Lectures 16-18 Flashcards
Provide three functions of the immune system.
Prevent entry of (or eliminate) infectious agents/pathogens
Minimise damage that pathogens may cause
Ensure infection is short-lived and leaves no permanent damage.
What two basic processes occur in immune responses?
Recognition of the pathogen.
Reaction to eliminate the pathogen.
What are the two types of immune responses?
Innate or adaptive.
Give an overview of innate immunity.
First line of defence
Non-specific and rapid, same response on each encounter with an antigen.
Innate immune response systems include:
- External barriers (e.g., skin or mucous membranes).
- Receptors that recognise a broad spectrum of pathogens.
Give an overview of adaptive immunity.
It’s acquired immunity based on creating an immunological memory.
It’s pathogen specific (e.g., measles, after infection you have life long immunity).
Works on the formation of antibodies in response to antigens.
Basis of vaccinations.
Name 8 cells specific to the innate immune response.
Innate Immunity (Rapid Response):
1. Macrophage
2. Dendritic Cell
3. Mast Cell
4. Natural Killer Cell
5. Complement Proteins
6. Neutrophil
7. Eosinophil.
8. Basophil
Name 4 cells that are specific to adaptive immune response. Give a brief description of them too.
Adaptive Immunity (Slow Response):
1. B Cell: Lymphocytes that produce antibodies specific to antigens; also function as antigen-presenting cells and form memory cells for long-term immunity.
2. T Cell: Lymphocytes with various roles in adaptive immunity, subdivided into:
- CD4+ (Helper T Cell): Coordinate immune responses by activating B cells and other immune cells.
- CD8+ (Cytotoxic T Cell): Kill infected or cancerous cells by recognizing antigens presented on MHC class I molecules.
3. γδ T Cell: A less common type of T cell with innate and adaptive immune features; they respond to non-peptide antigens and play a role in tissue surveillance.
4. Natural Killer T Cell: Hybrid cells that share characteristics of both innate NK cells and adaptive T cells; they recognize lipid antigens presented by CD1d molecules and can quickly respond to pathogens.
Name two cells that overlap both the innate and adaptive immune responses. Give a brief description of them too.
- Dendritic Cells: Serve as a bridge by presenting antigens to T cells, initiating adaptive immunity.
- Natural Killer T Cells: Exhibit characteristics of both systems, with rapid response and antigen specificity.
What are Macrophages?
Large phagocytic cells that engulf and digest pathogens, debris, and dead cells; also release cytokines to recruit other immune cells.
What are Dendritic cells?
Specialized antigen-presenting cells that capture antigens and present them to T cells, bridging innate and adaptive immunity.
What are Mast cells?
Involved in allergic reactions and inflammation; release histamine and other chemicals to recruit immune cells to the infection site.
What are Natural killer cells?
Lymphocytes that destroy infected or cancerous cells by inducing apoptosis without the need for prior activation.
What are Complement proteins?
A group of proteins that enhance the ability of antibodies and phagocytic cells to clear pathogens through a cascade of reactions.
What are Neutrophils?
Highly abundant phagocytic cells that are the first responders to infection, ingesting and killing microbes.
What are Eosinophils?
Granulocytes that combat multicellular parasites (e.g., helminths) and are involved in allergic reactions.
What are Basophils?
Granulocytes that release histamine and other chemicals during allergic and inflammatory responses.
What are B cells?
Lymphocytes that produce antibodies specific to antigens; also function as antigen-presenting cells and form memory cells for long-term immunity.
What are T cells?
Lymphocytes with various roles in adaptive immunity, subdivided into:
CD4+ and CD8+ cells
What are CD4+ cells? (Helper T cell)
Coordinate immune responses by activating B cells and other immune cells.
What are CD8+ cells? (Cytotoxic T cell)
Kill infected or cancerous cells by recognizing antigens presented on MHC class I molecules.
What are γδ T cells?
A less common type of T cell with innate and adaptive immune features; they respond to non-peptide antigens and play a role in tissue surveillance.
What are Natural killer T cells?
Hybrid cells that share characteristics of both innate NK cells and adaptive T cells; they recognize lipid antigens presented by CD1d molecules and can quickly respond to pathogens.
Outline the types of cells in the immune cells and their relation to circadian clocks.
Numerous different immune cells have been demonstrated to possess clockwork machinery.
Circulating immune cells possess clocks.
Tissue resident cells which respond to inflammatory insult have clocks.
Also ILCs and CD8+ T cells have clocks.
What are APCs?
Antigen Presenting Cells
When was the first evidence of intrinsic clocks in macrophages found? Where were they?
2009 and they were peritoneal
Outline the Keller et al (2009) study into Per2 and Rev-erba in mice.
METHODS:
- Used Per2::Luc reporter mice.
- Took the spleen, lymph nodes and macrophages.
- Put them under photon multiplier tubes to measure bioluminescence over time.
- Used qPCR to look at two genes, Per2 and Reverba
RESULTS:
- 24 hour rhythms were shown.
- The oscillations in the macrophages were in antiphase with each other.
Outline the Kelly et al (2009) study into the influence of clock timing on EX VIVO macrophage activity.
METHODS:
- Cultured macrophages from the spleen and introduced lipopolysaccharide.
- When macrophages detect this, they produce cytokines.
- 4 hours later they used ELISA to quantify TNF alpha response.
- Repeated this every 4 hours over 72 hours.
RESULTS:
- There is NOT a consistent response across time; there is a higher response at some points and lower at others.
Thus, a macrophage intrinsic clockwork regulates circadian TNF-alpha secretion upon LPS stimulation.
Outline the Kelly et al (2009) study into the influence of clock timing on IN VIVO macrophage activity.
METHODS:
- Took a live mouse and injected LPS into the peritoneal cavity and 4 hours later used ELISA to quantify cytokine responses.
- Changed the time of day that LPS was injected and measured the differences.
RESULTS:
- Found that at CT12, active phase, there were higher levels of cytokine response compared to CT0.
Thus, time of day does impact the innate response of macrophages in vivo.
Outline the Gibbs et al (2012) study into the role of the macrophage clock in vivo.
METHODS:
- Used Cre-Lox system to delete BMAL1 in LysM expressing cells.
- This was targeting strategy to remove it from macrophages (as it has LysM).
- Used PER2::Luc reporter to track rhythms in WT vs LysM-BMAL1-/- over 10 days.
- Introduced Lipopolysaccharides (LPS) to induce an inflammatory response from the macrophages - measured the IL-6 as a result.
RESULTS:
- WT there is still BMAL and KO there was no BMAL (method worked).
- Macrophages without BMAL1 were arrhythmic.
- In WT, there was diurnal variation and higher responses to LPS at CT12 than CT0.
- In LysM-BMAL1-/- KO, there was a loss of temporal gating of immune response; there was no significant difference between CT0 and CT12.
Thus the circadian clock regulates in vivo responses in macrophages.
Outline the Gibbs et al (2012) study into REV-ERB alpha and its role in clock and immunity.
METHODS:
- Took WT and LysM-BMAL1-/- mice.
- Compared levels of PER2, BMAL1, CRY1 REV-ERB alpha, REV-ERB beta and DBP.
- Then compared cytokine levels (IL-6) in response to LPS between WT and REV-ERBalpha-/- at CT0 and CT12.
RESULTS:
Levels of Clock Gene Expression in BMAL1-/-:
- BMAL1 KO leads to a reduction in rev-erb alpha and rev-erb beta expression.
- This indicates that REV-ERB alpha’s activity is disrupted when BMAL1 is absent.
- The rhythmic expression (temporal gating) of other clock genes (e.g., per2, cry1) and immune-related genes is also altered.
REV-ERBalpha-/-:
- In REV-ERBalpha-/- you lost the temporal gating, in WT it is present, showing that REV-ERB is critical for time dependent regulation of inflammatory responses.
THUS, this shows that REV-ERB is a key link between the clock and immunity.
What is the NLRP3 inflammasome a critical component of?
Innate Immune System.
What is the two-step activation process of NLRP3 inflammasome?
- Primed by pro-inflammatory stimuli (e.g., LPS).
- Secondary ignals (danger associated molecular pattern, DAMPs) trigger further activation.
What does activation of the NLRP3 inflammasome elicit?
Maturation of IL1Beta and IL18.
In what manner is NLRP3 expressed and what controls it?
A diurnal manner under the control of REV-ERBalpha.
What did Chromatin Immunoprecipitation (ChIP) reveal about REV-ERB-alpha and NLRP3?
That REV-ERBalpha can directly bind to NLRP3 promoter region.
Outline the Findings of the Pourcet et al (2018) study into REV-ERBalpha and the NLRP3 inflammasome.
This work showed that REVEVB alpha regulated the NLRP3 inflammasome, which is a critical component of the immune response.
In WT mice, the NLRP3 transcriptome peaks in the dark phase.
In the KO condition, you lose the peak expression of NLRP3.
Thus REV-ERB is regulating it’s expression via direct binding to the NLRP3 promoter.
Assays shows it can directly bind.
Outline the Pourcet et al (2018) study into Fulminant hepatitis.
METHODS:
- Animals were given the trigger for Fulminant hepatitis and then treated at different circadian times ranging from day time to night time.
RESULTS:
- There is a variation in response depending on the time of day of treatment.
- Treated at night means more likely to survive.
- Worse outcome if treated during the day.
Response was mediated by REV-ERB alpha.
Describe the basic jet-lag protocol in mice.
6h phase advance once per week (by shortening the dark period).
Equivalent to flying from Chicago to Manchester every week.
Describe the Castanon-Cervantes et al (2010) study into the effects of ‘Jet Lag’ on macrophage function.
METHODS:
- Implement a jet lag protocol in which there is one 6h phase advance per week (6h reduction in dark phase).
- Compare pro-inflammatory response by peritoneal macrophages to LPS challenge vs controls.
RESULTS:
- Circadian disruption worsens outcomes.
- There was an increased pro-inflammatory response in the peritoneal macrophages across all cytokines.
Describe monocytes.
Monocytes are versatile immune cells that act as first responders to infections and tissue damage.
They bridge the innate and adaptive immune systems by removing harmful materials, producing inflammatory signals and differentiating into macrophages and dendritic cells.
Their role is essential in both fighting infections and maintaining immune balance.
Describe mast cells.
Mast cells are highly versatile immune cells with a pivotal role in the body’s defence against pathogens, allergic responses and tissue repair.
While they are essential for protecting the body, their dysregulation can result in allergic diseases and chronic inflammation.
Their strategic location in tissues and potent mediators make them a critical component of the immune system.
What is the relation between the clock and monocytes? (Nguyen et al., 2013)
The clock within monocytes can impact how they traffic to sites of inflammation.
What is the relation between the clock and mast cells? (Nakamura et al., 2017).
They have a robust clock so if you KO the clock there is no longer a time of day gating of histamine release.
Summarise the 4 main points regarding clocks and immunity.
Clocks within innate immune cells influence the amplitude of the cellular response to a challenge.
Clocks influence whole animal responses to acute pathogenic challenge.
In general, deletion or disruption of the clock negatively impacts how a cell responds to an inflammatory stimulus.
REV-ERB alpha directly regulates inflammatory mechanisms independent of clockwork machinery.
Where are T cells and B cells derived from?
Haematopoietic stem cells in the bone marrow.
Where do B cells mature?
In the bone marrow.
Where do T cells mature?
In the thymus.
What differentiates CD4+ cells into subsets?
Transcription factors.
