H15: PYROPTOSIS IN DISEASE
15.1 INTRODUCTIE
15.1.1 CRITICAL CARE - SEPSIS - MODS
Sepsis and multiorgan dysfunction syndrome
= the leading causes of death in intensive care units, and the incidence is still rising.
= Pro- and anti cytokine present → body does not know what to do
Lack of Effective Treatment
- Xigris, the sole FDA-approved treatment for severe sepsis, was withdrawn in 2011.
- The absence of effective treatments poses a significant challenge in managing these conditions.
Clinical Trial Failures
- Many clinical studies have failed to produce successful treatments.
- This highlights the complexity and heterogeneity of critical illnesses, necessitating further research.
Incidence Statistics
- Approximately 52 million cases of sepsis occur worldwide annually.
- Sepsis accounts for one-fifth of ICU admissions.
- A significant portion of ICU patients with sepsis, will die bcs of multiple
organ failure
ANIMAL MODELS
Lack of sepsis animal models
= crucial for understanding sepsis mechanisms, testing new treatments, identifying biomarkers, studying
host-pathogen interactions, and developing therapeutic approaches.
● Barrier to developing effective therapies for sepsis.
● Despite the use of hundreds of different animal models over the years, no single model perfectly
replicates the human condition of sepsis, which complicates the translation of research findings into
clinical practice.
Common experimental sepsis models
● TNF-induced SIRS (Systemic Inflammatory Response Syndrome)
● Endotoxemia: introducing endotoxins into blood to induce inflammatory response
● Cecal Ligation and Puncture (CLP): tying off and puncturing the cecum to induce polymicrobial sepsis
● Colon Ascendens Stent Peritonitis (CASP): placing a stent in the ascending colon to create a continuous
leak of intestinal contents into the peritoneal cavity, leading to peritonitis and sepsis.
→ Each of these models offers unique advantages and limitations for studying the pathophysiology of sepsis
and testing potential treatments.
,ISSUES WITH GENETIC BACKGROUND
Differences in response of Black6 mouse how they respond to LPS and TNF.
→ Passenger mutations confound interpretation of all genetically modified congenic mice!
● Scientists assume that any observed differences between the modified mice and their controls are due
to the targeted genetic changes (induced by Crispr/Cas9).
● However, if there are passenger mutations present, these unintended mutations can also affect the
phenotype of the mice.
● This leads to misattribution: they attribute observed effects to the targeted genetic modification,
when they are actually caused by passenger mutations
MOLECULAR INTERVENTION STRATEGY?
1) Anti-TNF therapy: block upstream → but other receptors take over
○ Inhibits inflammatory response associated with sepsis
○ But blocking TNF alone isn’t sufficient because other inflammatory receptors can continue
inflammatory response
2) Block hubs → more successful
○ Instead on targeting single molecules like TNF, they focus on “hubs”
○ Hubs = critical nodes in the network that integrate multiple signaling pathways
○ → This approach allows targeting critical nodes in molecular pathways, potentially offering
broader and more impactful therapeutic effects interventions.
HETEROGENEITY OF SEPSIS PATIENTS
1. Diverse Clinical Presentations: Varying symptoms, severity, and organ dysfunction.
2. Complex Pathophysiology: Multiple pathways and individual responses
3. Treatment Response Variability: Different reactions to standard treatments.
→ Accounting for this heterogeneity is crucial for personalized care and treatment effectiveness.
→ Still no cure
,15.1.2 SIMPLIFIED WORKING MODEL FOR CRITICAL ILLNESS
Basic Working Model
● Dying cells → go into necrosis → release cellular contents → trigger immune reaction → induce
inflammation
● If necrotic cells have important barrier functions (like lung epithelial cells) → barrier will disrupt →
allow pathogens to infiltrate and infect the host.
● This sets off an auto-amplifying loop, worsening the immune response and inflammation.
Need for Intervention
● Because of the auto-amplifying loop → need to intervene at various levels to mitigate the progression
of critical illness.
Drugs
● Infection: antibiotics, antiviral drugs
● Inflammation: NSAIDs
● Necrosis: necrosis inhibitors
, 15.1.3 AIM - ROAD PLAN (CHATGPT)
1) RELEVANCE EXPERIMENTAL SEPSIS MODEL?
Aim road plan = pinpoint converging molecular nodes in sepsis that allow therapeutic intervention
Disputed Genes in Sepsis
● Casp1 & -11 (Caspase 1 and 11)
● Casp7 & -3 (Caspase 7 and 3)
● RIPK1 (Receptor-Interacting Protein Kinase 1)
● Panx1 (Pannexin 1)
● TLR4 (Toll-Like Receptor 4)
● GPX4 (Glutathione Peroxidase 4)
● IL1β & -18 (Interleukin 1 beta and 18)
● MLKL (Mixed Lineage Kinase Domain-Like Protein)
Embryo Transfer in SPF Facility
● Genetically modified mice with KO of 1 of the disputed genes
● Embryos with the disputed genes are transferred into a Specific Pathogen-Free (SPF) facility
In Vivo Screen:
→ 3 Models
1) LPS (Lipopolysaccharide): This model uses bacterial endotoxins to induce a sepsis-like inflammatory
response.
2) TNF (Tumor Necrosis Factor): This model involves administering TNF to trigger systemic inflammation
similar to sepsis.
3) CLP (Cecal Ligation and Puncture): This model simulates polymicrobial sepsis by causing intestinal
perforation and leakage of gut bacteria into the peritoneal cavity.
→ 2 Readouts
● Hypothermia: A common symptom of sepsis in mice
● Survival
Outcome
The research aims to generate and study specific mouse models, such as:
● Casp1/11-/
● IL1β/18-/-
Summary: investigate the role of specific genes in sepsis using genetically modified mice. The approach
includes generating these models through embryo transfer in a pathogen-free environment, testing them with
various sepsis models, and evaluating outcomes based on hypothermia and survival rates. This strategy aims to
uncover the genetic factors contributing to sepsis and inform the development of new treatments.
15.1 INTRODUCTIE
15.1.1 CRITICAL CARE - SEPSIS - MODS
Sepsis and multiorgan dysfunction syndrome
= the leading causes of death in intensive care units, and the incidence is still rising.
= Pro- and anti cytokine present → body does not know what to do
Lack of Effective Treatment
- Xigris, the sole FDA-approved treatment for severe sepsis, was withdrawn in 2011.
- The absence of effective treatments poses a significant challenge in managing these conditions.
Clinical Trial Failures
- Many clinical studies have failed to produce successful treatments.
- This highlights the complexity and heterogeneity of critical illnesses, necessitating further research.
Incidence Statistics
- Approximately 52 million cases of sepsis occur worldwide annually.
- Sepsis accounts for one-fifth of ICU admissions.
- A significant portion of ICU patients with sepsis, will die bcs of multiple
organ failure
ANIMAL MODELS
Lack of sepsis animal models
= crucial for understanding sepsis mechanisms, testing new treatments, identifying biomarkers, studying
host-pathogen interactions, and developing therapeutic approaches.
● Barrier to developing effective therapies for sepsis.
● Despite the use of hundreds of different animal models over the years, no single model perfectly
replicates the human condition of sepsis, which complicates the translation of research findings into
clinical practice.
Common experimental sepsis models
● TNF-induced SIRS (Systemic Inflammatory Response Syndrome)
● Endotoxemia: introducing endotoxins into blood to induce inflammatory response
● Cecal Ligation and Puncture (CLP): tying off and puncturing the cecum to induce polymicrobial sepsis
● Colon Ascendens Stent Peritonitis (CASP): placing a stent in the ascending colon to create a continuous
leak of intestinal contents into the peritoneal cavity, leading to peritonitis and sepsis.
→ Each of these models offers unique advantages and limitations for studying the pathophysiology of sepsis
and testing potential treatments.
,ISSUES WITH GENETIC BACKGROUND
Differences in response of Black6 mouse how they respond to LPS and TNF.
→ Passenger mutations confound interpretation of all genetically modified congenic mice!
● Scientists assume that any observed differences between the modified mice and their controls are due
to the targeted genetic changes (induced by Crispr/Cas9).
● However, if there are passenger mutations present, these unintended mutations can also affect the
phenotype of the mice.
● This leads to misattribution: they attribute observed effects to the targeted genetic modification,
when they are actually caused by passenger mutations
MOLECULAR INTERVENTION STRATEGY?
1) Anti-TNF therapy: block upstream → but other receptors take over
○ Inhibits inflammatory response associated with sepsis
○ But blocking TNF alone isn’t sufficient because other inflammatory receptors can continue
inflammatory response
2) Block hubs → more successful
○ Instead on targeting single molecules like TNF, they focus on “hubs”
○ Hubs = critical nodes in the network that integrate multiple signaling pathways
○ → This approach allows targeting critical nodes in molecular pathways, potentially offering
broader and more impactful therapeutic effects interventions.
HETEROGENEITY OF SEPSIS PATIENTS
1. Diverse Clinical Presentations: Varying symptoms, severity, and organ dysfunction.
2. Complex Pathophysiology: Multiple pathways and individual responses
3. Treatment Response Variability: Different reactions to standard treatments.
→ Accounting for this heterogeneity is crucial for personalized care and treatment effectiveness.
→ Still no cure
,15.1.2 SIMPLIFIED WORKING MODEL FOR CRITICAL ILLNESS
Basic Working Model
● Dying cells → go into necrosis → release cellular contents → trigger immune reaction → induce
inflammation
● If necrotic cells have important barrier functions (like lung epithelial cells) → barrier will disrupt →
allow pathogens to infiltrate and infect the host.
● This sets off an auto-amplifying loop, worsening the immune response and inflammation.
Need for Intervention
● Because of the auto-amplifying loop → need to intervene at various levels to mitigate the progression
of critical illness.
Drugs
● Infection: antibiotics, antiviral drugs
● Inflammation: NSAIDs
● Necrosis: necrosis inhibitors
, 15.1.3 AIM - ROAD PLAN (CHATGPT)
1) RELEVANCE EXPERIMENTAL SEPSIS MODEL?
Aim road plan = pinpoint converging molecular nodes in sepsis that allow therapeutic intervention
Disputed Genes in Sepsis
● Casp1 & -11 (Caspase 1 and 11)
● Casp7 & -3 (Caspase 7 and 3)
● RIPK1 (Receptor-Interacting Protein Kinase 1)
● Panx1 (Pannexin 1)
● TLR4 (Toll-Like Receptor 4)
● GPX4 (Glutathione Peroxidase 4)
● IL1β & -18 (Interleukin 1 beta and 18)
● MLKL (Mixed Lineage Kinase Domain-Like Protein)
Embryo Transfer in SPF Facility
● Genetically modified mice with KO of 1 of the disputed genes
● Embryos with the disputed genes are transferred into a Specific Pathogen-Free (SPF) facility
In Vivo Screen:
→ 3 Models
1) LPS (Lipopolysaccharide): This model uses bacterial endotoxins to induce a sepsis-like inflammatory
response.
2) TNF (Tumor Necrosis Factor): This model involves administering TNF to trigger systemic inflammation
similar to sepsis.
3) CLP (Cecal Ligation and Puncture): This model simulates polymicrobial sepsis by causing intestinal
perforation and leakage of gut bacteria into the peritoneal cavity.
→ 2 Readouts
● Hypothermia: A common symptom of sepsis in mice
● Survival
Outcome
The research aims to generate and study specific mouse models, such as:
● Casp1/11-/
● IL1β/18-/-
Summary: investigate the role of specific genes in sepsis using genetically modified mice. The approach
includes generating these models through embryo transfer in a pathogen-free environment, testing them with
various sepsis models, and evaluating outcomes based on hypothermia and survival rates. This strategy aims to
uncover the genetic factors contributing to sepsis and inform the development of new treatments.
