration in HCT116, SW480 and DLD-1 colorectal tumor cell lines, while not affecting the non-tumoral cell line IEC-6. In summary, piperine inhibits the canonical Wnt signaling pathway and displays anti-cancer effects on colorectal cancer cell lines.More than 94% of colorectal cancer cases have mutations in one or more Wnt/β-catenin signaling pathway components. Inactivating mutations in  The canonical Wnt signaling pathway is an important mechanism to control different phenomena since embryonic development throughout adulthood such as axis patterning, determination of tissues, stem cell renewal, cellular polarization and migration. Deregulation of this pathway causes several disorders such as cancer, neurodegenerative and neuropsychiatric diseases.1. The phosphorylation of β-catenin by CK1α and GSK3 marks it for ubiquitination and posterior degradation by the proteasomal machinery2. The binding of Wnt ligands to its receptors triggers the signaling cascade that disassembles the β-catenin destruction complex3. Wnt signaling activation induces Disheveled (Dvl) mediated Axin membrane translocation, which binds to the cytoplasmic portion of LRP5/65. Due to the membrane position of the degradation complex, GSK3 and CK1α start to phosphorylate LRP5/6 leading to β-catenin stabilization in the cytoplasm6. In that sense, β-catenin stabilization also occur through Axin proteolysis catalyzed by Tankyrases (TNKs)7. Then, β-catenin translocates to the nucleus where it will bind TCF/LEF transcription factors.When the Wnt ligand is not bound to the receptors Frizzled and Low-density lipoprotein receptors 5/6 (LRP5/6), cytosolic β-catenin is degraded by a multiprotein complex, named destruction complex, formed by Axin, Adenomatosis polyposis coli (APC), Protein phosphatase 2A (PP2A), Glycogen synthase kinase 3 (GSK3) and Casein kinase 1α (CK1α)8. Alternatively the presence of the Wnt extracellular inhibitor, DKK1, or the deletion of the TCF4 results in Wnt signaling blockage, leading to a failure in the normal development of proliferative crypts10.The finding that APC could negatively regulate β-catenin provided further support for the connection between Wnt and cancer12. According to the Cancer Genome Atlas more than 94% of cases of colorectal cancer have mutations in at least one component of the Wnt signaling pathway13. Hence, inhibiting the pathway downstream of these gain of function mutations is an interesting strategy to impair tumor progression.One of the best-characterized mechanisms controlled by Wnt signaling is the cellular proliferation in the intestinal crypt and maintenance of intestinal stem cells. Mutations in APC or β-catenin (CTNNB1) genes result in Wnt signaling overactivation and promote intestinal hyperplasia16. Our group uncovered the mechanism of action of distinct flavonoids on targeting Wnt signaling19.The treatment of different diseases, including cancer, has benefited from numerous classes of drugs isolated from plants natural products. Many interesting small molecules with biological activity to treat diseases have been published in recent yearsPiper nigrum) and long pepper (Piper longum), originally from the family Piperaceae21. Studies have shown that piperine has diverse pharmacological properties, including anticonvulsive activity22, antioxidant activity23, anti-inflammatory25, liver protective26, neuroprotective27 and acts as an antimicrobial agent28. Piperine also exhibits potential to treat depressive disorders and to enhance memory in animal models30. In addition to all these properties, piperine also exerts an anticancer effect31.Among the natural substances of alkaloid origin with pharmacological properties, piperine is outstanding. This substance is an example of a true alkaloid, derived from lysine, responsible for the spicy taste of black pepper  with 0.2% DMSO resulted in 12-fold increase in luciferase activity. Treatment of RKO pBAR/Renilla cell line with 50 or 100 μM piperine decreased the luciferase activity by 40% and 85%, respectively  as 34 μM by nonlinear regression of previous SW480 pBAR/Renilla data means  or dnTCF4 VP16 . Piperine treatment at 50 and 100 μM inhibited the Wnt signaling reporter activity basal levels of pCS2 transfected HEK293T cells by 60% , a topoisomerase I inhibitor39, besides taxol and vimblastine, which interact with tubulin41. Screenings for new molecules led to the discovery of novel alkaloids that have shown promising antineoplastic and anti-proliferative effects in different types of cancer cell lines. Among these compounds, piperine is included as one of the most studied42. The anti-cancer effects of piperine were shown for lung, prostate, breast, melanoma and colorectal cancers, however, very few papers showed the mechanisms of actions of piperine on cell machinery. In this study, we show that piperine inhibits Wnt/β-catenin signaling even if we activate the pathway with a constitutively activated form of β-catenin (S33A mutated) or with a constitutively activated form of TCF, independent of β-catenin binding  in HT1080 human fibrosarcoma cell line and 4T1 breast cancer cell line, DU145 prostate cancer and HOS and U2OS osteosarcoma cell lines. Our results show that piperine impairs migration of HCT116, SW480 and DLD-1 colorectal cancer cell lines and does not interferes with the migration rate of IEC-6 a non-tumoral intestine cell line.Several studies reported anti-invasive or anti-migratory effects of piperine in different cancer cell lines49. Nevertheless, this study did not show on which level piperine acts. Our results show through epistasis experiments that piperine inhibits Wnt signaling activated by constitutively activate form of TCF (dnTCF4 VP16) and also inhibits β-catenin nuclear localization in HCT116, a colorectal cancer cell line that harbors a mutated β-catenin form that overactivates Wnt signaling. Piperine Wnt signaling inhibition property is probably one additional effect that contributed for tumor growth impairment reported in previous publications. Recently, two works showed that piperine binds directly to DNA. Haris and co-workers revealed the molecular mechanism of interaction of piperine with DNA showing that piperine binds to DNA minor groove50. Another work showed that piperine binds to various G-quadruplex DNA sequences, including the c-myc promoter, one of Wnt signaling pathway target genes51. These recent findings, together with our epistasis experiment using dnTCF4 VP16 indicate that piperine could act through different pathways and could even have different targets in the Wnt/β-catenin signaling cascade. Our data suggests that piperine inhibits the translocation of β-catenin to the nucleus and might suppress the binding of TCF/LEF to the DNA, or even by direct binding to the promoter and downregulating Wnt target genes.Only one study evaluated the effects of piperine on Wnt signaling, however this study analyzed the effects of a co-treatment of piperine and curcumin on Wnt signaling in breast cancer cell linesNowadays, natural compounds with multiple targets are being revisited and studied for anti-cancer effects. Piperine displays anti-cancer effects in different cancer cell lines, probably by interfering with Wnt/β-catenin signaling, among others. Thus, a new path is opened for understanding the molecular mechanisms by which alkaloids suppress tumor progression.2. Dimethylsulfoxide (DMSO), anti-β-catenin and anti-α-tubulin were purchased from Sigma-Aldrich . Secondary antibodies were purchased from Life Technologies . Cell lines used were HEK293T, L-cell, L-Wnt3a, HCT116, DLD-1, SW480 and IEC-6 (ATCC). Wnt signalling reporters cell lines, RKO pBAR/Renilla and SW480-pBAR/Renilla were a kindly donated by Dr. Xi He . The alkaloid piperine used in this study was extracted from Piper nigrum, purified and identified at Research Institute for Natural Products in the Federal University of Rio de Janeiro (IPPN-UFRJ-Brazil).All cell lines were maintained in DMEM F12 culture medium supplemented with 10% fetal bovine serum and 100U/mL and 100 µg/mL penicillin/streptomycin  at 37 °C and 5% COPiper nigrum52. Methanolic extracts from seeds of P. nigrum were submitted to successive column chromatography using silica gel and gradient of solvents with increasing polarity. Piperine was isolated as yellow cristal and its identity was confirmed by comparison of the 1H NMR data with the literature20.Piperine was isolated from the seeds of The experiment was carried out in a high efficiency liquid chromatograph (HPLC) of the Shimadzu-LC20AD brand with photodiode array detection (CLAEPDA), Kromasil 100-5C18 column (4.6 mm by 250 mm), SPD-M20A detector (Diode Array) at 345 nm wavelength and 1 mL/minute constant flow. Piperine sample was diluted in acetonitrile. Automatic injection programming was performed so that the injected sample volume corresponded to 20 μL. Mobile phases used were: 60% acetonitrile and 40% water in the isocratic method. The running time was 15 min. The retention time band 7.15 min , UFRJ. To obtain the mass spectra the sample was dissolved in high purity methanol. The data was acquired in the positive mode using the Electrospray ionization method (ESI) on a scale of 96.77 to 1,200 m/z. The spectrum was analyzed using the mMass 5.5.0 software. The calculated [M + H] + molecular mass corresponding to piperine is 286.1438 and the found mass is 286.1432 . L-cell conditioned medium (L-cell CM) was used as negative control. DMSO 0.2% was also added as the vehicle control. In SW480-pBAR/Renilla cells there was no need for Wnt3a treatment since the pathway is overactivated in this lineage. After 24 h of treatment, Firefly and Renilla luciferase activities were detected according to the manufacturer’s protocol . 1.2 × 104 HEK293T cells were cultured on 96-well plates until they reach 70% confluence, cells were transfected with lipofectamine 3,000  and 100 ng TOP-Flash plasmid, 10 ng CMV-Renilla-luciferase, and 100 ng of the vector pCS2, or pCS2-β-catenin WT, or pCS2-β-catenin S33A, or pCS2-dnTCF4 VP16. dnTCF4-VP16 construct expression results in the translation of a fusion protein that contains the transcription activation domain of herpes simplex virus (VP16) and a truncated TCF4 that cannot bind to β-catenin (dnTCF4)54. dnTCF4 itself is a dominant negative form of TCF4, however, when fused with VP16, it becomes constitutively active, since it activates the Wnt/β-catenin in a β-catenin independent manner. 18 h after transfection, cells were treated for 24 h, using 10, 30, 50 or 100 μM of piperine. Firefly and Renilla luciferase activities were detected according to the manufacturer’s protocol .1.2 × 104 cells/well in 96-well tissue culture plates and cultured for 24 h before treatment with 20, 30, 50, 100 or 200 μM piperine or the vehicle DMSO for 0, 24, 48, or 72 h. MTT was added to each well at a final concentration of 0.25 mg/mL for 1 h before cell harvesting. The formazan reaction product was dissolved with DMSO and quantified spectrophotometrically at 570 nm using a UV–visible system. (Modulus II microplate multimode reader).3--2,5-diphenyl tetrazolium bromide (MTT) (Sigma-Aldrich) was used to assay mitochondrial activity in viable cells. Cells were plated at a concentration of 1.2 × 104 cells were plated on the previous day and treated with 50, 100 or 200 μM of piperine for 24 h. Click-iT EdU (Life Sciences) assay was performed according to manufacturer’s protocol. DMSO was used as a vehicle to solubilize the alkaloid and was added to control cultures conditions at 1%.For cell proliferation assay, 5.0 × 1055, containing 0.1% Triton X-100, 0.1% citrate buffer and 0.1 mg/mL RNase and 50 μg/mL propidium iodide . After 15 min incubation, we analyzed the cell suspension for DNA content by flow cytometry using a FACSCalibur flow cytometer . The relative proportions of cells with DNA content diploid G0/G1 (2n), S phase (> 2n but < 4n), and G2/M phase (4n) were acquired and analyzed using CellQuest software. Cell dissociation procedure does not affect fluorescence under the experimental conditions that were used in this study or in any others of which we are aware.RKO and SW480 cell lineages were cultured for 48 h with medium alone (untreated), DMSO, 50 µM piperine or 100 µM piperine. Subsequently, supernatant of each condition was collected, and cells were detached with trypsin at room temperature. After centrifugation, cells were resuspended in 0.5 mL ice-cold Vindelov solutionRenilla cells were fixed in 4% paraformaldehyde, washed with PBS, and permeabilized with 0.1% Triton X-100. Samples were then blocked for 1 h with 5% normal goat serum (Gibco). A rabbit anti-β-catenin (1:200) primary antibody was incubated overnight. Specific secondary antibody Alexa 546  (1:2000) were incubated for 1 h at room temperature. After PBS washes, DAPI staining  (Sigma) was performed for 10 min, and then slides were mounted with Fluormount (Sigma) and observed at confocal microscope (Leica TCS SP5).RKO pBAR-Lysate samples from piperine treated cells at 50 and 100 μM were harvested in Triton buffer . Protein was quantified by the Bradford method, and 20 μg of the total lysate was loaded in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane . Membranes were pre-blocked in 5% nonfat dry milk in Tris-buffered saline, 0.001% Tween 20 for 1 h and then incubated overnight with anti-β-catenin , anti-α-tubulin , anti-c-Myc , anti-phospho Histone-H3 , anti-β-actin  primary antibodies previously diluted in Tris-buffered saline, 0.001% Tween 20, 1% nonfat milk. Secondary antibodies conjugated with horseradish peroxidase  diluted in the same buffer were used to probe the membranes, and the reaction was visualized using SuperSignal West Pico and SuperSignal West Femto chemiluminescent substrate.The sgRNA designed to target “GAGTGGTAAAGGCAATCCTG” was cloned into the lentiCRISPRv2 according to Feng Zhang laboratory manual. HEK293T cells were transfected with the lentiCRISPRv2 cloned plasmid and clonally expanded with selection medium containing puromycin. CTNNB1 knockout (KO) efficiency was examined by Western blot. LentiCRISPRv2 plasmid was obtained from Addgene (Plasmid #52961).HCT116, SW480, DLD-1 and IEC-6 cell cultures were cultivated in 12-well plates until confluence and treated with 50 or 100 μM of piperine, together with Ara-C, an inhibitor of cellular proliferation. A line down the center of each well was scraped with a pipette tip. Images were taken at 0 h and 24 h after treatment, scratches area were measured, and wound closure was calculated by subtraction of the area measured after 24 h of incubation by the initial scraped area. Each experiment was carried out in triplicate, and three fields were counted per well.p value) were determined by paired Mann–Whitney test .Each experiment was performed at least three times. The luciferase and MTT assays were performed in triplicate. Cell staining quantification was performed by counting the number of DAPI and the number of non-nuclear and nuclear β-catenin stained cells in randomly chosen microscope fields, and then the percentage of nonnuclear and nuclear cells of the total cells was calculated. Statistical analysis were performed using the unpaired Student’s t test. In the MTT assays, we used two-way ANOVA following a Bonferroni post-test . Standard error and significance (Supplementary file1 (PDF 882 kb)


In this study, the pharmacokinetic properties of LMT-28 including physicochemical property, in vitro pharmacokinetic profiles such as liver microsomal stability and Madin-Darby canine kidney (MDCK) cell permeability assay were examined. For in vivo pharmacokinetic studies, pharmacokinetic parameters using BALB/c mice were calculated.LMT-28 has been synthesized and is being developed as a novel therapeutic IL-6 inhibitor. The physicochemical properties and app; 9.7×10−6 cm/s) showed that LMT-28 possesses a moderate-high cell permeability property across MDCK cell monolayers. The plasma protein binding rate of LMT-28 was 92.4% and mostly bound to serum albumin. The metabolic half-life (t1/2) values of LMT-28 were 15.3 min for rat and 21.9 min for human at the concentration 1 μM. The area under the plasma drug concentration-time curve and Cmax after oral administration (5 mg/kg) of LMT-28 were 302±209 h·ng/mL and 137±100 ng/mL, respectively.The logarithm of the partition coefficient value  and the apparent permeability coefficient values (PThese data suggest that LMT-28 may have good physicochemical and pharmacokinetic properties and may be a novel oral drug candidate as the first synthetic IL-6 inhibitor to ameliorate mammalian inflammation. The discovery and development of a new drug can save millions of lives and enhance the quality of numerous lives. Although the pharmaceutical and biotechnical researches and developmental efforts may lead to successful approval, there are also many hurdles in drug discovery and development . Most ofActivation of the interleukin-6 (IL-6) and signal transducer and activator of transcription factor-3 (STAT3) signaling pathway have been studied and IL-6, a phosphorylated glycoprotein, is a major causative factor which plays an important role in inflammatory disease . IL-6 plTo discover a novel synthetic small molecule as a new drug candidate for IL-6 blocking, LMT-28  has beenin vitro and in vivo pharmacokinetic profiling including physicochemical properties were characterized to support future development and clinical trials.The aim of this study was therefore to evaluate pharmaco kinetic profiles of LMT-28 as a novel IL-6 inhibitor, because there are no reports about characterizing it as a synthetic small molecule that inhibits IL-6 and ameliorates inflammation in mice by functioning through direct interaction with gp130. Furthermore, both LMT-28 .Male BALB/c mice (CD-1) were purchased from Orient-Bio Company . Animals were kept in an air conditioned room at a temperature of 20°C to 25°C, humidity (50%±3%), and 12 h day/night cycles with specific free condition. Food and water were supplied HepG2 cells were seeded in MEM media  with 10% (v/v) feta bovine serum, streptomycin (100 U/mL), and penicillin . After seeding, cells were starved overnight, treated with LMT-28 for 1 h, and stimulated with IL-6  for 10 min. Cells were treated with cell lysis buffer  for 30 min on ice. Cell lysates were separated on an 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, then transferred to nitrocellulose membranes . The membranes were blocked overnight in Tris-buffered solution  containing 5% nonfat dry milk at 4°C and then incubated for 1 h with the appropriate primary Abs against p-STAT3  or α-tubulin in Tris-buffered solution  containing 5% nonfat dry milk at room temperature. The membranes were washed  and incubated for 1 h with horseradish peroxidase-conjugated secondary Abs diluted to 1:6,000. After 10 washes, the membranes were incubated with enhanced chemiluminescence reagents  and chemiluminescent signals were visualized using X-ray film.The shake-flask method was applied to measure the equilibrium solubility of LMT-28 ,17. ExceMetabolic stability using rat and human liver microsomes  of LMT-28 was performed according to previous reported protocol . In briein vitro intrinsic clearance of LMT-28 were calculated by next equations in vitro and in vivo by previous reports [in vitro and in vivo pharmacokinetic properties of LMT-28 were investigated and the pharmacokinetic properties of LMT-28 were confirmed in this study.LMT-28, a new drug candidate, with the mechanism of action as a novel therapeutic IL-6 inhibitor is under development. IL-6 inhibition by LMT-28 has been demonstrated to be effective  reports  and confThe physicochemical properties of LMT-28 were drug-like. The equilibrium solubility of LMT-28 was 0.39±0.12 mg/mL. This was slightly soluble in water system, however, it was a reasonable solubility for a new drug candidate. More importantly, new drug candidates may require a balance between hydrophilicity and lipophilicity . Log P vIn liver microsomal stability, LMT-28 was eliminated at a moderate rate by both human and rat cytochrome P450 metabolic enzymes. The half-life value of rat liver microsomal stability was 15.3 min and that of human was 21.9 min. In addition, there were interspecific differences between human and rat liver microsomal stability and metabolic stability of human liver microsome was slightly higher than that of rat. These data suggested the bioavailability in humans can be increased compared to that in rats. In addition, the differences between human and rat might be caused by the genomic differences of cytochrome P450 genes between human and rodent ,26.In this study, MDCK cells were used to examine cell per meability, drug absorption, and tissue disposition. Even though Caco-2 cell is one of the gold standard methods to evaluate drug properties, MDCK cells are also widely used for estimating both drug absorption and disposition to tissues ,23. In aapp was 9.7±1.8 (×10−6 cm/s) at 10 μM. Furthermore, in the plasma protein binding assay, LMT-28 showed moderate plasma protein binding properties (92.4%). These results suggested that most of LMT-28 bound to plasma protein for transport in blood circulation. Taken together, it is reasonable to suggest LMT-28 is a good drug candidate for novel therapeutic IL-6 inhibition.LMT-28 had very good permeability properties in the MDCK cell permeability assay. The PIn vivo pharmacokinetic studies using BALB/c mice, the area under the plasma concentration-time curve values of LMT-28 during 8 h (AUC8h) after oral administration (5 mg/kg) was 292±202 h·ng/mL and AUC∞ after integration into infinity was 302±209 h·ng/mL. The sampling time (8 h) was over 7 times of terminal half-life  and these AUC values for 8 hours (AUC8h) were able to cover approximately 96.7% of the total AUC (AUC∞). Thus, in the case of oral administration, the sampling time and the AUC value for 8 hours were sufficient to assess in vivo pharmacokinetics of LMT-28. The peak plasma concentration (Cmax) was 137±100 ng/mL and the time to reach Cmax (Tmax) was 0.80±0.67 h. After oral administration of LMT-28 to BALB/c mice, Cmax and Tmax showed good drug-like properties. Compared with the AUC value after intravenous administration, the bioavailability (F) of LMT-28 was calculated to be approximately 38.2%, and this may be regarded as a reasonable value considering that LMT-28 has been developed as a novel oral drug candidate for the first synthetic IL-6 inhibitor.in vivo system of mice. These results suggest that LMT-28 is a good oral drug candidate for the first synthetic IL-6 inhibitor.In summary, LMT-28 showed a moderate liver microsomal stability in both rat and human, and the bioavailability in human may be increased due to their interspecific differences between rat and human liver microsomal stabilities. In addition, the high cellular permeability of LMT-28 across monolayers of MDCK cells may be associated with sufficient exposure to systemic blood circulation following oral administration of LMT-28 in the