Guanylate cyclase C (GUCY2C) is a transmembrane receptor expressed on the luminal aspect of the intestinal epithelium. Its ligands include bacterial heat-stable enterotoxins responsible for traveler's diarrhea, the endogenous peptide hormones uroguanylin and guanylin, and the synthetic agents, linaclotide, plecanatide, and dolcanatide. Ligand-activated GUCY2C catalyzes the synthesis of intracellular cyclic GMP (cGMP), initiating signaling cascades underlying homeostasis of the intestinal epithelium. Mouse models of GUCY2C ablation, and recently, human populations harboring GUCY2C mutations, have revealed the diverse contributions of this signaling axis to epithelial health, including regulating fluid secretion, microbiome composition, intestinal barrier integrity, epithelial renewal, cell cycle progression, responses to DNA damage, epithelial-mesenchymal cross-talk, cell migration, and cellular metabolic status. Because of these wide-ranging roles, dysregulation of the GUCY2C-cGMP signaling axis has been implicated in the pathogenesis of bowel transit disorders, inflammatory bowel disease, and colorectal cancer. This review explores the current understanding of cGMP signaling in the intestinal epithelium and mechanisms by which it opposes intestinal injury. Particular focus will be applied to its emerging role in tumor suppression. In colorectal tumors, endogenous GUCY2C ligand expression is lost by a yet undefined mechanism conserved in mice and humans. Further, reconstitution of GUCY2C signaling through genetic or oral ligand replacement opposes tumorigenesis in mice. Taken together, these findings suggest an intriguing hypothesis that colorectal cancer arises in a microenvironment of functional GUCY2C inactivation, which can be repaired by oral ligand replacement. Hence, the GUCY2C signaling axis represents a novel therapeutic target for preventing colorectal cancer. Identified nearly 30 years ago as a signaling network hijacked by diarrheagenic bacteria to stimulate intestinal secretion , cGMP si2+ transients, and nitric oxide. In turn, cGMP effectors include cGMP-dependent protein kinases (PKGs), cGMP-gated ion channels, and phosphodiesterases (PDEs). The spatiotemporal parameters of intracellular cGMP transients are a function of synthesis by guanylate cyclases and degradation by phosphodiesterases. In the spirit of brevity, we refer readers to thorough reviews of guanylate cyclase signaling  to the second messenger, cGMP , 13. Theignaling , 14. Her− and HCO+/H+ exchanger 3 (NHE3), preventing Na+ absorption from the lumen (Linzess™) ((Trulance™) (The GUCY2C isoform belongs to the particulate family of guanylate cyclases. While other cyclases (including soluble guanylate cyclase and the particulate guanylate cyclases A and B) have a widespread tissue distribution, GUCY2C is largely restricted to the intestinal tract , 16. It al lumen –20. Addihe lumen –20. The he lumen , 22. Twoinzess™) –25 an STulance™) , 27 an aulance™) .D. virginiana) and the European eel (A. japonica), respectively , 3029, 3ectively –33. The ectively –37, and ectively . Structuectively , 39, 40.ectively . Howeverectively . Despiteectively .Guanylin peptides have gained increasing attention as drug templates for the treatment of gastrointestinal disorders. In addition to the aforementioned linaclotide and plecanatide, both already FDA-approved, a second uroguanylin analog, dolcanatide has shown promise in ameliorating intestinal inflammation in rodent models , 44. FroThe primary effectors of cGMP are the cGMP-dependent protein kinases (PKGs), PKGI, and PKGII. PKGs belong to the serine/threonine class of protein kinases and consist of three domains: (1) an N-terminal domain necessary for homodimerization, autoinhibition, and subcellular localization, (2) a regulatory domain consisting of two cGMP binding pockets, and (3) a catalytic domain containing the ATP and substrate-binding pockets . cGMP biThe cyclic nucleotides cGMP and cAMP are degraded to 5′-GMP and 5′-AMP by a family of enzymes called phosphodiesterases (PDEs). Eleven PDEs have been identified, each with varying tissue distribution, subcellular localization, and affinity for cGMP and cAMP. For example, PDE-4,−7, and−8 have higher affinity for cAMP, PDE-5,−6, and−9 for cGMP, and PDE-1,−2,−3,−10, and−11 hydrolyze both –55. PDEsThe most apparent role of cGMP signaling in the intestine can be appreciated from human populations harboring mutations in GUCY2C, resulting in hyper- or hypo-secretion syndromes. In the recently described familial GUCY2C diarrhea syndrome (FGDS), a single missense mutation in the catalytic domain of GUCY2C produces hyperactivation of the receptor in response to ligand , 59, 60.−/−  –82. In tn injury . Correspial cGMP , 85. TheSeveral studies demonstrate that cGMP opposes proliferation by arresting the cell cycle. This was observed nearly two decades ago in colorectal cancer cell lines treated with the GUCY2C ligands, ST and uroguanylin, 8-Br-cGMP (a cell permeable cGMP), or the PDE inhibitor, zaprinast . Subsequ2+ opposes proliferation through activation of plasma membrane-bound calcium-sensing receptors (CaRs), and entry through cyclic-nucleotide-gated ion channels. Stimulation of GUCY2C with ST recruits CaRs to the cell surface and activates cGMP-gated channels, resulting in Ca2+-mediated cytostasis  generated by metabolically-active crypt cells . Cells dmin/+ mice exhibit tetraploidy  and DNA oxidation  or by the paracellular route, which is regulated by junctional complexes that bind the lateral walls of epithelial cells together . These j−/− relative to wild type littermates (−/−) accelerated the onset of the disease  a sterile inner layer, rich in secreted immunoglobulin A and bioactive molecules , that protects the epithelium from direct bacterial contact, and (2) an outer layer home to bacterial flora , 139. Thodentium . Mice laus layer . In turnE. coli and incidence of colorectal cancer. Geographic regions with endemic enterotoxgenic E. coli , which produce the virulence factor and GUYC2C agonist, ST, have far lower rates of colon cancer  and cellular  elementPathological conditions, including chronic inflammation and neoplastic transformation, promote the recruitment of activated fibroblasts in a mutually reinforcing feedback loop. Secretion of TGFβ by injured, inflamed, or neoplastic epithelium activates fibroblasts, inducing changes in their proliferation, migration, adhesion, secretory, and matrix remodeling properties , 150–152cGMP signaling opposes epithelial-mesenchymal interactions underlying tumorigenesis. Several studies have described mechanisms by which intestinal cGMP signaling inhibits cancer cell migration, invasion, and microenvironment remodeling –159. cGMBeyond its role as an intracellular second messenger, cGMP also acts as a paracrine signaling molecule in the intestine. Activation of GUCY2C produces intracellular cGMP accumulation, as well as cGMP release into the extracellular environment –162. ThiColorectal cancer remains the second leading cause of cancer death and fourth most incident cancer in the United States . Geneticmin/+ mice , suggesting that a paracrine field of GUCY2C silencing is responsible for cGMP loss and tumor susceptibility. This latter paradigm supports the reconstitution of cGMP signaling with exogenous ligand replacement, and forms the basis for clinical trials exploring oral GUCY2C agonists as a chemopreventative strategy in humans . The relThe GUCY2C-cGMP signaling axis has emerged as a key regulator of epithelial homeostasis in the intestine. Initially described as a the regulator of fluid and electrolyte secretion, cGMP is now recognized for its roles in modulating epithelial proliferation, DNA integrity, barrier function, microbiome composition, epithelial-mesenchymal cross talk, and other aspects of epithelial function. Dysregulation of these circuits underlies intestinal transformation, and perhaps unsurprisingly, loss cGMP signaling has emerged as a common feature of colorectal tumors. The precise role of cGMP signaling in the pathophysiology of colorectal cancer remains an open-ended question, but its tumor-suppressive properties are diverse. As such, suppression of cGMP signaling may be a necessary step in tumorigenesis because it lifts a block on proliferation, microenvironment remodeling, and the accrual of DNA mutations necessary for transformation. Supporting this notion, endogenous GUCY2C activating ligands are lost in early in transformation, and also from chronically inflamed epithelium, suggesting a mechanistic basis for this recognized risk factor for colorectal cancer. Preclinical data from several laboratories demonstrates that re-activation of cGMP signaling opposes tumor formation, and the availability of FDA-approved cGMP-elevating agents underscores the tractability of this approach. Given these observations, the GUCY2C-cGMP axis represents a logical, mechanism-based target for colorectal cancer prevention.JR wrote the manuscript with input, critical feedback, and revisions by SW.SW is the Chair (uncompensated) of the Scientific Advisory Board and a member of the Board of Directors of Targeted Diagnostics & Therapeutics, Inc., which has a license to commercialize inventions arising from his work. Also, he receives research funding from, and has been a compensated speaker for, Synergy Pharmaceuticals, Inc. Further, he is Chair of the Board of Directors of Feelux Company, Ltd. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.


Epithelial–mesenchymal transition (EMT) is a tightly regulated process by which epithelial cells lose their hallmark epithelial characteristics and gain the features of mesenchymal cells. For podocytes, expression of nephrin, podocin, P-cadherin, and ZO-1 is downregulated, the slit diaphragm (SD) will be altered, and the actin cytoskeleton will be rearranged. Diabetes, especially hyperglycemia, has been demonstrated to incite podocyte EMT through several molecular mechanisms such as TGF-β/Smad classic pathway, Wnt/β-catenin signaling pathway, Integrins/integrin-linked kinase (ILK) signaling pathway, MAPKs signaling pathway, Jagged/Notch signaling pathway, and NF-κB signaling pathway. As one of the most fundamental prerequisites to develop ground-breaking therapeutic options to prevent the development and progression of diabetic kidney disease (DKD), a comprehensive understanding of the molecular mechanisms involved in the pathogenesis of podocyte EMT is compulsory. Therefore, the purpose of this paper is to update the research progress of these underlying signaling pathways and expound the podocyte EMT-related DKDs. As a severe kidney complication of diabetes mellitus (DM), diabetic kidney disease (DKD) is the major cause of disability and death in patients with diabetes. Intuitively, renal fibrosis and proteinuria are two of the most significant pathological changes of DKD. Recent findings suggest that numerous pathways are activated during the course of DKD and that these pathways individually or collectively play a role in the induction and progression of renal failure.,The podocyte is a kind of terminally differentiated cells which covers the GBM over the glomerular capillary. It consists of three morphologically and functionally different segments, the cell body, the major processes and the extending fingerlike foot processes (FPs).,Epithelial–mesenchymal transition is a tightly regulated process by which epithelial cells lose their hallmark epithelial characteristics and gain the features of mesenchymal cells. For podocytes, expression of nephrin, podocin, P-cadherin and ZO-1 is downregulated, the cell-to-cell junctions (SD) will be altered, and the actin cytoskeleton will be rearranged.Epithelial–mesenchymal transition in podocytes indicates a serious of differentiation compared with general epithelial cells mostly whereby the specificity of SD adaptor proteins. For instance, one of the criteria for type-2 EMT is a cadherin switch from E-cadherin to N-cadherin. While the podocytes do undergo a cadherin switch in EMT diseased state, this is from P-cadherin to N-cadherin. Other characteristic signals, such as high cell matrix interactions and high migration capacity, also show remarkable difference against typical epithelial cells. Because of these specific points, some scientists claim that the concept of EMT as an important pathomechanism driving podocyte injury is highly controversial. From their idea, differentiated podocytes are visceral epithelial cells that arise from epithelial precursors during renal development. Mature podocytes lack classic characteristics of epithelial cells  and they have established features of mesenchymal cells (such as expression of vimentin). Therefore, podocyte differentiation involves a physiological EMT, and it is unclear how differentiated podocytes could undergo EMT. For example, May et al.However, recently most of the scientists prefer to regard podocyte dedifferentiation as a process of EMT. Actually, differentiated podocyte still indicates most of the epithelial cell characters. Differentiated podocyte is polarized with low invasive capacity and anchorage dependence. Its SD is a functional-modified tight junction. Additionally, podocyte do undergo an analogous type-2 EMT in response to hyperglycemia and TGF-β. LiIn conclusion, it is widely accepted recently that podocytes do undergo EMT process under high glucose condition. Actually, the specialization of podocyte EMT also increases the research value of podocytes.in vitro and in vivo, could be the inducible factor of podocyte EMT through TGF-β/Smad classic pathway and multiple other pathways such as Wnt/β-catenin signaling pathway, Integrins/ILK signaling pathway, MAPKs signaling pathway, PI3K/AKT/mTOR signaling pathway, Jagged/Notch signaling pathway, and NF-κB signaling pathway  in podocytes is observed in patients with most progressive kidney diseases, indicating that TGF-β is the key regulator of ECM protein synthesis in podocytes. During EMT process, TGF-β1 contributes to excessive deposition of fibrotic materials, rewriting the expression of several epithelial cell recognition and organizational proteins.The classic signaling pathway of TGF-β in EMT is as follows. TGF-β first integrates TGF-β receptor type II (TβRII) and TGF-β receptor type I (TβRI) to form ligand receptor complex. Binding of TGF-β1 to the TβRII receptor is accompanied by phosphorylation and subsequent activation of TβRI within its cytoplasmic domain. This association results in the downstream phosphorylation and activation of its classic signaling mediators, Smad2 and Smad3. Phosphorylated Smad2/3 combines with Smad4 to form a Smad complex in cytoplasmic domain and get nuclear translocation.Alu-containing), Ski (Sloan-Kettering Institute proto-oncogene), and TGIF (TG-interacting factor) prevent gene transcription through inhibition of R-Smads.,,In TGF-β/Smad classic pathway, the I-Smads inhibit R-Smads phosphorylation by blocking their access to TβRI and by promoting the degradation of the receptor complexes. The co-repressors SnoN  pathwayWnt signaling pathway is an essential signaling pathway in living organisms. Glycosylated cysteine-rich Wnt proteins play a variety of cellular functions involving cell fate, proliferation, migration, polarity, and death,in vivo to cause podocyte EMT.,In canonical Wnt/β-catenin signaling pathway, β-catenin protein is inhibited by the Axin complex in the cytoplasm without the Wnt signaling factor. Axin complex is consisted of the axis protein Axin, the tumor gene suppressor adenomatosis polyposis coli (APC), casein protein kinase 1 (CK1) and glycogen synthase kinase-3β (GSK-3β), among which CK1 and GSK-3β may cause β-catenin amino-terminal phosphorylation and inactivation. When phosphorylated, β-catenin can be recognized and led to ubiquitin-mediated proteasomal degradation by β-TrCP.in vitro and in vivo, most of which is interacted with other signal pathways. ILK dependency β-catenin nuclear translocation was observed in both puromycin (PAN) intervened podocyte and FSGS samples.,in vitro. Mice were injected with doxorubicin and Wnt1 plasmid intravenously to construct classic podocyte EMT injury models and β-catenin activation animal models. Downregulation of glomerular SD proteins (nephrin) and proteinuria was observed in model mice.In vitro model, intervening podocytes with recombinant Wnt3a could cause activation of β-catenin accompanying the decline in adhesion between podocytes and collagen I/IV, which demonstrated the existence of podocyte EMT.Activation of Wnt signaling and over expression of β-catenin has been reported in podocyte EMT-related experiments ,,Integrin-linked kinase is an intracellular serine/threonine protein kinase which interacts with the cytoplasmic domains of the β-integrins and cytoskeleton to mediate the integrin, growing factor, and Wnt signaling pathways in podocyte transdifferentiation.in vivo triggered the integral incident of EMT process. Indubitably, inhibition of ILK expression could also obstruct podocyte EMT and the coming DKD induced by TGF-β.,The effect of ILK has recently been identified through the interactions with other classic signaling pathways such as TGF-β/Smad and Wnt/β-catenin signaling pathway. Under high glucose condition, ILK could individually or cooperatively highlight the expression of α-SMA in podocyte, which is a remarkable index of mesenchymal cells. Upregulated level of ILK and α-SMA are both significant hallmarks in the progression of podocyte EMT.,Mitogen-activated protein kinases (MAPKs) are the common pathway for the whole physiological reactions from extracellular stimuli to nuclear transcription. By influencing the gene transcription and regulation, MAPKs thereby affect the biological behavior of cells, including inflammation, cell differentiation, cell growth, and cell death.in vitro studies have shown that high levels of glucose could activate the p38 MAPK signaling pathway in podocytes and induce the phosphorylation of p38 MAPK, which promotes the production of fibronectin by the mesangial cells.,Both JNK pathway and p38 MAPK pathway play a critical role in TGF-β induced podocyte EMT. Stambe et al. have shown that p38 MAPK could be a downstream signaling in TGF-β1 induced podocyte-related renal interstitial fibrosis (RIF). Blockade of p38 MAPK has been reported to reduce ECM production.The Jagged/Notch signaling pathway is also considered an important regulator for EMT induction, despite several reports that Notch signaling is insufficient to completely induce EMT for it requires crosstalk with other signaling molecules.,,Notch signaling requires coordination with other signals to promote podocyte EMT. Previous theories thought Notch signaling was a downstream target molecule of Wnt signaling pathway, Notch signaling pathway is a Wnt-mediated pathway, but recent studies have found that Notch is more like an independent podocyte EMT signaling pathway.,,,As a transcription factor, NF-κB plays a key role in the expression of genes involved in EMT initiation and progression. NF-κB induces and maintains EMT in model systems through two mechanisms, upregulation of EMT master-switch transcription factors2+ deprivation.NF-κB signaling pathway plays an important role in podocyte dedifferentiation and podocyte EMT. Qi et al.In this review, we discussed and summarized most of the known molecular mechanisms and downstream interactions between TGF-β/Smad classic pathway and multiple other pathways during the occurrence and development of podocyte EMT and associated DKD under high glucose condition. We also highlighted the significant cytokines linking these pathways to the process of EMT. As to podocytes, podocyte do undergo an analogous type-2 EMT in response to hyperglycemia and TGF-β. In other words, these special EMT also increase the research value of podocytes. Therefore, this review may a beneficial supplement for our further research on podocyte dysfunction and podocyte-related DKDs.


Jiawei Fengshining (JWFSN) is a new formula originated from Fengshining, a classic formula for the treatment of rheumatoid arthritis (RA). The mechanism of JWFSN in the treatment of RA is still unclear. The aim of this study was to evaluate the effect of JWFSN formula on the inflammatory mediator levels in the serum and the TGF-β1/Smad pathway in the synovium and to explore the underlying mechanisms of JWFSN formula to ameliorate synovial hyperplasia and apoptosis inhibition of synovium in rats with RA. β1, p-Smad2, p-Smad3, and Smad7 protein expressions in synovial tissue were measured by western blot assay. In addition, human rheumatoid arthritis fibroblast-like synoviocytes cell line MH7A was treated with 20% JWFSN-containing serum to obtain in vitro data.  SPF female Wistar rats were randomly divided into 6 groups: the blank control group, the model control group, the positive drug group, and the low-, medium-, and high- dose JWFSN groups, with 8 rats in each group. Enzyme-linked immunosorbent assay (ELISA) was used to detect inflammatory mediators, anti-inflammatory mediators, and rheumatoid factor (RF). The pathological condition and apoptosis of the synovial tissue were detected by hematoxylin and eosin (HE) and TUNEL staining, respectively. TGF-β1/Smad signaling pathway in CIA rats. In vitro JWFSN treatment increased the apoptosis of MH7A cells and decreased cell viability. Additionally, JWFSN treatment inhibited the TGF-β1/Smad signaling pathway in MH7A cells. Interestingly, kartogenin (TGF-β1/Smad pathway activator) treament reversed the effects of JWFSN treatment.  The administration of JWFSN was found to ameliorate synovial hyperplasia and promote apoptosis; increase the serum contents of anti-inflammatory mediators; reduce inflammatory mediators and RF contents; and inhibit the TGF-β1/Smad signaling pathway. JWFSN may ameliorate inflammatory factors' abnormality, synovial hyperplasia, and apoptosis inhibition of synovium via the TGF- Rheumatoid arthritis (RA) is an inflammatory autoimmune disease. RA is distinguished by chronic synovitis of joints and is known to have the following pathological characteristics: synovial hyperplasia, inflammatory cell infiltration, pannus production, and damage of bones and cartilages, which finally lead to the destruction and lack of function of a whole joint . This diβ1) is a multifunctional cytokine that regulates cell growth, inflammation, and angiogenesis [β1. The TGF-β1/Smad pathway has been shown to be associated with synovial hyperplasia in RA and considered as a potential target for the treatment of RA [Transforming growth factor-beta1 (TGF-ogenesis . The Smant of RA –13.Fengshining (FSN) is a famous Traditional Chinese Medicine prescription used for the treatment of rheumatoid arthritis, which is sourced from Mr. Bai Qingzuo, one of the four famous Chinese medicine practitioners in the Shanxi province [Notopterygium incisum, Radix Angelicae Biseratae, Caulis Sinomenii, Rhizoma Wenyujin Concisum, Rehmannia glutinosa, Clematis chinensis Osbeck, Saposhnikovia divaricata, Ephedra sinica, Corydalis yanhusuo, Cyathula officinalis, Curcuma zedoaria, Rhizoma Sparganii, Ligusticum chuanxiong, Zingiber officinale, Glycyrrhiza uralensis, Cinnamomum cassia, and Amomum villosum. Moreover, Periploca forrestii Schltr and Gaultheria leucocarpa var. yunnanensis are used in Traditional Chinese Medicine (TCM) known as “Miao medicine” and have a long history of use in the treatment of rheumatism, RA, and joint pain [Jiawei Fengshining (JWFSN), based on the foundation of classic prescription “FSN” and “Miao medicine.” In this study, we aimed to investigate the potential effects of JWFSN on TGF-β1/Smad signaling pathway in vivo and in vitro.province . FSN is int pain –17. ThusNotopterygium incisum 18 g, Radix Angelicae Biseratae 18 g, Caulis Sinomenii 15 g, Rhizoma Wenyujin Concisum 15 g, Rehmannia glutinosa 15 g, clematis chinensis Osbeck 12 g, Saposhnikovia divaricata 12 g, Ephedra sinica 12 g, Corydalis yanhusuo 12 g, Cyathula officinalis 12 g, Curcuma zedoaria 9 g, Rhizoma Sparganii 9 g, Ligusticum chuanxiong 9 g, Zingiber officinale 9 g, Glycyrrhiza uralensis 6 g, Cinnamomum cassia 6 g, Amomum villosum 6 g, Periploca forrestii Schltr 15 g, and Gaultheria leucocarpa var. yunnanensis 15 g. These samples were purchased from Tongrentang Pharmacy in Chengdu, Sichuan province. The raw medicinal herbs were immersed in eight-time volumes of distilled water for 30 min and then boiled for 50 min. Thereafter, five-time volumes of distilled water were added into the residue and decocted twice for 25 min. All the collected supernatants were filtered through 8 layers of gauze, condensed into an extractum by rotary evaporators.The JWFSN is an aqueous preparation from 19 medicinal herbs: in vivo studies [n = 48, weighing 190 g to 230 g) were provided by Chengdu Dashuo Experimental Animal Co., Ltd. Rats were allowed to acclimatize for at least one week before the experiment started. Bovine type II collagen  was emulsified in complete Freund adjuvant  with the ratio of 1 : 1 to obtain a collagen emulsion. 0.2 ml of collagen emulsion was injected into the rat-tail root. Rats in the control group were injected with an equal volume of saline. At day 7, another 0.2 ml of collagen emulsion was injected into the rat-tail root avoiding the original pinholes. Rats in the control group were again injected with equal volume saline [The CIA rat model has many similarities with the symptoms in human rheumatoid arthritis and is therefore widely used in RA-related  studies . Female e saline . Oral trAll the experimental rats were randomly divided into 6 groups, with 8 rats in each group. Group I: the blank control group, receiving saline treatment; group II: the model control group, suffering CIA and received no treatment; group III: the positive drug group, where CIA rats were treated with 2.33 mg/kg leflunomide ; group IV: the low-dose JWFSN group, where CIA rats were treated with 9.12 g/kg JWFSN; group V: the medium-dose JWFSN group, where CIA rats were treated with 18.24 g/kg JWFSN, and group VI: the high-dose JWFSN group, where CIA rats were treated with 36.48 g/kg JWFSN.μm filter membrane and inactivated at 56°C for 30 min, and finally stored in the ultralow temperature freezer .Rats were divided into two groups: negative control group and JWFSN treament group, with 10 rats in each group. Rats in the JWFSN treament group were treated with 182.4 g/kg/d JWFSN for 3 days. Rats in the negative control group were treated with an equivalent volume of normal saline (NS). After 3 days of treatment, blood was collected from the eyeball and centrifuged to gain the serum. Then, the serum was disinfected using the 0.22 2.Human rheumatoid arthritis fibroblast-like synoviocyte cell line MH7A was obtained from Procell Life Science & Technology Co., Ltd. . MH7A cells were cultured in the DMEM/high glucose medium  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin . Cells were incubated in the cell incubator  at 37°C under 5% CO−6 M kartogenin [Cells were divided into 4 groups. The blank control cells (BC) were cultured as normal. The negative control cells (NC) were cultured in the medium containing 20% normal serum. The JWFSN-treament cells (JWFSN) were cultured in the medium with 20% JWFSN-containing serum. The kartogenin + JWFSN treament cells (KGN + JWFSN) were pretreated using 10rtogenin  for 2 h γ (IFN-γ), interleukin-1β (IL-1β), interleukin-4 (IL-4), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α) were measured using ELISA kits .The rat serum rheumatoid factor (RF), interferon-μm thick paraffin sections were prepared. The pathological changes of tissues were observed under light microscope following hematoxylin and eosin (HE) staining. The apoptotic cells were stained using a TUNEL assay kit . Images were captured with a microscope .Rats were sacrificed at day 28 after arthritis induction. The synovial tissues of ankle and knee joints were taken out immediately and fixed with 4% paraformaldehyde for 3 d, decalcified with 5% formic acid, and 5 β1 , and β-actin . The blots were washed and incubated with goat anti-rabbit IgG H&L  secondary antibody. The protein bands were visualized using an ECL chemiluminescence kit  and quantified using Image-ProPlus 6.0 software .Protein was extracted from the synovial tissues and MH7A cells using a protein extraction kit  and quantified with the BCA kit . Equal amounts of protein were mixed with the loading buffer, boiled for 15 min at 95°C, and loaded into a 10% SDS-PAGE gel. After resolving the proteins by SDS-PAGE, the bands were transferred to PVDF membranes. Then, they were blocked for 1 h with 5% skimmed milk powder at room temperature and incubated overnight with primary antibodies against p-Smad2 , p-Smad3 , Smad7, TGF-Paraffin-embedded synovial tissue sections were dewaxed through an ethanol gradient and incubated with 3% hydrogen peroxide in methanol for 10 min at room temperature to quench endogenous peroxidases and then boiled in 0.01 M sodium citrate buffer (pH = 6) for antigen retrieval, washed with PBS, and blocked with 10% normal goat serum  for 20 min at 37°C. The sections were incubated overnight with primary antibodies against XIAP , Bcl-2 , and Bax  at 4°C and then incubated with the goat anti-rabbit IgG H&L (HRP polymer)  secondary antibody for 30 min at 37°C. After immersing in DAB  for coloration, the sections were rinsed with distilled water, counterstained with hematoxylin, dehydrated, and mounted. The stained sections were observed under a microscope . The integrated optical density (IOD) was quantified using Image-ProPlus 6.0 software , and the mean optical density (MOD) of each sample was calculated.3/well). Cells were incubated with 10% CCK-8 solution for 1 h, and the absorbance of cells at 450 nm was measured using a microplate reader after incubation.Cell Counting Kit-8 (CCK-8)  assay was used to measure cell proliferation. MH7A cells in the logarithmic phase of growth were seeded into 96-well plates (6 × 10Annexin V-APC/PI staining and flow cytometry (FCM) were performed to detect apoptosis of MH7A cells. In brief, cells were double-labeled with Annexin V-APC/PI  for 15 min at room temperature in the dark. Then, FACSCalibur™ Flow Cytometer flow cytometry  was used to analyze the cell apoptosis within 1 h.P values <0.05 were considered statistically significant.Statistical analyses were performed using SPSS 20.0 software . Data were presented as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to compare differences among multiple groups, followed by LSD's post hoc test. As shown in γ, IL-1β, TNF-α, IL-4, IL-10, and RF in serum of CIA rats. As shown in γ, IL-1β, TNF-α, and RF in the model group was significantly higher than those in the control group. However, compared with those in the model group, the levels of the abovementioned inflammatory mediators and RF were significantly decreased in the JWFSN-high, positive drug, and JWFSN-medium groups. The levels of IL-4 and IL-10 in the model group were significantly lower than those in the control group. Compared with those in the model group, the levels of the abovementioned anti-inflammatory mediators were significantly increased in the JWFSN-high, positive drug, and JWFSN-medium groups.To observe the effect of JWFSN on production of inflammatory mediators, anti-inflammatory mediators, and rheumatoid factor, we examined the expression levels of INF-Pathological changes in the synovial tissue were measured with HE staining . CompareThe results of apoptotic rate in synovial tissues of ankle and knee joints indicated that there was no significant difference between the control and model groups. The apoptotic rate of JWFSN-low, JWFSN-medium, and JWFSN-high and positive drug groups were increased compared with model group .These results indicated that JWFSN could improve synovial hyperplasia and promote apoptosis of synovial tissues in CIA rats.To characterize the mechanism of JWFSN-induced apoptosis in synovial tissue cells, we examined the expression levels of XIAP, Bcl-2, and Bax in ankle joints' synovial tissues of CIA rats by IHC . The resβ1, p-Smad2, and p-Smad3 in synovial tissue of ankle joint and knee joint in the model group were significantly increased, and the expression level of Smad7 was significantly decreased. Compared with model group, TGF-β1, p-Smad2, and p-Smad3 levels of ankle joint and knee joint synovial tissue of JWFSN-high and positive drug groups were significantly decreased, and the expression level of Smad7 was significantly increased. This result indicated that the TGF-β1/Smad signaling pathway was overactivated in RA rats, and the high concentration of JWFSN could reverse.As shown in β1, p-Smad2, p-Smad3, and Smad7 in MH7A cells. As shown in β1, p-Smad2, p-Smad3, and Smad7 between the blank control group and the negative control group. Compared with the negative control group, the expression of TGF-β1, p-Smad2, and p-Smad3 in the JWFSN treatment group was significantly decreased, and the expression level of Smad7 was significantly increased. However, after pretreatment with KGN, the expression of TGF-β1, p-Smad2, and, p-Smad3 in the KGN + JWFSN treatment group was significantly increased, and Smad7 was significantly decreased. This result indicated that JWFSN could inhibit the activation of TGF-β1/Smad signaling pathway, while KGN treatment reversed the effects of JWFSN.To further testify the signal transduction pathway of JWFSN, western blot assay was performed to detect the expression of TGF-β1/Smad pathway.To further investigate the effect of JWFSN on MH7A cells, CCK8 and FCM assays were performed. As shown in TCM-based therapies for RA are popular in China . In thisα, IFN-γ, and IL-1β are significantly elevated in the peripheral blood of patients with RA [β and TNF-α in the model group were significantly higher than those in the control group, while the levels of serum IL-4 and IL10 were significantly lower than those in the control group. The anti-inflammatory factors IL-4 and IL10 are inhibited by the increased inflammatory factors IFN-γ, IL-1β, and TNF-α, which proved that the CIA model prepared during the experiment was successful. Rats were given JWFSN decoction for 28 days, inhibited the expression of inflammatory factors IL-1β and TNF-α, and increased the expression of anti-inflammatory factors IL-4 and IL-10. The effect of rheumatism in the JWFSN-middle and JWFSN-high dose groups is particularly obvious, suggesting that the therapeutic effect of JWFSN on CIA rats may be through direct or indirect reduction of TNF-α, IFN-γ, and IL-1β expression and improve IL-4 and IL-10. It may be one of the mechanisms of rheumatoid arthritis in the treatment of rheumatoid arthritis.The release of proinflammatory factors and the inhibition of anti-inflammatory factors are important characteristics of rheumatoid arthritis. Studies have found that TNF- with RA –22. IL-4 with RA , 24. TheConcerning H&E and TUNEL staining experiments, we found that the synovial tissues were improved inflammatory cell infiltration and synovial hyperplasia in CIA rats following JWFSN administration. Moreover, the administration of JWFSN also promoted synovial cells from apoptosis. These results indicated that JWFSN could alleviate the damage of ankle and knee joins synovial tissues. The Bcl-2 family proteins are critical regulators of the mitochondrial apoptotic pathway . An incrβ1 is mediated through both TGF-βR1 and TGF-βR2 receptors to induce phosphorylation of Smad2 and Smad3 [β1 can induce Smad7 expression, an inhibitory Smad family member, to negatively regulate TGF-β/Smad signaling pathway via a feedback regulation [β causes articular subchondral bone destruction in the rheumatoid arthritis rat/mouse models, and conditional deletion of TGF-βR2 in nestin-positive cells effectively halted progression of RA joint damage [β1, p-Smad2, and p-Smad3 were decreased, and the expression of Smad7 was increased in CIA rats following JWFSN administration. Additionally, JWFSN inhibited TGF-β1/Smad signaling pathway activity, decreased cell viability, and promoted apoptosis in MH7A cells. Interestingly, KGN reversed the effects of JWFSN treatment. These results showed that TGF-β1/Smad signaling pathway might be a target for the treatment of rheumatoid arthritis by JWFSN.It is well accepted that TGF-nd Smad3 –34. Moregulation , 36. Xu t damage . Zhou ett damage . Converst damage . The wesβ1/Smad pathway. We found that JWFSN ameliorate synovial hyperplasia and apoptosis inhibition. These effects might be associated with downregulation of TGF-β1, p-Smad2, and p-Smad3, and upregulation of Smad7.At the present study, we investigated the effects and the possible mechanism of JWFSN on CIA rats, with a focus on the TGF-β1/Smad signaling pathway in RA. This result provides a theoretical basis for the treatment of RA by JWFSN.Our results demonstrated that JWFSN inhibited synovial hyperplasia and promoted apoptosis via TGF-


Human T cell leukemia virus type 1 (HTLV-1), the etiological agent of adult T-cell leukemia/lymphoma (ATLL) and the demyelinating neuroinflammatory disease known as HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP), was the first human retrovirus to be discovered. T-cells, which represent the main reservoir for HTLV-1, have been the main focus of studies aimed at understanding viral transmission and disease progression. However, other cell types such as myeloid cells are also target of HTLV-1 infection and display functional alterations as a consequence. In this work, we review the current investigations that shed light on infection, transmission and functional alterations subsequent to HTLV-1 infection of the different myeloid cells types, and we highlight the lack of knowledge in this regard. In early 80’s, several works described HTLV-1 endemicity in Japan, Caribbean, South America, and central Africa . Even thin blood . HTLV-1 in blood , 8. The in blood , 10. Besin blood , these tA number of inflammatory diseases such as uveitis , 13, art+, and to a lesser extent CD8+ T-cells represent the main target of HTLV-1 in vivo, and they present the highest PVL [+ T-cells, HTLV-1 infection may affect other cell types, and that T-cells functional alteration may be the top of the iceberg, as a result of earlier or subtler modifications of others cells types or immune compartments. Since it is at the forefront of induction and maintenance of immune responses, myeloid compartment may deserve a special interest, through its unique ability to polarize naïve T-cells into either cytotoxic, inflammatory, regulator or tolerant T-cell effectors [CD4hest PVL , 16. Thehest PVL , 18 and hest PVL , 20. Finffectors –25. ThusIn this work, we review the current literature investigating the role of myeloid cells during HTLV-1 infection, and we highlight the lack of knowledge that impairs researchers from fully understanding HTLV-1 infection and, potentially, the differential mechanism of disease evolution.Myeloid cells derive from a common myeloid progenitor whose differentiation gives rise to several cell-type forming the myeloid compartment. This includes dendritic cells present in blood (named as myeloid or myDC throughout the text) and in the different mucosa, plasmacytoid dendritic cells (pDC), and monocytes, that can further differentiate into macrophages or dendritic cells Fig. . As sent+ and CD4+ T-cells) isolated from HAM/TSP blood samples were shown to share the same HTLV-1-integration site. Same results were obtained using cells from STLV-1 infected Japanese macaques. These results demonstrate a primary infection of stem-cell lineage [Recently, HTLV-1 DNA was also detected in hematopoietic stem cells (HSC) in vivo , 34. HSC lineage , 34.To validate these in vivo results, several groups performed in vitro exposure of blood macrophages, myDC, monocytes, monocytes derived DC (MDDC) or pDC to HTLV-1 , 35–43. In contrast to MDDC, investigations using pDC represented a challenge due to their sparse representation in human blood. Nevertheless, one study reported in vitro infection of blood pDC by cell-free HTLV-1 viral particles, with a permanent viral production even after several week of pDC culture . This reWhile several groups were able to show in vitro infection of MDDC , 53, 54 Since in vitro infection is abortive, how viral DNA could be detected in monocytes and pDC after their purification from patient’s blood remains to be understood. One possible explanation relies on the fact that viral DNA was detected in HSC , 34. TheAs expected, all in vitro infected DCs were shown to produce HTLV-1 viral particles, detected in the supernatant using Gag p24 detection kit  or in ce+ T-cells, compared to a similar experiment performed in mice without DC depletion [The role of DC infection in HTLV-1 dissemination is also supported by investigations performed in animals. After dendritic cells depletion, mice infection with a chimeric HTLV-1 virus pseudotyped with the murine leukemia virus (MLV) envelop showed lower HTLV-1 proviral load in CD4epletion . In addiepletion . Thus, iof an active HTLV-1 replication, are detected in the blood obtained either from asymptomatic HTLV-1 carriers, and from ATLL or HAM/TSP patients [HTLV-1 transmission requires an interaction between the target cell and an infected cell. Transfusion of cellular blood components of HTLV-1 infected donors results in infection of recipients, whereas recipients of non-cellular blood compartment are not infected , 61. Imppatients . Thus thpatients . Thus, ipatients , and unlpatients –68, it rIn vivo observations were confirmed by in vitro studies showing that cell–cell contact was required for HTLV-1 transmission between T-cells, either through (i) viral synapse formation between infected T-cells and target T-cell , (ii) thIn the context of breast-feeding or sexual intercourse, infected T-cells present in the maternal milk or in the semen are exposed to the luminal side of intestine or genital mucosa, mainly composed of epithelial cells. However, HTLV-1 infected T-cells were shown to be unable to infect in vitro epithelial cells or to cross the epithelial barrier , thus prAltogether a model in which dendritic cells play a central role during primo-infection for HTLV-1 dissemination in new individuals can be proposed. In the case of breast-feeding or sexual transmission Fig. a, capturAlthough not truly infected by HTLV-1, monocytes bearing viral DNA inherited from HSC differentiation , 34 may Besides its viral transmission to T-cells, HTLV-1 presence and expression in myeloid cells may alter their function. However, compared to what is known regarding T-cells functional perturbation little is known concerning these changes . MoreoveLittle is known about modifications in macrophage’s cytokines secretion upon HTLV-1 infection. Balistrieri et al.  demonstr+CD14+) compared to monocytes from healthy donors, while the frequency of classical monocytes (CD16−CD14+) was not affected. Of note, frequency of non-classical monocytes (CD16+CD14−) was not investigated in this study. However, others using more precise identification of monocytes showed that frequency of non-classical (CD16+CD14−) monocytes was increased, while the frequency of classical monocytes was decreased in HTLV-1 infected individuals (AC and HAM/TSP) [+ monocytes  have been reported to be more prone to differentiation into dendritic cells and could be the monocyte subset committed to DC differentiation in vivo.Nascimento et al.  observedHAM/TSP) . Interes+ T-cells degranulation compared to monocytes from healthy donors or from asymptomatic carriers [+ infected T-cells, suggesting that direct interaction of activated monocytes  with CD4+ lymphocytes potentiate reactivation of viral replication, thus skewing monocyte immune function in favor of viral replication.Direct alteration of monocytes from HAM/TSP patients was also reported and was associated with an increased ability to induce CD8carriers , 85. Thi+ conjugates in samples from HAM/TSP patients compared to uninfected samples, and to the loss of monocytes populations after 18 h culture of PBMCs from HAM/TSP patients [Yet the mechanism responsible of such monocytes activation is not known. Furthermore, whether these defects are linked to their proviral load in vivo is unknown, although a negative correlation between virus burden in intermediate monocytes and their phagocytic function was observed . Alternapatients . This su+ monocyte compartment by HTLV-1, as discussed above. Furthermore, MDDC derived from monocytes of ATLL patients have a reduced ability to present antigen and have altered capacities to stimulate proliferation of allogenic T-lymphocytes [+ and CD8+ T-lymphocytes [Study performed in monocytes obtained from ATLL patients showed they poor ability to differentiate into Monocytes Derived Dendritic Cells in vitro, probably as a result in alterations of the CD16phocytes , 93. In phocytes , althougphocytes , 91  might be a key factor in immune system maturation, and especially regarding the role of myeloid cells in controlling the viral adaptive immune responses, further investigations should be focused on understanding the role of myeloid cells in HTLV-1 spreading and disease progression.


Immunity against pathogens evolved through complex mechanisms that only for sake of simplicity are defined as innate immunity and adaptive immunity. Indeed innate and adaptive immunity are strongly intertwined each other during evolution. The complexity is further increased by intrinsic mechanisms of immunity that rely on the action of intracellular molecules defined as restriction factors (RFs) that, particularly in virus infections, counteract the action of pathogen gene products acting at different steps of virus life cycle.Here we provide an overview on the nature and the mode of action of restriction factors involved in retrovirus infection, particularly Human T Leukemia/Lymphoma Virus 1 (HTLV-1) infection. As it has been extensively studied by our group, special emphasis is given to the involvement of the MHC class II transactivator CIITA discovered in our laboratory as regulator of adaptive immunity and subsequently as restriction factor against HIV-1 and HTLV-1, a unique example of dual function linking adaptive and intrinsic immunity during evolution. We describe the multiple molecular mechanisms through which CIITA exerts its restriction on retroviruses. Of relevance, we review the unprecedented findings pointing to a concerted action of several restriction factors such as CIITA, TRIM22 and TRIM19/PML in synergizing against retroviral replication. Finally, as CIITA profoundly affects HTLV-1 replication by interacting and inhibiting the function of HTLV-1 Tax-1 molecule, the major viral product associated to the virus oncogenicity, we also put forward the hypothesis of CIITA as counteractor of HTLV-1-mediated cancer initiation. Immunity against pathogens and pathogen adaptation to their host coevolved and keep evolving in symbiosis as a continuous process with mutualistic and antagonistic features to guarantee protection of the host as a species and selection of the pathogen for the best fitting without killing the host . MechaniRFs were first identified as inhibitors of Human Immunodeficiency Virus 1 (HIV-1) infection, targeting various stages of viral life cycle, from capsid uncoating to viral budding , 9  anchors budding viral particles at the late post-integration stages of replication on the surface of infected cells, preventing the release of HIV-1 and other enveloped viruses , 15. HIVTripartite motif proteins (TRIMs) are an E3 ligase family critical in many cellular functions, including the regulation and coordination of innate immunity and antiviral responses. They are characterized by a conserved tripartite motif, known as “RBCC”, comprising 3 functional domains: a RING, responsible for the E3 ubiquitine-ligase activity of the protein, one or two B-box(es) and a coiled-coil (CC) domain. The integrity of the TRIM motif is essential for their homo-multimerization and cellular localization , 34. SevMore recently, the family of potential RFs against HIV has substantially increased, indicating how much we still have to unveil about the complexity of molecules involved in intrinsic immunity .As described above, the discovery of RFs and the description of their mechanism of action have been mostly derived by studies on HIV-1 retrovirus. The other extremely important member of human retroviruses affecting human health is HTLV-1, the first described human retrovirus, and the etiologic agent of a severe and still untreatable form of adult T cell leukemia/lymphoma (ATL) , 54. HTLtax gene [HTLV-1 infects approximately 10–20 million people world-wide, with high prevalence in the South of Japan, in sub-Saharan Africa, the Caribbean islands. Infection is also common in some regions of South America, the Middle East and Austro-Melanesia . HTLV-1 tax gene  and by ttax gene . Converstax gene . Cumulattax gene . Recent tax gene –75.Cumulatively, these studied may in part explain the behavior of the classical adaptive immune response against HTLV-1, which is predominantly mediated by a strong CTL response against Tax-1 in the first phases of infection. Such response is believed to counteract virus spreading. Silent infection can proceed for decades. The subsequent adaptation and/or escape of the virus from host immunity is associated to down regulation of Tax expression and low response against HBZ , 77 as iHBZ coding region than in other genes, such as tax, may in part explain why HBZ is constantly expressed in ATL and during HTLV-1 infection [As mentioned before, differently from HIV-1, HTLV-1 produces only very low levels of cell-free infectious virions, since it is transmitted by cell-to-cell contact. Interestingly, the genetic diversity of HTLV-1 is much lower than that of HIV-1  even thonfection . The relnfection . Based onfection  in a vernfection , 83.Comparatively less information is available for other families of RFs other than APOBEC in HTLV-1 infection and associated diseases.Recently, Leal et al.  by usingAlthough HTLV-1infects preferentially T cells, also cells of myeloid lineage, which play critical roles in the host innate response against viral infection, are targeted by HTLV-1 , 60, 88.Studies related to a possible effect of tetherin, also called BST-2, on HTLV-1 infectivity have indicated that, unlike HIV-1, HTLV-1 does not express a protein to downregulate the expression levels of tetherin to overcome its restriction. Indeed, tetherin is highly expressed in chronically HTLV-1-infected cells and colocalizes with viral particles at the site of cell to cell contact. Nevertheless, silencing of tetherin impacts only minimally on infectivity of HTLV-1 although cell-to-cell transmission is certainly more relevant for HTLV-1 spreading as compared to HIV . Based oIn recent years, several studies have shown the importance of micro RNAs (miRNA) in HTLV-1 infection and associated disease pathogenesis . InteresAnother host factor endowed with anti-viral function for HTLV-1 is the MHC class II transcriptional activator, originally discovered in our laboratory as the major coordinator of expression of all MHC class II genes –96, By pBesides its prominent role in the regulation of adaptive immune response, the first evidence that CIITA may act as an RF emerged in the context of HIV-1 infection, when we found it was acting as negative transcriptional regulator of HIV-1 expression in T cells. Here, CIITA inhibited virus replication by competing with the viral transactivator Tat for the binding to the Cyclin T1 subunit of the positive transcription elongation complex (P-TEFb) . More reThe first evidence that CIITA exerted an inhibitory function also on HTLV retroviruses dates back to 2004 when we demonstrated that CIITA blocks HTLV-2 virus replication both in T cells and B cells . That CIThe great plasticity of CIITA molecule and the different mechanisms exerted by this host factor to counteract retroviral infections has been further confirmed and extended by our recent findings that CIITA binds directly to HTLV-1 Tax-1 . BesidesAlthough several studies have focused at identifying restriction factors and elucidate their antiviral mechanisms on HTLV-1 infection, much investigation is still required to delineate a structured framework similar to the one described for the RFs in HIV-1 infection. Controversial informations are part of this still reduced knowledge that results from an intrinsic limitation in studying HTLV-1-infected cells as compared to HIV-1-infected cells, due to the time frame through which HTLV-1 infection develops, the mode of viral transmission and the distinct pathological outcomes of infection. Nevertheless, some evidence of objective involvement of RFs in the control of HTLV retrovirus life cycle exists as well as preliminary important distinction on the putative mechanism of these RFs with respect to their mechanisms of action against HIV-1 (Table As far as CIITA, its restricted tissue distribution to lymphoid and myelomonocytic cells, both susceptible targets of HTLV-1 infection, in conjunction with its inducible expression by IFNγ, similar to other RFs, and its strong inhibitory activity on HTLV-1 as well as HTLV-2 and HIV-1 (Table


E software. Our study revealed a panel of changes in protein expression linked to HTLV-1 infection. Upregulation of heat shock proteins and downregulation of canonical histone expression were observed in monocytes from HTLV-1-infected patients. Moreover, expression of cytoskeleton proteins was increased in monocytes from HTLV-1-infected patients, mainly in those from HAM/TSP, which was confirmed by flow cytometry and fluorescence microscopy. Importantly, functional assays demonstrated that monocytes from HAM/TSP patients present higher ability for adhesion and transmigration thought endothelium than those from AC and uninfected individuals. The major changes on monocyte protein profile were detected in HAM/TSP patients, suggesting that these alterations exert a relevant role in the establishment of HAM/TSP.Human T-cell lymphotropic virus type-1 (HTLV-1) is the etiological agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The endothelial breakdown and migration of leukocytes, including monocytes, to the spinal cord are involved in HAM/TSP development. Monocytes from HTLV-1-infected individuals exhibit important functional differences when compared to cells from uninfected donors. Using proteomic shot gun strategy, performed by nanoACQUITY-UPLC system, we analyzed monocytes isolated from peripheral blood of asymptomatic carriers (AC), HAM/TSP and uninfected individuals. 534 proteins were identified among which 376 were quantified by Expression The neurodegeneration affects mainly the thoracic spinal cord section, which leads to slowly progressive spastic paraparesis with low back pain, in addition to bowel, urinary and sexual dysfunctions4. About 5–10% of total infected population, referred as symptomatic patients, develop either ATLL or HAM/TSP5; however, some studies have shown that HTLV-1-asymptomatic carriers (AC) present important clinical manifestations, such as paresthesia, paresis, nocturia, arthralgia, gingival bleeding, erectile dysfunction and opportunistic infections7. High prevalence areas include Latin America, Southern Japan, Central and Western Africa7. Brazil is the largest endemic country for HTLV-1 infection, and its prevalence among blood donors ranges from 0.04 to 1%7.Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL) malignancy+ T lymphocytes are the predominant target for HTLV-1 infection9. HTLV-1-infected T lymphocytes display higher levels of spontaneous proliferation and activation markers than their uninfected counterparts11. HAM/TSP disease depends on endothelial breakdown and migration of activated and/or infected T lymphocytes into the spinal cord13. Analyses of lesions from HAM/TSP patients revealed the presence of infected CD8+ T cells, B cells14, dendritic cells (DCs)15, macrophages16 and monocytes18, suggesting they may play a role during disease onset.CD419. HAM/TSP patients present lower frequencies of classical monocytes than uninfected individuals21. Our group demonstrated that monocytes from HTLV-1-infected individuals, AC and HAM/TSP patients, presented reduced ability for in vitro differentiation into DCs when compared to monocytes from uninfected donors22. However, HTLV-1 proviral load in freshly isolated monocytes was not associated with this finding, suggesting that impaired DC differentiation reflects alterations other than direct infection of monocytes22. Here we investigated the hypothesis that monocytes from HTLV-1-infected patients could exhibit distinct molecule profiles, thus contributing to alteration of their phenotype and function.Monocytes constitute a multifunctional heterogeneous cell population associated with immune response, tissue repair and homeostasis23.HTLV-1-infected cells have been described as exhibiting alterations in protein expression profiles, such as the reduction in choline, phosphocholine, spermine and glutathione production, and an increase in creatine, dopamine, arginine and adenosine monophosphate levelsFor the first time, monocytes obtained from AC peripheral blood, HAM/TSP and uninfected individuals were studied using proteomic analysis. Proteomic analysis is a powerful tool to understand HTLV-1 pathophysiology, indicating candidate biomarkers for diagnosing, staging, and tracking HTLV-1-associated diseases, and our results evince that HTLV-1 infection impact monocyte protein profile during HAM/TSP disease.This study enrolled HAM/TSP patients and AC , and uninfected individuals as a control group  were isolated using Ficoll-Paque density gradient centrifugation (Sigma-Aldrich), and then washed three times with phosphate buffer saline (PBS). Monocytes were purified by negative selection using Dynabeads Untouched Human Monocytes Kit (Invitrogen). After that, cells were washed with PBS and treated with lysis solution for LC-MSE supplemented with protease inhibitor cocktail tablets (Roche). Extracts were sonicated for 5 minutes, centrifuged at 15,000 × g for 15 minutes at 4 °C, and the supernatants were recovered. Protein concentration for each extraction was determined by BCA method (Sigma-Aldrich), and samples were stored at −80 °C.4HCO3 using 3-kDa cutoff Amicon filter (Millipore), the protein extract supernatant samples were quantified. Monocyte lysate supernatant (2 µg/μL) in 50 mM NH4HCO3 was mixed to RapiGEST 0.2% (w/v) (Waters) and incubated at 80 °C for 15 minutes.Samples were reduced by the addition of dithiothreitol (100 mM) at 60 °C for 30 minutes, followed by incubation with iodoacetamide solution (300 mM) at room temperature (RT) for 30 minutes. Digestion was performed with porcine trypsin  at 37 °C overnight. To precipitate and remove the surfactant RapiGEST and stop digestion, 5% TFA (v/v) solution was added. Samples were vortexed, incubated for 90 minutes at 37 °C, and then centrifuged at 6 °C for 30 minutes. Supernatants were dried down in vacuum centrifuge24, and pellets were resuspended in anion exchange column loading buffer  pH 3.2). Aliquots of each sample were transferred to Waters Total Recovery vials and injected onto a nanoUPLC system coupled to Synapt HDMS mass spectrometer (Waters).Eight samples from each group  were pooled before protein digestion. Following four times buffer exchange to 50 mM NH4HCO2 and 5 to 50% (v/v) acetonitrile according to Silva et al.25.Nanoscale on-line LC separation of tryptic peptides from the monocyte lysate was performed using nanoACQUITY UPLC system (Waters) equipped with strong cation-exchange (SCX) column  (Waters), pre-column  (Waters) and analytical reversed-phase column  (Waters). The pooled samples from each group (8 µg) were initially injected by an auxiliary nanoACQUITY system pump (ASM) onto SCX column using loading buffer solution with a 5 µL/minute flow rate for 10 minutes. After injection, a step gradient was performed by loading 9 µL of 8 salt plugs onto SCX columns. The plug solutions varied from 50 to 350 mM NHThe peptides were separated onto the analytical column using linear gradient with a 300 nL/minute flow rate. Mobile phase A was 0.1% (v/v) formic acid in water, while B was 0.1% (v/v) in acetonitrile. The gradient consisted of 5 to 50% mobile phase B for 57 minutes, followed by an increase to 85% for 3 minutes, and rinse with 85% during 3 minutes. Then mobile phase B decreased from 85% to 5% in 3 minutes, and the column was finally re-equilibrated to the initial condition for 15 minutes. Analytical column temperature was kept at 35 °C.+ 2 H]2+  = 785.8426 were used as lock mass correction for post-acquisition data calibration. For nano LC-MSE analysis acquisition mode, MS data were collected by alternating low-energy (at constant collision energy of 4 eV) and elevated-energy (ramped from 15 to 55 eV) conditions. Continuum spectra acquisition time, in each mode, was 0.8 s with a 0.02 s interscan delay.Mass spectrometer (MS) was operated in “V mode” with typical resolving power of at least 10,000 full-width half-maximum (FWHM). All analyses were performed in positive nanoelectrospray ion mode. TOF mass analyzer was calibrated with a 100 fmol glu-fibrinopeptide (GFP) solution delivered from ASM using a constant 600 nL/minute flow rate to the reference sprayer of NanoLock Spray source with a 30-seconds frequency. GFP fragment ions from m/z 50 to 2000 and double-charged precursor ion [ME were processed by ProteinLynx Global Server (PLGS) 2.5.1 (Waters). The embedded IdentityE algorithm identified proteins, and data searches were performed against Human UniProt database (released July_2013). Randomized sequences were appended to the original database for estimating false-positive discovery rate. The following parameters were selected for protein searches: one missed trypsin cleavage site; cysteine carbamidomethylation as fixed modification; N-terminal acetylation; asparagine and glutamine deamidation and methionine oxidation were considered variable modifications. Precursor and fragment ion tolerances were determined automatically as 10 and 20 ppm, respectively. We considered as positive protein identification match the detection of at least three fragment ions per peptide, seven fragment ions per protein, and one peptide per protein. False-positive discovery rate was set at a maximum of 1% according to the established search algorithm26. For assessment of differential expression of the protein among uninfected, AC and HAM/TSP pooled samples, three independent 2D-LCMSE runs were performed. Final protein output tables from all replicates generated by ion accounting algorithm integrated in PLGS were merged by MassPivot 2.2.327 used to estimate the constitutive protein that presented no significant difference in relative abundance, and the lowest coefficient of variation (CV) of the three most intense ions (top3) in all replicates. Normalization of the total amount of proteins was based on LogE intensity of exact mass retention time (EMRT) clusters according to Silva et al. (2005)28 using ExpressionE embedded in PLGS. To analyse quality control of LCMSE data TIBCO Spotfire program was used. Scaffold (v. 3.3.2) was used to remove redundancy identifications and to analyse the quantitative protein profile of each pool of samples by Venn diagram. The ID confidence range used was over 95%. Gene ontology classification was performed with Panther 8.029.MS data obtained from LCMS6 cells/well in RPMI-1640 medium supplemented with 10% FBS, penicillin and streptomycin (Gibco) for 2 h at 37 °C under 5% CO2 in a humidified atmosphere. Non-adherent cells were removed by extensive washing and fixed in 2% paraformaldehyde (Sigma-Aldrich) overnight at 4 °C. Cells were permeabilized with permeabilization buffer (eBioscience) for 30 minutes at RT; then, washed and labeled with rabbit anti-human-gelsolin  (SantaCruz Biotechnology) or anti-human-histone-2B antibody   overnight at 4 °C. Cells were washed and stained with anti-rabbit-IgG Alexa647 conjugated  or with anti-rabbit-IgG Alexa488 conjugated antibody  (Life Technologies). After 1 h at RT, cells were washed and stained with phalloidin Alexa Fluor488  at RT for 30 minutes. Cells were washed and labeled with 4′,6-diamidino-2-phenylindole . Coverslips were rinsed and mounted with Fluoromount-G medium (eBioscence). Cell images were captured through microscopy (Leica TCS-SP5), using 63X magnification objectives. Image analysis was performed by ImageJ_v1.46.To ensure validation of the data, PBMC obtained from individuals other than those who composed the monocyte pool of the proteomic analysis were used in the fluorescence microscopy experiments. PBMCs were plated in glass coverslips at 5 × 105 cells/mL were washed with PBS + FBS 5% and labeled with monoclonal antibody anti-human-HLA-DR APC   at 4 °C. After 30 minutes, cells were washed, fixed with 2% paraformaldehyde and permeabilized for 30 minutes at RT. Cells were washed and stained with rabbit anti-human-gelsolin antibody  overnight at 4°C. Thereafter, cells were washed and labeled with anti-rabbit-IgG Alexa488 conjugated antibody  for 30 minutes at RT. Cells were washed, and the fluorescence was examined using FACSCalibur (BD). Monocytes were analyzed after gating based on forward versus side scatter and HLA-DR expression. Analysis was performed using Summit V4.0.Monocytes from individuals other than those who composed the monocyte pool of the proteomic analysis were used in the flow cytometry experiments. 5 × 105 cells/mL. The monocytes were obtained from individuals other than those who composed the monocyte pool of the proteomic analysis were used in the adhesion and transmigration assays. Monocytes were labelled with 1 µM CFSE (Invitrogen) for 15 minutes, washed three times and resuspended in medium supplemented with 10% FCS. 2 × 105 cells were transferred onto hBMECs monolayers and incubated for 3 h at 37 °C under 5% CO2 in humidified atmosphere. Adherent cells were quantified by lysing cells with 0.1 M NaOH and measuring the fluorescence intensity using SpectraMax Paradigm reader (Molecular Devices). Data are expressed as mean ± SEM of arbitrary units.Human Brain Microvascular Endothelial Cells (hBMECs) were seeded on plate wells at a concentration of 3 × 105 cells/insert, and monolayers reached confluence. 2 × 105 CFSE-labelled monocytes were transferred onto monolayers and incubated for 12 h at 37 °C under 5% CO2 in humidified atmosphere. Transmigrated monocytes were recovered and counted by flow cytometer using 40,000 Flow-Check Fluorospheres (Beckman Coulter).Monocyte transmigration assays were performed using Millipore Millicell inserts . hBMEC were seeded on the upper side of the filter at a concentration of 3 × 10tax and the human β-globin genes, as previously described by Silva et al.30, were used in independent reactions with 5 µl of DNA. HTLV-1 proviral load was calculated as tax copies/(β-globin copies/2), and results are shown as infected monocytes per 100,000 cells. Qualitative PCR was performed with 10 µl of DNA in 50 µl reactions using the HotStar Taq Plus PCR kit (Qiagen), following manufacturer’s instructions, using the same primers for HTLV-1 tax and human β-globin genes. Amplification cycle consisted of: enzyme activation at 95 °C for 5 min, 45 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s, and a final extension step at 72 °C for 10 min. PCR products were electrophoresed in 2% agarose gel stained with GelRed® (Biotium) in 1× Tris-Borate-EDTA buffer (Invitrogen) at 100 V for 90 min. Amplification of HTLV-1 tax results in a 159 bp PCR product.DNA was extracted from monocytes with the QIAamp DNA blood mini kit (Qiagen), and DNA was eluted in 30 µl. HTLV-1 proviral load was determined by quantitative PCR in a Rotor-Gene Q instrument (Qiagen), using the Rotor-Gene Probe PCR kit (Qiagen), according to manufacturer’s instructions. Primers and 5′-FAM and 3′-TAMRA-labeled TaqMan® probes (Sigma-Aldrich) for the HTLV-1 p-values ≤0.05 were considered statistically significant.Statistical analysis was performed by Mann–Whitney U test, using GraghPad Prism5, and E, in triplicates. Three replicates data sets were analyzed for each sample group, and prior to performing quantitative analysis. 159,490 EMRT clusters were identified; among the replicate injections, the average CV of the measured signal intensity was under 7% in all three samples. Reproducibility of retention times for the clusters resulted in less than 14%, and they were mostly under 6% , variable modifications (15.9%) and in-source fragmentation rate (5.2%). Results obtained for the dynamic range of the experiment including all quantified molecules showed nearly four orders of magnitude, indicating reasonable distribution of high and low molecular mass proteins  and HSP70 , while HSP90β3 presented decreased levels in comparison to monocytes from uninfected donors  proteins. These proteins are associated to replication control, transcription and repair processes. Our data showed that HMGB1 and HMGB2 were more abundant in samples from HTLV-1-infected individuals, markedly in HAM/TSP patients  higher in monocytes from seven HTLV-1-infected individuals from donors other than those who composed the monocyte pool of the proteomic analysis Fig. .Figure 5Finally, we investigated whether the modifications in cytoskeleton-related proteins observed in monocytes obtained from HTLV-1-infected patients could be translated into higher ability of adhesion and/or migration. The first step was to compare the adhesion of monocytes to monolayers of hBMECs among the three groups. Monocytes obtained from HAM/TSP patients exhibited higher ability of adhesion to hBMECs than those from AC and uninfected individuals Fig. , suggestin vivo35. Thus, we explored whether the monocytes alterations in protein profile and the functions related with these molecules observed was associated to the infection of these cells. The PBMC proviral load in AC was 3.76 ± 3.49 and HAM/TSP was 10.70 ± 3.35. Fresh monocytes were positive for HTLV-1 in 30% of samples from HTLV-1-seropositive individuals and the proviral load was estimated in 3.3 ± 9.97 in one thousand cells  in total HTLV-1 carries (AC + HAM/TSP) is similar to uninfected individuals, approximately, 84% and 89%, respectively20. However, de Castro Amarante et al. demonstrated that the frequency of classical monocytes (CD14+CD16neg) population was reduced in HTLV-1-infected patients21.Tattermusch In vivo, was observed that classical moNnocytes (CD14+CD16neg) can convert into intermediated monocytes (CD14+CD16+)37. Moreover, both subsets can be differentiated into DCs under inflammatory or non-inflammatory microenvironment38. Recently, Hadadi et al., demonstrated that classical and intermediated monocytes exhibited a similar secretion of IL-6, IL-8 and TNF-α after LPS stimulation. The authors also observed that the classical and intermediated monocytes produced similar levels of IL-1β and exhibited the same capacity to activate caspase-1 in response to LPS39. Although, the HAM/TSP patients present higher frequencies of intermediated monocytes than uninfected individuals20, functionality these cells could be the same or very similar HTLV-1 infection. Furthermore, an argument in favor of our data is the significant similarity between monocytes obtained from AC and HAM/TSP patients. Thus, our findings may be related to the monocytes’ role during HTLV-1 infection.Monocytes have a plastic nature and pleiotropic functions. 40. HSPs are associated to the mechanism of HTLV-1 infection through cell-to-cell contact since anti-HSP70 neutralizing antibody significantly inhibits syncytium formation42. HSP90 is described as a binding partner of HTLV-1 Tax protein, and the treatment of HTLV-1-infected cells with HSP90 inhibitors repress cell proliferation and induced death43.We observed that purified monocytes from HTLV-1-infected individuals express higher levels of HSP70 and HSP90 than monocytes from uninfected donors. Likewise, the HTLV-1-infected monoblastoid cell line also exhibits high levels of HSP7044. Tax was detected in intracellular medium of infected-cell or plasma samples from HAM/TSP and AC patients45. Moreover, it was demonstrated that HTLV-1-infected cells can release exosomes containing Tax and viral mRNA transcripts46. These data suggest that proteins derived from HTLV-1-infected cells can be delivered to uninfected cells and induce modulation of cell function.Our results also suggest that HTLV-1 infection might influence the structure of nucleosome. Canonical and variant histone subtypes were downregulated in monocytes derived from HTLV-1-infected individuals, markedly in HAM/TSP patients’ samples. Tax protein promotes downregulation of H1 incorporation into chromatin, leading to activation of histone acetyltransferases, and consequent transcription48. Gelsolin expression is altered in cancer, inflammation, idiopathic interstitial pneumonia and Alzheimer’s disease49. In addition, gelsolin also influences macrophage infiltration to nerve distal to the crush injury site; its absence reduces macrophage infiltration50.The cellular mechanism that triggers the migration of mononuclear phagocytes to spinal cord is still unknown. Our results demonstrated a differential expression of cytoskeleton proteins in monocytes from HTLV-1-infected individuals. Quantitative protein analyses demonstrated higher levels of actin-binding proteins in monocytes from HTLV-1-infected patients, as well as the cytoskeleton protein gelsolin, mainly in those from HAM/TSP patients. Myosin and tropomyosin were also present in increased levels in monocytes from HAM/TSP patients. We further investigated gelsolin because its active form regulates some functions of actin filaments, including severing, capping, nucleating and phagocytosis51, and acts as a pro-inflammatory molecule52. Furthermore, HMGB1 activates human endothelium, inducing the expression of adhesion molecules53. Corroborating this, PBMCs from HAM/TSP patients can adhere with higher affinity to activated endothelium than cells from AC and uninfected individuals54.Monocytes from HAM/TSP patients exhibited higher ability to adhere to hBMECs monolayers than monocytes from AC and uninfected donors. This phenomenon may be associated to altered cytoskeleton protein expression and HMGB1 production. We also have shown increased levels of HMGB1 in monocytes derived from HTLV-1-infected individuals, mainly in HAM/TSP patients. HMGB1 can be released by activated macrophages and monocyteset al. identified an increase in Tryptophan–tRNA ligase transcripts (WARS gene) and IL-15 in monocytes form HAM/TSP36. The Tryptophan–tRNA ligase also was found in our samples , and was related with cytoskeletal reorganization of endothelial cells55, but it activity in monocytes remain unknown. For IL-15 it was demonstrated that this cytokine induces a cytoskeleton rearrangement, alternating cell shape and phagocytosis capacity in neutrophils. In monocytes the IL-15 increase the cell adherence through the Small GTPase activation56. In our analysis we observed an increase of Small GTPases in AC group (Supplementary Table The Tattermusch 58. During HIV infection, monocyte trafficking across the blood-brain-barrier depends on cytoskeletal modifications, including increased levels of cortactin58. Cortactin was detected only in monocytes from HAM/TSP. It contributes to the organization of the actin cytoskeleton and cell structure and plays a role in the regulation of cell migration, corroborating our functional data. Our results indicate that alterations in cytoskeleton protein profile in monocytes from HAM/TSP patients might be involved in transmigration to spinal cord.The proteomics and functional results suggest that increased cytoskeleton protein expression can be related to augmented adhesion ability. During neuroinflammation, leukocytes emigrate from peripheral blood to the CNS across the blood-brain-barrier, which depends on cell adhesion to the microvasculature, followed by crawling and transmigrationcohort of HTLV-1-infected patients, exploring alterations in cytoskeleton protein expression as biomarker for neuroinflammation.In conclusion, this study was the first to demonstrate that monocytes obtained from HTLV-1-infected patients exhibit distinct protein profiles compared to cells from uninfected individuals. Our results suggest that during HTLV-1 infection the altered environment induces profound modifications that can contribute to: alteration in immune response that makes HTLV-1 carriers more susceptible to opportunistic infections; impairment of DC differentiation in HTLV-1-infected patients; presence of mononuclear phagocytes in spinal cord lesions and development of neurological disease. This study points a new perspective to researches involving a Supplementary information


Innate immune signaling and programmed cell death are intimately linked, and many signaling pathways can regulate and induce both, transcription of inflammatory mediators or autonomous cell death. The best-characterized examples for these dual outcomes are members of the TNF superfamily, the inflammasome receptors, and the toll-like receptors. Signaling via the intracellular peptidoglycan receptors NOD1 and NOD2, however, does not appear to follow this trend, despite involving signaling proteins, or proteins with domains that are linked to programmed cell death, such as RIP kinases, inhibitors of apoptosis (IAP) proteins or the CARD domains on NOD1/2. To better understand the connections between NOD signaling and cell death induction, we here review the latest findings on the molecular regulation of signaling downstream of the NOD receptors and explore the links between this immune signaling pathway and the regulation of cell death. Sensing of pathogen-associated molecular patterns (PAMPs) is the initiating step in an efficient immune reaction to a bacterial, viral or parasitic threat. The intracellular receptors nucleotide-binding oligomerization domain-containing protein 1 and 2, NOD1 and NOD2, are members of the pattern recognition receptors (PRR) and recognize intracellular bacterial peptidoglycans. The PRR family consists of a range of cytoplasmic or transmembrane stress sensors that recognize PAMPs and damage-associated molecular patterns (DAMPs).PRRs are divided into two main groups based on their cellular localization: the transmembrane/endosome-associated PRRs, consisting of toll-like receptors (TLRs) and C-type Lectin receptors, and the cytosolic PRRs which are further divided into the RIG-1-like receptors, AIM2-like receptors and the NOD-like receptors (NLRs) . NLRs arThe NLRs consists of four subfamilies, based on the nature of their N-terminal effector domain: The NLRC subfamily is characterized by one or multiple N-terminal caspase activation and recruitment domains (CARDs) that allow direct interaction with other CARD-containing proteins. Among the NLRCs, NOD1 and NOD2 represent the two best characterized members and are sensors of intracellular bacterial peptidoglycan , b. The The second key member of the NLR family are the NLRPs, which are best known for their role in the formation of large oligomeric complexes, the inflammasomes. Inflammasomes mediate the processing, activation and secretion of pro-inflammatory cytokines as well as the induction of pyroptosis through the recruitment and activation of caspase-1 . NLRPs c+ and CD8+ T cells  , tumor nOnce expressed, NOD1 and NOD2 reside in the cytosol and localize to bacterial entry sites at the plasma membrane . Both reExpression levels of NOD1 and NOD2 are held in check through constant degradation by the proteasome. This is counteracted by several chaperones including HSP90, SGT1  and HSP7A candidate E3 ubiquitin ligase that was shown to ubiquitinate NOD2 and target it for proteasomal degradation is TRIM27 . NOD2 waUpon its discovery, various groups reported that NOD1 is activated by lipopolysaccharides (LPS) and mediates the activation of NF-κB in a MyD88-independent manner . HoweverShigella flexneri  have been demonstrated  . The recBrucella abortus are blunted in NOD1/2-deficient mice. The underlying mechanisms are still largely unclear, however, experiments conducted with a dominant-negative form of TRAF2 suggested that this process is TRAF2-dependent. The ER stress sensor IRE1 and TRAF2 have been previously shown to interact in overexpression studies and in yeast-two-hybrid screenings  . TranscrWhile the major outcomes of the NOD1 and NOD2 pathway primarily occur via the activation of NF-κB transcription factors and cytokine production, NOD1 and NOD2 activation can also lead to autophagy induction to clear a bacterial threat. This is in line with NOD2 localization at early endosomes, and the role of NOD1/2 in intestinal homeostasis. Activated NOD has been shown to interact with the autophagy protein ATG16L1 at the site of bacterial entry, although whether RIPK2 is involved in this process is under debate. Two studies show the involvement of RIPK2 in autophagy induction , while aAs mentioned above, RIPK2 is the central adaptor kinase in the NOD pathway. RIP kinases represent a class of serine/threonine kinases that play essential roles in the regulation of innate immune signaling. Their functions depend on the highly conserved N-terminal kinase domains and distinct C-terminal interaction motifs. Amongst the RIP kinases, RIPK1 and RIPK3 are the best-characterized members, which are being extensively studied due to their involvement in cell death and their role in chronic diseases and cancer. RIPK1 contains a C-terminal death domain (DD) that mediates direct binding to death receptors of the TNF receptor superfamily members including TNFR1, Fas, and TRAIL, and to adaptor proteins such as FADD or TRADD. Upon binding, oligomeric protein complexes are formed that can regulate survival or cell death. Under specific conditions, RIPK1 associates with RIPK3 through their RIP homotypic interaction motif (RHIM) to activate the pseudokinase mixed lineage kinase domain-like (MLKL) to induce necroptosis, a pro-inflammatory form of programmed cell death .Receptor-interacting serine/threonine-protein kinase 2 (RIPK2) represents the next best-characterized member of the RIP kinase family. Compared to RIPK1 and RIPK3, RIPK2 does not have a RHIM or a DD and is therefore unable to interact with these death receptor complexes. RIPK2 is composed of an N-terminal kinase domain, an intermediate domain of unknown function, and a C-terminal CARD that mediates binding to NOD1/2 via homotypic CARD-CARD interactions . StructuRIPK2 is indispensable for NOD-mediated activation of the NF-κB and MAPK pathways and its recruitment to NOD2 occurs via CARD-CARD interaction . An acidThe NOD2-RIPK2 interface differs from that between NOD1 and RIPK2. Overexpression of both NOD2 CARDs is required for a constitutive NF-κB response . Even thRIPK2 was originally identified as a serine-threonine kinase based on sequence homology , but wasThe only substrate of RIPK2 that has been described so far, is RIPK2 itself. Upon activation by dimerization via the CARD domains of NOD1/2, RIPK2 autophosphorylates on S176 in the activation loop of the kinase domain  and on YIn vitro auto-phosphorylation assays indicated that catalytically inactive mutants could still be phosphorylated by purified full-length RIPK2, suggesting that autophosphorylation occurs in trans , it was observed that besides S176, multiple additional serine residues within the activation loop can be phosphorylated . More phin trans , which rin trans .S. flexneri, while the S176E mutant resulted in reduced cytokine levels  could still activate NF-κB signaling, although this occurred independently of NOD2 engagement with MDP . On the e levels . In conte levels .While the importance of the kinase activity of RIPK2 remains somewhat dubious, it has become clear that its ubiquitination is a critical determinant of downstream signaling. Ubiquitin is a small, 8.5 kDa protein that can be covalently attached via its C-terminus to lysine residues of target proteins or to the N-terminus of one of the seven lysine residues of a substrate-attached ubiquitin. The diverse biological outcomes of protein ubiquitination are dependent on the complex interplay between the sites of the ubiquitination, chain length, chain type, chain branching as well as posttranslational modifications on ubiquitin itself .Mycobacterium tuberculosis infections in macrophages. RIPK2 was stably ubiquitinated, and this ubiquitination was required for optimal cytokine production , as well as X-linked IAP (XIAP), have all been reported to regulate NOD signaling . The IAPin vivo in cIAP1 and cIAP2-deficient mice but normal signaling in ex vivo stimulated BMDMs can be explained by a secondary autocrine loop that drives cIAP-dependent NF-κB and MAPK activation through TNFR1  -2, -5, and -6, which are key adaptors in the TNFR1 signaling pathway contain a RING domain with E3 ligase activity. All three of these proteins have been linked to NOD signaling . So far The E3 ligase Pellino3 was identified as another positive regulator of the NOD2 pathway, by mediating K63-linked ubiquitination of RIPK2. BMDMs from Pellino3-deficient mice displayed a lower activation of NF-κB and MAPK pathways and produce fewer cytokines after stimulation with MDP . Of notein vitro ubiquitination assays and pulldown experiments (E3 ubiquitin-protein ligase Itchy homolog (ITCH) was also reported to be a direct E3 ligase for RIPK2, by adding K63-linked ubiquitin chains in eriments . BMDMs fL. monocytogenes , alterations in the intestinal barrier lead to increased intestinal permeability and translocation of PAMPs to the bloodstream, a phenomenon named metabolic endotoxemia . It is ain vivo after intraperitoneal administration of MDP (Given the involvement of NOD2 and RIPK2 in a range of diseases, inhibition of RIPK2 could have an application in inflammatory diseases driven by dysregulated NOD signaling pathways. Kinase inhibitors with significant activity toward RIPK2 are already approved for clinical use, such as the multi-tyrosine kinase inhibitor ponatinib and the EGFR inhibitor gefitinib . Over thn of MDP . Furthern of MDP . These ein vivo (An alternative approach to inhibit the NOD pathway is to antagonize the critical E3 ligases IAPs. However, compounds that target cIAPs and XIAP are not tolerated in the clinic as they induce an inflammatory response in vivo . Until rin vivo . Recentlin vivo . SimilarAs discussed in detail above, signaling downstream of NOD1/2 harbors many proteins and protein domains that are closely associated with cell death signaling. A link between NOD signaling and cell death induction seemed therefore likely ever since NOD signaling was studied.NOD1, NOD2, and RIPK2 harbor one or multiple highly conserved CARDs, which are known to recruit caspases, the key mediators of apoptosis. It is therefore not surprising that all these proteins have initially been associated with caspase binding and with programmed cell death. Indeed, overexpression studies with NOD2 showed that it could bind to multiple caspases via its CARD, and was able to directly activate caspase-9 and induce apoptosis . This waAn indirect link between NOD signaling and apoptosis was suggested in early studies on RIPK2, which showed that overexpressed RIPK2 could potentiate CD95-induced apoptosis via caspase-8 and caspase-10 . ATP binThe strongest evidence that suggests direct involvement of NOD signaling pathways in regulating caspase functions stem from observations that NOD1 and NOD2 can induce IL-1β through NF-κB and MAPK pathways in multiple human and mouse cell populations, including myeloid-derived cells . MoreoveA surprising finding was presented later, when it was shown that Bid, a well-characterized pro-apoptotic member of the Bcl2 family, was shown to be required for NOD signaling, as cells and mice deficient in Bid were not able to react to MDP . HoweverWhile there seems to be no direct link between NOD signaling and apoptosis, there is a strong link between NOD signaling and autophagy, the disassembly of damaged or unnecessary cellular components, that can result in death. In the context of NOD signaling, autophagy is more likely to be a cellular defense mechanism for bacterial clearance rather than a cell death mechanism.Taken all together, initial experiments that linked NOD signaling with cell death could not be confirmed when endogenous protein levels and physiological ligands were used in later experiments. While overexpression studies are an important tool to determine molecular mechanisms of cell signaling, it has become clear that one has to be cautious when assessing the effects of overexpressed proteins on cell death. The last decade has seen many advances in establishing the links between innate immune signaling pathways and cell death, using mainly myeloid cells and relevant ligands. It has become clear that the link between NOD signaling and cell death is not as straight forward as initially thought, despite the indisputable involvement of cell death-related proteins and cell death-promoting domains.The title of this research topic is “Connecting the dots between inflammatory signaling and the working of cell death.” Here we have dissected the molecular mechanisms of signaling downstream of the intracellular PGN receptors NOD1/2. We have found that a critical point of difference between the NOD pathway and other innate immune signaling pathways is its failure to connect these dots. This is somewhat surprising. Not only do most inflammatory signaling pathways directly or indirectly induce cell death, but also have early studies implicated that activation of the NOD signaling pathway results in caspase activation and apoptosis. The development of new reagents and model systems has led to studies using endogenous proteins and specific means to stimulate the NOD pathways, as well as the use of relevant cell types. This is in contrast to earlier studies that were largely based on overexpression of members of the NOD pathway. In these newer work, the initial findings that NOD1/2 activation leads to any form of cell death could not be confirmed.So what is different between the NOD pathway and other cell death-inducing inflammatory pathways? One reason could be that the NOD pathway is not exclusively pro-inflammatory at all. The best evidence is the strong association of NOD2 mutations with Crohn’s disease: These are prominently loss of function mutations within NOD2, suggesting that NOD signaling has an anti-inflammatory role. Conversely, hyperactivation of the NOD pathway is described in other inflammatory diseases and elevated RIPK2 activation, a hallmark of NOD signaling, is observed in many pathologies, intriguingly also in patients with IBD. Therefore, the NOD pathway rather plays an immunomodulatory role, rather than a pro-inflammatory. A cell that induces inflammation needs to be shut down rapidly to avoid hyperactivation of an inflammatory response, and a potent way to do so is to induce programmed cell death in this cell. If NOD signaling is, however, not as inflammatory at all, there is no need to self-destruct and hence the missing link between NOD signaling and cell death.Despite the missing link between cell death and NOD signaling, this pathway has emerged as an important contributor to human pathologies. Therefore, significant efforts have been put toward better understanding the molecular mechanisms of NOD signaling. The focus for the development of therapeutics interfering with NOD signaling has been the kinase RIPK2, and several ATP competitive inhibitors have been developed by commercial and academic entities. The most recent data show convincingly, however, that the kinase activity of RIPK2 is dispensable for downstream signaling, and the critical role of RIPK2 is its scaffolding function in the pathway. Therefore, the understanding of protein-protein interactions and the ubiquitin network on RIPK2 and other members of the NOD pathway is pivotal for the development of novel therapeutics in this space.VH generated the figures. VH, CS, and UN wrote the review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.