Question

Cell Death and Western Blot Salmonella enterica is a pathogenic bacterium that can be found in undercooked meat and eggs. Ingestion of Salmonella can result in severe intestinal inflammation, resulting in diarrhea, abdominal pain, vomiting, fever, and potential death if left untreated. In the United States alone, over 100,000 people are infected with Salmonella annually. Studies of Salmonella infection progression in mice led to an interesting observation: although inflammation is typically characterized by recruitment of immune cells, Salmonella actually causes a decrease in immune cells at early time points prior to induction of inflammation. This led researchers to hypothesize that perhaps Salmonella is causing cell death in the immune system. To address this question, you decide to perform in vitro experiments with Salmonella and Bone Marrow Derived Macrophages (BMDMs). BMDMs are a very useful cell to study macrophage biology because there are a large number of these cells in mice, they are easy to culture, and they are true macrophages as opposed to immortalized macrophage cell lines which have been manipulated towards a cancerous phenotype. Very simply, BMDMs are monocytes isolated from the bone marrow (usually from mice) and stimulated with Macrophage Colony Stimulating Factor (MCSF) to induce differentiation into a macrophage. BMDMs infected with Salmonella result in robust cell death and release of several inflammatory cytokines. Of interest, you observe very high levels of IL-1β released as the BMDMs die. Apoptosis is a form of programmed cell death initially characterized in 1972. Every day, between 50-70 billion cells die within the human body, usually via apoptosis. As you may imagine, the decision to kill a cell is a very important decision, and therefore, apoptosis is very tightly regulated by a number of key steps. If a cell loses its ability to undergo apoptosis, it may develop into cancer. Conversely, hyperactive apoptosis can result in neurodegenerative diseases such as Alzheimer's disease. Perhaps the loss of immune cells in the intestine is caused by apoptosis. Apoptosis can be initiated primarily by two pathways: extrinsic and intrinsic. As its name implies, the extrinsic pathway occurs when proteins in the extracellular space signal through receptors present on the cell. There are two main initiators of the extrinsic pathway of apoptosis: (1) TNF signaling to the TNF receptor and (2) Fas ligand (FasL) signaling to Fas. TNF is a cytokine produced mainly by myeloid cells following PRR signaling, while FasL is a transmembrane protein on the surface of cytotoxic T cells. Both the TNF receptor and Fas have special intracellular signaling domains called Death Domain (DD) that initiate a signaling cascade resulting in activation of Caspase-8. In contrast, the intrinsic pathway occurs following recognition of DNA damage, or by damage to cellular mitochondria. Cellular damage results in the release of cytochrome c from the mitochondria to the cytosol where it can interact with Apoptotic Protease Activating Factor 1 (APAF1), forming a large multimeric macromolecule known as the apoptosome. The apoptosome is then capable of activating Caspase-9. 1. What is the role of IL-1β in the immune system? 2. If Salmonella is indeed causing apoptosis in our BMDM culture system, what do you predict is the most likely pathway: TNF-extrinsic, FasL-extrinsic, or intrinsic? Why? 3. Do you think it would be advantageous for Salmonella to induce apoptosis in host cells? Why or why not? 4. Do you think it would be advantageous for the infected host animal to initiate apoptosis in infected cells? Why or why not?

Name: cell death and western blot salmonella enterica is a pathogenic bacteria that can be found in undercooked meat and eggs ingestion of salmonella can result in severe intestinal inflammation r 02414
Uploaded: 2022-09-21T12:37:42-08:00
Duration: 1 min 9 s
Channel: Sri K
Description: cell death and western blot salmonella enterica is a pathogenic bacteria that can be found in undercooked meat and eggs ingestion of salmonella can result in severe intestinal inflammation r 02414

Cell Death and Western Blot

Salmonella enterica is a pathogenic bacterium that can be found in undercooked meat and eggs. Ingestion of Salmonella can result in severe intestinal inflammation, resulting in diarrhea, abdominal pain, vomiting, fever, and potential death if left untreated. In the United States alone, over 100,000 people are infected with Salmonella annually.

Studies of Salmonella infection progression in mice led to an interesting observation: although inflammation is typically characterized by recruitment of immune cells, Salmonella actually causes a decrease in immune cells at early time points prior to induction of inflammation. This led researchers to hypothesize that perhaps Salmonella is causing cell death in the immune system.

To address this question, you decide to perform in vitro experiments with Salmonella and Bone Marrow Derived Macrophages (BMDMs). BMDMs are a very useful cell to study macrophage biology because there are a large number of these cells in mice, they are easy to culture, and they are true macrophages as opposed to immortalized macrophage cell lines which have been manipulated towards a cancerous phenotype. Very simply, BMDMs are monocytes isolated from the bone marrow (usually from mice) and stimulated with Macrophage Colony Stimulating Factor (MCSF) to induce differentiation into a macrophage.

BMDMs infected with Salmonella result in robust cell death and release of several inflammatory cytokines. Of interest, you observe very high levels of IL-1β released as the BMDMs die.

Apoptosis is a form of programmed cell death initially characterized in 1972. Every day, between 50-70 billion cells die within the human body, usually via apoptosis. As you may imagine, the decision to kill a cell is a very important decision, and therefore, apoptosis is very tightly regulated by a number of key steps. If a cell loses its ability to undergo apoptosis, it may develop into cancer. Conversely, hyperactive apoptosis can result in neurodegenerative diseases such as Alzheimer's disease. Perhaps the loss of immune cells in the intestine is caused by apoptosis.

Apoptosis can be initiated primarily by two pathways: extrinsic and intrinsic. As its name implies, the extrinsic pathway occurs when proteins in the extracellular space signal through receptors present on the cell. There are two main initiators of the extrinsic pathway of apoptosis: (1) TNF signaling to the TNF receptor and (2) Fas ligand (FasL) signaling to Fas. TNF is a cytokine produced mainly by myeloid cells following PRR signaling, while FasL is a transmembrane protein on the surface of cytotoxic T cells. Both the TNF receptor and Fas have special intracellular signaling domains called Death Domain (DD) that initiate a signaling cascade resulting in activation of Caspase-8. In contrast, the intrinsic pathway occurs following recognition of DNA damage, or by damage to cellular mitochondria. Cellular damage results in the release of cytochrome c from the mitochondria to the cytosol where it can interact with Apoptotic Protease Activating Factor 1 (APAF1), forming a large multimeric macromolecule known as the apoptosome. The apoptosome is then capable of activating Caspase-9.

1. What is the role of IL-1β in the immune system?
2. If Salmonella is indeed causing apoptosis in our BMDM culture system, what do you predict is the most likely pathway: TNF-extrinsic, FasL-extrinsic, or intrinsic? Why?
3. Do you think it would be advantageous for Salmonella to induce apoptosis in host cells? Why or why not?
4. Do you think it would be advantageous for the infected host animal to initiate apoptosis in infected cells? Why or why not?

Added by Mario R.

Biology for AP Courses

Julianne Zedalis, John Eggebrecht

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Solved by Expert Sri K

09/21/2022

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Step 1: IL-1β is a crucial cytokine in the immune system that is important for inflammation and resistance against Salmonella infection. Show more…

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Cell Death and Western Blot Salmonella enterica is a pathogenic bacterium that can be found in undercooked meat and eggs. Ingestion of Salmonella can result in severe intestinal inflammation, resulting in diarrhea, abdominal pain, vomiting, fever, and potential death if left untreated. In the United States alone, over 100,000 people are infected with Salmonella annually. Studies of Salmonella infection progression in mice led to an interesting observation: although inflammation is typically characterized by recruitment of immune cells, Salmonella actually causes a decrease in immune cells at early time points prior to induction of inflammation. This led researchers to hypothesize that perhaps Salmonella is causing cell death in the immune system. To address this question, you decide to perform in vitro experiments with Salmonella and Bone Marrow Derived Macrophages (BMDMs). BMDMs are a very useful cell to study macrophage biology because there are a large number of these cells in mice, they are easy to culture, and they are true macrophages as opposed to immortalized macrophage cell lines which have been manipulated towards a cancerous phenotype. Very simply, BMDMs are monocytes isolated from the bone marrow (usually from mice) and stimulated with Macrophage Colony Stimulating Factor (MCSF) to induce differentiation into a macrophage. BMDMs infected with Salmonella result in robust cell death and release of several inflammatory cytokines. Of interest, you observe very high levels of IL-1β released as the BMDMs die. Apoptosis is a form of programmed cell death initially characterized in 1972. Every day, between 50-70 billion cells die within the human body, usually via apoptosis. As you may imagine, the decision to kill a cell is a very important decision, and therefore, apoptosis is very tightly regulated by a number of key steps. If a cell loses its ability to undergo apoptosis, it may develop into cancer. Conversely, hyperactive apoptosis can result in neurodegenerative diseases such as Alzheimer's disease. Perhaps the loss of immune cells in the intestine is caused by apoptosis. Apoptosis can be initiated primarily by two pathways: extrinsic and intrinsic. As its name implies, the extrinsic pathway occurs when proteins in the extracellular space signal through receptors present on the cell. There are two main initiators of the extrinsic pathway of apoptosis: (1) TNF signaling to the TNF receptor and (2) Fas ligand (FasL) signaling to Fas. TNF is a cytokine produced mainly by myeloid cells following PRR signaling, while FasL is a transmembrane protein on the surface of cytotoxic T cells. Both the TNF receptor and Fas have special intracellular signaling domains called Death Domain (DD) that initiate a signaling cascade resulting in activation of Caspase-8. In contrast, the intrinsic pathway occurs following recognition of DNA damage, or by damage to cellular mitochondria. Cellular damage results in the release of cytochrome c from the mitochondria to the cytosol where it can interact with Apoptotic Protease Activating Factor 1 (APAF1), forming a large multimeric macromolecule known as the apoptosome. The apoptosome is then capable of activating Caspase-9. 1. What is the role of IL-1β in the immune system? 2. If Salmonella is indeed causing apoptosis in our BMDM culture system, what do you predict is the most likely pathway: TNF-extrinsic, FasL-extrinsic, or intrinsic? Why? 3. Do you think it would be advantageous for Salmonella to induce apoptosis in host cells? Why or why not? 4. Do you think it would be advantageous for the infected host animal to initiate apoptosis in infected cells? Why or why not?