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If you are trying to control something in the real world using your computer, the serial port is perhaps the easiest means of. Self-hosted services that use the Net.TCP transport can control several advanced settings, such as ListenBacklog and MaxPendingAccepts, which govern the behavior of. Serial communications can be done via either direct to physical serial port connected to the computer or via a USB to serial converter interface. If the device do require a serial port and your computer don’t have any, you can make use of such converters easily. This type of communication aren’t as much easy as other similar tasks such as working with logic drive on computer via c# and need use of specific kind of communication protocol. One interesting thing that you might need to remember that, when the physical serial port are being used, it doesn’t have any PID or VID. But if you are using any specific type of devices which facilitate this kind of communication via USB interface, you can retrieve their PID/VID respectively and communicate accordingly. Lets have a look into them. Retrieve List Serial Ports: OK, lets first see whether we can detect the serial ports from within our application. As a prerequisite, you need to make sure that, while the application is running, the windows user must need to have access to the ports. The following C# code examples will return a list of Serial port names connected to the computer. List< string> Get. All. Ports(). . First step to start serial port communication is to open the port, then send/receive necessary data and finally close the port(s). Lets see an example how we can open and close ports. System. IO. Ports. Serial. Port my. Port = new System. IO. Ports. Serial. Port(. However, it is very important that, you have prior knowledge what kind of data the connected device is expecting. For this, you will need the corresponding firmware API command lists. Here, I will give a simple prototype how the send/receive data workflow will be. System. Timers. public class Comm. Timer. . Then, we will write an array of bytes to the port. This array can contain several hex values to represent a single command. Here, I have used one . After writing, and before you start reading the response, it’s always good to wait for a while, thus add a slight delay, which helps to make up the time required between receiving and sending reply for the device. In this time, normally, windows do ques your work instruction and sends to devices. But, it may not happen because of CPU scheduling issue etc. So, better to check whether any response came or not. If not, force windows to perform this action now by . Serial Port Communication Asp Net WebLet me know if you want some more similar tutorials or have any questions. Happy coding : ). Serial to Ethernet Converter Software TCP/Com. Multifunction serial to ethernet (and ethernet to serial) interface software. Easily access a serial device via a TCP/IP or UDP network or redirect Ethernet/IP data to real or “Virtual” RS2. This allows you to use existing Windows- based serial RS2. TCP/IP network. TCP/Com software is extremely simple but powerful. It can be used to solve a wide variety of device interfacing and RS2. TCP/IP conversion problems, such as the following: Turn a PC into a Serial Device Server (Terminal Server)Send or receive data over a TCP/IP port with any serial communications program. Open COM ports located on another computer in your network as if they were local. Use Ethernet network (or the Internet) as a giant serial RS2. Serial Tunneling)Feed data from one physical RS2. RS2. 32 serial communications applications. Map a TCP/IP port to a different TCP/IP port. Use TCP/Com as a Serial Device Server (Learn More)Share serial devices over a network (or even the internet)Use TCP/Com as a Virtual Serial Port Splitter Cable (Learn More)Windows does not allow multiple programs to access the same COM port. Bypass this restriction by using TCP/Com to split a single COM port into multiple virtual COM ports. This allows all your serial communications programs to access the same data. Intuitive Multi- Document Interface.
Are you desperately seeking for a serial over IP software to access COM port device connected directly to the network? Serial to Ethernet Connector brilliantly solves. In the TCP/Com window just select the com port number and the serial communications parameters for your serial device and enter an IP address and a socket number. Then activate! It's that easy! Com port data will appear at the IP address and data from an IP port will appear at a real or virtual com port. Recent Updates. Version 7. June 2. 01. 4) Fixed rare issue with . A bug fix that addresses the remote pings feature is also included. Version 7. 0. 2 minor bug fix. Version 7. 0. 0 Updated Virtual COM Port drivers. The Serial Port - Software Setup: 8 Steps. In order to set it up, first you have to find it on your computer. Long long ago, almost all computers had two serial ports, called . The USB to serial converter has arrived, and it can be used instead. It is usually a dongle which plugs into a USB port, with a nine pin male connecter at the other end. The third seial port, here labelled COM4, belongs to the USB to serial adapter I have plugged in. If you find a nine pin male connector (two rows, five and four, pins sticking out inside a metal shell) it has a serial port built in. Right click on . Click on the . Click on the . The list of printer and Serial ports available will be shown. Make a note of those, you will need them in the steps that follow. Serial Communication in Windows. Environment: Source code. This article is meant to give you a jump start on doing serial communication in Windows (NT family). The article will provide a class called CSerial. Comm. Helper that you can use directly to do serial communication in your application. The class that is provided here with this article does uses overlapped IO. You do not need to know much about serial communication or overlapped IO for this article. However, you need to know some about the synchronization objects such as Events and some Windows APIs like Wait. For. Single. Object and Wait. For. Multiple. Object, and so forth. Also, some basic understanding of Windows threads is required—such as thread creation and termination. Introduction. In order for your computer to do serial communication, the computer has to have a serial port. Most computers have at least one serial port, also known as a COM port (communication port), and are generally called COM1, COM2, and so on. Then there are the device drivers for the serial ports. If you think it over, all that you need to do in serial communication is either send data or receive data. In other words, you are doing input/output (IO) to the serial port. The same IO is done with disk- based files. Hence it is no surprise that the APIs for reading and writing to a file apply to serial ports as well. When you send data to the serial port, it's in terms of bytes but when it leaves the serial port it is in the form of bits. Similarly, when the data arrives at the serial port, it's in bit format and when you get data you get it in bytes. Without any further discussion, let's get started. Opening the COM Port. The first and the foremost step in doing a serial communication is to open the desired port. Let's say you have your device hooked to COM1; you can open the COM port using the following API. HANDLE m. We want to open the file (the COM port) in an overlapped fashion—that's why the sixth parameter is FILE. We will get into the details of overlapped IO a bit later. As you must have guessed from the name, the Create. File() API can be used to create a file (disk- based); it also can be used to open an existing file. To Windows, a serial port or a disk- based file both are IO devices. So, to open an existing file (serial port), all we need to know the name of the device (COM1) and pass the creation flags as OPEN. However, if the system could not open the COM port, it would return INVALID. And you can get the reason by calling Get. Last. Error(). One of the common errors when opening a COM port is that the COM port is already opened by some other application; in that case, you would get ERROR. Similarly, if you mistakenly opened a COM port that does not exist , you would get ERROR. For example, let's say you want to send . When you want to send the data across the serial port, you need to write to the serial port just as you would write to a file. You would use following API: i. Ret = Write. File (m. So, you need to read the data. Again, you would use the following API: ab. Ret = : :Read. File(m. We will get to all this later. All this sounds very simple. Right? Now, let's start digging into issues. Issues with Serial Communication. Just now I said that, in response to your . But the problem here is that you don't know when the device is going to respond. Or, will it ever respond? When should you start to read from the port? One option is that as soon as you made the call to Write. File, you make a call to Read. File. If no data is there, you need to make a read call again, later on. This leads to what is called polling. You keep polling the port for data. This model does not really seem to be a good one. It would be nice if somehow the system notified you when data has arrived and only then would you make a call to Read. File. This is an event- driven approach and fits well into Windows programming. And the good news is that such a model is possible. Another issue with the serial communication is that because it always occurs between two devices, the two devices need to agree on how they talk to each other. Each side needs to follow certain protocols to conduct business. Because it's the serial port that actually carries out the communication, we need to configure the serial port. There is an API available for that exact same purpose. Following is the API: Set. Comm. State ( HANDLE h. File, LPDCB lp. DCB)The first parameter is the handle to the COM port and the second parameter is what is called a device control block (DCB) . The DCB is a struct defined in winbase. For example, we need to specify the baud rate at which the COM port operates; you need to set the Baud. Rate member of the struct. Baud rate is usually 9. But the two devices have to use the same baud rate to conduct business. Similarly, if you want to use parity you need to set the Parity member of the struct. Again, the two devices have to use same parity. Some of the data members are reserved and have to be 0. I have found it easier to get the current DCB struct and then set those members that we are interested in changing. The following code gets the current DCB and sets some of the fields. DCB dcb = . But if you need to change the structure, you need to be very careful about the fields because changing the fields will affect the behavior of the serial communication; hence, you should be very sure about what you want to change. Event- Driven Approach. Let's return to our earlier problem with reading the data. If we do not want to keep polling the COM port for any data, we need to have some kind of event mechanism available. Fortunately, there is a way that you can ask the system to notify you when certain events happen. The API to use is: Set. Comm. Mask( HANDLE h. Handle,DWORD dw. Evt. Mask)The first parameter is the handle to the open COM port. The second parameter is used to specify a list of events that we are interested in. The events that need to be specified in the mask depend upon the application needs. For simplicity, let's say that we are interested in getting notified whenever a character arrives at the serial port. We would need to specify EV. Similarly, if we are interested in knowing when all the data has been sent, we also need to specify the EV. So, our call would look like this: Set. Comm. Mask( m. How would the system let us know that a particular event occurred? An obvious thing seems to be a callback mechanism. But there is no such mechanism available. Here is when things get a little tricky. In order for the system to let us know about the communication event occurrence, we need to call Wait. Comm. Event This function waits for the events specified in Set. Comm. Mask. But if you think a little more, it sounds like we are turning a notification mechanism back to a polling mechanism. Actually, it's even worse that than. Wait. Comm. Event blocks until an event occurs. So what's the use of Wait. Comm. Event? Well, the answer lies in overlapped IO. If you look at the Wait. Comm. Event signature, it looks like this: BOOL Wait. Comm. Event(HANDLE h. Comm. Port, LPDWORD dw. Evt. Mask,LPOVERLAPPED. Overlapped); The third parameter is the key here. Think of overlapped IO as asynchronous IO. Whenever a function makes a call and specifies the overlapped IO structure, it means that it is trying to do the current operation but, if you are not able to complete it immediately, let me know when you are done with this IO. The way the system lets you know about the completion is by setting a kernel event object that is part of the lp. Overlapped structure. So, all you do is spawn a thread and make the thread wait for that event object using one of the Wait. For. Single. Object() APIs. Let's look at the overlapped structure: typedef struct . This event is generally a manual reset event. When you make a call like Wait. Comm. Event(), passing the overlapped structure as the last parameter, and the system could not complete the call—meaning it did not see any characters at the port—it would return immediately but would return FALSE. If you now make a call to Get. Last. Error(), you would get ERROR. Also it means that whenever the characters arrive, the system will set the h. Event of the overlapped structure that you passed in. So, if your thread would wait for a single object on h. Event and you pass INFINITE, whenever your Wait function returns WAIT. The following pseudo code will explain it. You can read the data from the COM port, reset the event, and make the call to Wait. Comm. Event again, and so on. The above piece of code is simple; it uses the overlapped IO method to do its job. After we receive the indication that the data has arrived, we need to read the data. The important thing to note here is that the when data arrives at the serial port, it is copied over to the system buffer. The data is removed from the system buffer only when you have read the data using an API such as Read. File. Like any buffer, the system buffer has a limited size. So if you do not read the data from the buffers quickly enough, the system buffers can become full if more data is arriving. What happens to further data depends upon the configuration that you have set in the device configuration block (in a call to Set. Comm. State). Usually, the applications do some kind of handshaking at the application level but you can also make configurations such that the COM port does not accept any further data upon buffer- full events. But all that is beyond the scope of this discussion. If possible, it's always better to have applications themselves implementing some kind of handshaking—like not sending the next block of data until you get an okay for the first block. Generally this kind of handshaking is implemented using some sort of ACK/NAK and ENQ protocol. In order for us to read data, we need to use the Read. File() API. The Read. File API has to specify how much data to read. Let's say we are monitoring character arrivals and 1. As soon as the first character arrives at the port the system will set the overlapped structure's event object and our Wait. SIMATIC S7-200 stands for a reliable, fast and flexible controller in the micro automation area with a broad scale of modules. Programming was based on the easy to. SETUP STEP7 V5. 5 FOR WINDOWS 1. BITl. INK SOFTWARE PLCCONGNGHECAO. M. COMANDPLCCONGNGHECAO. COMWE DONATETHANKS YOU SO MUCH! Offers 50 GB of free storage space. Uploaded files are encrypted and only the user holds the decryption keys.
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Cannabidiol inhibits pathogenic T cells, decreases spinal microglial activation and ameliorates multiple sclerosis- like disease in C5. BL/6 mice. Abstract. BACKGROUND AND PURPOSECannabis extracts and several cannabinoids have been shown to exert broad anti- inflammatory activities in experimental models of inflammatory CNS degenerative diseases. Clinical use of many cannabinoids is limited by their psychotropic effects. However, phytocannabinoids like cannabidiol (CBD), devoid of psychoactive activity, are, potentially, safe and effective alternatives for alleviating neuroinflammation and neurodegeneration. EXPERIMENTAL APPROACHWe used experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) in C5. BL/6 mice, as a model of multiple sclerosis. Using immunocytochemistry and cell proliferation assays we evaluated the effects of CBD on microglial activation in MOG- immunized animals and on MOG- specific T- cell proliferation. KEY RESULTSTreatment with CBD during disease onset ameliorated the severity of the clinical signs of EAE. This effect of CBD was accompanied by diminished axonal damage and inflammation as well as microglial activation and T- cell recruitment in the spinal cord of MOG- injected mice. Moreover, CBD inhibited MOG- induced T- cell proliferation in vitro at both low and high concentrations of the myelin antigen. This effect was not mediated via the known cannabinoid CB1 and CB2 receptors. CONCLUSIONS AND IMPLICATIONSCBD, a non- psychoactive cannabinoid, ameliorates clinical signs of EAE in mice, immunized against MOG. Suppression of microglial activity and T- cell proliferation by CBD appeared to contribute to these beneficial effects. Keywords: cannabinoids, cannabidiol, microglia, T cells, EAE, MOGIntroduction. The anti- inflammatory properties of cannabinoids, constituents of the Cannabis sativa plant, have been appreciated since ancient times and have been supported experimentally, in models of inflammation in vitro and in vivo (Tanasescu and Constantinescu, 2. Indeed, immune cells express the elements of the cannabinoid system including endocannabinoid ligands, endocannabinoid enzymatic machinery and cannabinoid CB receptors, mostly of the CB2 type and many fewer of the CB1 type (Mackie, 2. Alexander et al. Moreover, expression of CB1 and CB2 receptors and the activity of the endocannabinoid system are regulated in response to inflammation, a finding that further confirms the involvement of the cannabinoid system in immune processes (Croxford and Yamamura, 2. Maresz et al., 2. Pietr et al., 2. 00. The clear and promising therapeutic potential of cannabinoids (Klein and Newton, 2. CB1- mediated, psychotropic effects of many of these materials. A good example is . The preferential CB2 receptor expression on immune cells offers an attractive opportunity to regulate the function of the immune system with CB2 receptor ligands. Studies on CB1/CB2 receptor knock- down mice revealed the existence of other possible, receptor and non- receptor, cannabinoid targets (J. Thus, cannabinoid compounds not acting on CB1 or CB2 receptors seem to offer new tools to manipulate inflammation. Cannabidiol (CBD) is one of these compounds and is the major Cannabis- derived non- CB1/CB2 receptor ligand (Showalter et al., 1. This compound is not only devoid of psychotropic effects but also is able to inhibit many central effects of CB1 receptor ligands, for example, the anxiogenic and psychotogenic activities of THC (Zuardi, 2. Interestingly, despite different pharmacological and behavioural effects, CBD shares with . These effects include suppression of humoral responses, such as release of cytokines, chemokines, growth factors, as well as suppression of immune cell proliferation, activation, maturation, migration and antigen presentation (Mechoulam et al., 2. In an earlier publication, we showed that CBD inhibited production of the cytokine IL- 6 and the chemokine CCL- 2 by activated microglial cells and in parallel activated intracellular anti- inflammatory pathways (Kozela et al., 2. Among the many types of neurodegenerative diseases in which inflammation is involved, multiple sclerosis (MS) is one of those clearly induced and driven by dysfunctional immune system activity. In MS, myelin autoreactive peripheral T cells migrate into the CNS and initiate cytotoxic, degenerative processes that include demyelination, oligodendrocyte cell death and axonal degeneration. These effects lead to neurological deficits such as visual and sensory disturbances, motor weakness, tremor, ataxia and progressive disability as the main clinical symptoms (Compston and Coles, 2. Infiltrating T cells are constantly reactivated within the CNS parenchyma by microglia. Microglial cells via chemokine and cytokine release and constant antigen presentation potentiate T- cell recruitment to the CNS and facilitate their polarization into cytotoxic phenotypes (Th. Th. 17). Moreover, via released chemokines such as CCL- 2, microglial cells recruit other immune cells of myeloid origin, specialized in epitope spreading and phagocytosis of myelin including monocytes, macrophages, B cells and dendritic cells (Jack et al., 2. Koning et al., 2. Depletion of microglia or impairment of their function can attenuate disease progression in experimental animal models of MS supporting their role in initiation and development of this disease (Huitinga et al., 1. Heppner et al., 2. Thus, suppression of microglia will potentially reduce inflammatory lesions and limit demyelination within the CNS. One of the best described and commonly used animal models of MS is experimental autoimmune encephalomyelitis (EAE) which is induced when the animals are immunized with myelin components, for example, myelin basic protein, proteolipid protein or myelin oligodendrocyte glycoprotein (MOG) or by passive transfer of autoreactive myelin specific T cells to produce demyelination and MS- like neurological and clinical signs (Shevach, 1. Using the MOG- induced EAE mouse model, we investigated if systemically given CBD at the time of symptomatic disease onset could affect the progression of the disease. We observed that CBD ameliorated the severity of the EAE in MOG- injected mice. Moreover, CBD attenuated microglia activation in MOG- immunized animals and inhibited MOG- induced proliferation of encephalitogenic T cells. Methods. Animals and experimental design. All animal care and experimental procedures complied with and were approved under the guidance and regulations of the Weizmann Institute of Science and Tel Aviv University. EAE was induced in 3. C5. 7BL/6 mice (Harlan Laboratories, Rehovot, Israel) by two subcutaneous injections of MOG3. MOG) on days 1 and 8, injected in the left and right flanks respectively. Each injection contained 3. Control mice received only CFA without MOG (Ctrl, n = 1. CBD (5 mg. Clinical disease scores were recorded daily until 3. At this time point, spinal cords were collected for further pathological and immunological studies. The signs of EAE were scored as follows: 0, no clinical signs; 1, loss of tail tonicity; 2, partial hind limb paralysis; 3, complete hind limb paralysis; 4, partial frontal limb paralysis; 5, total paralysis; 6, death (Lev et al., 2. In parallel, we used control mice which received only CFA (Ctrl) and were also injected with CBD on days 1. Ctrl + CBD group, each group n = 1. The days of EAE onset and respective CBD injections were chosen based on our previous experience with this model (Lev et al., 2. CBD (National Institute on Drug Abuse, Rockville, MD, USA) solution was prepared freshly before each of the treatments. The dose of CBD was chosen based on previous studies with systemic administration of the drug in which CBD at 5 mg. MOG synthesis was carried out by the Weizmann Institute Synthesis Unit, using a solid- phase technique on a peptide synthesizer (Applied Biosystems Inc., Foster City, CA, USA). Histology and immunocytochemistry. Histological analysis of spinal cord sections was used to define severity of inflammation and demyelination and the effect of CBD treatment on these parameters. Spinal cords were dissected 3. The presence of axonal pathology was supported by immunohistochemistry with anti- non- phosphorylated neurofilament H (SMI- 3. Sternberg Antibodies, Emeryville, CA, USA) on 8 . Subsequently, 4 . The assessment of immune cell infiltration was performed using immunocytochemistry on 4 . Thus, in further description of the stainings we refer to microglia/macrophage populations. The Iba- 1 protein is specifically expressed in microglia/macrophages and becomes up- regulated during the activation of these cells. Mac- 2/Galectin- 3 is another microglial/macrophage marker whose expression reflects specifically the activation state of these cells. For example, Mac- 2/Galectin- 3 expression is known to be increased in these cells following phagocytosis of damaged myelin and cell particles (Reichert and Rotshenker, 1. The immunostainings were visualized with respective secondary antibodies conjugated to Cy. Cy. 3 fluorochromes. Spinal cords from control, healthy mice that received 3 daily injections of either CBD or its vehicle were dissected and stained in parallel. Assessments of intensity and differences in immunofluorescence staining for Iba- 1 and Mac- 2/Galectin- 3 were performed using the Image Pro Plus analysis software (Media cybernetics, Bethesda, MD, USA) and collected as arbitrary units representing optical density per area. The statistics was carried out on per cent values calculated from optical density values and is given in the appropriate figure legend. In the case of T cells, CD3+ cells were counted (three areas per mouse, three to four mice per treatment) and the cell numbers were subjected to statistical analysis. Encephalitogenic T- cell line. The MOG3. 5- 5. 5- specific T- cell line was established from lymph node cells of C5. BL/6 female mice that had been primed 1. Activation of caspase- 1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function. Abstract. Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process is phagosome acidification, which regulates a number of functions of these organelles that allow them to participate in processes essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram- positive bacteria is regulated by the NLRP3- inflammasome and caspase- 1. Active caspase- 1 accumulates on phagosomes and acts locally to control the p. H by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3- inflammasome and caspase- 1 in host defense. A particularly important process in host defense is phagocytosis, the internalization of particles into organelles called . Despite this crucial role in immunity, the molecular mechanisms that regulate the functions of phagosomes remain poorly understood. What is known is that phagosomes in macrophages are dynamically remodelled during their . An important aspect of the maturation process is vacuolar acidification, which regulates the activity of the p. H- sensitive enzymes that are delivered from lysosomes and required to digest internalized cargo 5. The timely delivery of microbes into a mature and acidified phagolysosome is essential not only for microbial killing 6, but also to facilitate activation of certain innate immune signalling pathways 7. Moreover, the rate of phagosome acidification also regulates antigen processing and presentation by macrophages and dendritic cells (DCs) 8. Because of these pivotal roles in immunity, understanding the mechanisms that allow the host to remodel phagosomal compartments to optimize their microbicidal and hydrolytic activity is of fundamental importance. Acidification is key to many facets of phagosome function. It is a regulated process that begins almost immediately after the phagocytic cup has closed 9,1. H can drop from 7 to 4 in a matter of minutes. These rapid changes precede the fusion with acidic compartments and instead early acidification requires delivery of the vacuolar- H+- ATPase (V- ATPase)1. This proton transporting holoenzyme is recruited from endosomes and lysosomes, and assembled on the membrane of the nascent vacuole 9,1. However, how the p. H is then regulated remains poorly defined. V- ATPase activity in mammalian macrophages and DCs can be . Additionally, the NADPH oxidase has been suggested to counteract the V- ATPase and neutralize the phagosome p. H in certain cells 1. A number of pathogens have evolved mechanisms to evade these processes, including buffering their local environment in an attempt to maintain a beneficial neutral p. H. The nature of the mature phagosome is therefore determined by the complex interplay between the internalized microbe and the rapid remodeling of the organelle by the host in response to the different cargo. The observation that Toll- like receptors (TLRs) are recruited to some phagosomes 1. TLRs might survey the luminal contents and control vacuole maturation in a cargo- dependent and organelle autonomous manner to mediate these rapid changes. Although attractive as a mechanism for sensing and remodeling the phagosome depending on the type of internalized material, this function of the TLRs remains controversial, especially as the molecular details of how they might regulate this process remains obscure 2. The rapidity of the changes that occur after microbial engulfment argues against the fate of the phagosome being controlled at a transcriptional level. Rather we reasoned that local, post- translational modifications induced by innate immune signals were more likely to underlie the prompt remodeling of the phagosome that occurs after internalization of different microbes. To investigate this possibility, we chose to focus on the inflammasome, as it is an innate immune pathway whose terminal effectors are proteases that can rapidly modify select host components. We found that caspase- 1 is rapidly activated upon phagocytosis of Gram- positive microbes. Moreover, we demonstrate that activation of the NLRP3 inflammasome and its effector, caspase- 1, are instrumental in enabling the microbicidal activity of Staphylococcus aureus- containing phagosomes. Caspase- 1 acts on the NADPH oxidase (NOX2), and by controlling its activity it modifies the p. H of the vacuole. These findings identify an essential role of caspase- 1 in locally coordinating the environment of phagosomes. We propose that this cell autonomous defense function of caspase- 1 acts in concert with its role in pro- inflammatory cytokine release and pyroptosis and thus positions it as a master regulator of innate immunity. Results. Caspase- 1 accumulates on phagosomes containing S. While investigating inflammasome activation during S. Caspase- 1 exists as both an inactive pro- enzyme and, after auto- proteolytic cleavage in an inflammasome platform, an active protease. As these methods could not distinguish pro- caspase- 1 from active caspase- 1 we used the Fluorescent Labelled Inhibitor of Caspases (FLICA) reagent, which forms a covalent bond with the active protease 2. Figure 1c- f). Active caspase- 1 accumulated around bacterial phagosomes (Figure 1c). The FLICA staining required internalization of S. The localization of caspase- 1 after S. The accumulation of active caspase- 1 on phagosomes was cargo- dependent; latex bead phagosomes did not stain with FLICA, despite the presence of total caspase- 1 as detected by antibody staining (Supplementary Fig. Gram- negative bacterium Escherichia coli was not associated with detectable caspase- 1 activation in unprimed cells (Supplementary Figure 1d,e). Together these data indicate that caspase- 1 was activated and then specifically recruited to S. Activation was dependent on the NLRP3 inflammasome, as cells deficient in Ice (caspase- 1), or the inflammasome components Asc and Nlrp. Figure 2a). A similar reduction of FLICA staining was observed when confocal microscopy was used to assess both the total FLICA staining and the amount of phagosome- associated active caspase- 1 (Figure 2b). These data confirmed that the FLICA staining was specific and indicated that the inflammasome was required for the phagosome- associated caspase- 1. Phagocytosis of S. Although caspase- 1 could not be detected in cell supernatants at early time points, we did observe the appearance of caspase- 1 p. Figure 2c). To further define the kinetics, we used flow cytometry to monitor YVAD- FLICA. Phagocytosis of both live and heat- inactivated (HI) S. Early caspase- 1 activation did not require priming initiated by NF- . Similarly, it did not require TLR signaling as it was unimpaired in Myd. Early caspase- 1 activation was also not attenuated by Bafilomycin A, an inhibitor of the V- ATPase, indicating that vacuolar acidification did not play a role at this early point (Figure 2g- h). Instead we observed that phagocytosis of S. However, and consistent with previous reports, caspase- 1 activation was normal in gp. ROS that activates the inflammasome was not the phagocyte oxidase (NOX2) (Supplementary Figure 2a). Notably, addition of DPI, either alone or with Bafilomycin A, did not completely block caspase- 1 activation indicating that there are other additional, but currently unidentified factors, that contribute to activate the inflammasome early during phagocytosis. Further arguing against a role for ROS in early E. Thus phagocytosis triggered ROS and early caspase- 1 activation occurs during the uptake of select microbes. In contrast, late or sustained caspase- 1 activation (> 1 hour after infection) was observed only with live and not heat killed S. Consistent with previous reports, S. The sustained activation of caspase- 1 and IL- 1. Late caspase- 1 activation and IL- 1. Together these data indicate that caspase- 1 activation begins immediately after internalization of S. The p. H of phagosomes containing certain live bacteria including S. Therefore we first measured acidification using HI bacteria which are unable to neutralize the phagosome. For this purpose the rate of acidification was monitored using a highly sensitive and accurate ratiometric assay (Figure 3a,b) in cells pretreated with either a pan- caspase (ZVAD) or caspase- 1 specific inhibitor (YVAD). Both ZVAD and YVAD inhibited acidification of S. Similar results were observed when p. H was measured by quantifying delivery of p. Hrodo. Caspase inhibition reduced phagosome acidification in all macrophages tested including bone marrow- derived macrophages, peritoneal macrophages, and the macrophage cell lines J7. RAW 2. 64. 7 (Figure 3d). Additionally, ZVAD also retarded acidification of phagosomes containing live bacteria (Supplementary Figure 3b). Caspase- 1 inhibition blocks the acidification of S. As an alternative, we explored whether caspase- 1 might directly regulate the accumulation of protons in S. To test this we used a phagosome p. H dissipation assay 2. Supplementary Figure 3e). This assay can estimate the dynamic regulation of p. H by monitoring the acute changes induced after pharmacological manipulation of macrophages containing phagosomes that have already acidified. As an example, and similar to previous reports using other bacteria 9,2. V- ATPase with Bafilomycin A resulted in rapid and total dissipation of the phagosome p. H (Figure 3e), confirming that the p. H of the S. Addition of ZVAD (data not shown) and YVAD (Figure 3f) resulted in partial dissipation of the p. H of phagosomes containing both live and HI S. |
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