Introduction
Over activation of T-cells and macrophages results in symptoms of HLH
Familial Hemophagocytic Lymphohistiocytosis (fHLH) is an immune condition caused by an over activation and excessive formation of macrophages and T- lymphocytes. Symptoms of fHLH result from decreased regulation of the macrophages, allowing excessive cytokine release. The cytokine causes excessive inflammation in multiple regions of the body, including the spleen, liver, brain, and bone marrow. (Zhang, 2021). Additional symptoms include prolonged and high fever, splenomegaly, cytopenias, or low blood cell counts (Janka et al., 2005).
Familial HLH is caused by loss-of-function mutation in PRF1
A mutation in the PRF1 gene is one of the causes of fHLH (Zhang, 2021). There are two recognized protein domains in this gene: the Membrane Attack Complex/ Perforin Domain as well as the Calcium dependent C2 Domain (Paysan-Lafosse et al. 2022). We will focus on the MACPF_2 domain for this discussion.
Loss of PRF1 decreases perforin pore formations and associated cytotoxic responses
The MACPF_2 domain is the pore forming domain, where perforin subunits are encoded. The quaternary structure of perforin is the pore (Osinka et al., 2014). This protein localizes at the cellular membrane when the immune response is activated. The function of pore formation is involved in leukocyte mediated cytotoxicity (Paysan-Lafosse et al. 2022). When the recessive, loss- of- function mutation occurs as it does in fHLH, cytotoxins are unable to enter and degrade the infected cell. This leads to an increase in natural killer cell and T- cell activation, causing the excessive cytokine release (Osinka et al., 2014).
Protein Domains Across Species
The MACPF_2 domain is well conserved amongst vertebrates, and is only found in organisms with an immune system (Paysan-Lafosse et al. 2022).
PRF1 Phylogeny
A maximum likelihood phylogenetic tree for Perforin was generated using Mega11. Mammalian PRF1 is most closely related, but, given the well conserved nature of the protein domains, Danio Rerio is still a candidate for any proposed study of PRF1 mutation. (Tamura, Stecher, and Kumar 2021)
The relationship between hyperferritinemia and PRF1 mutation is unclear
One possible symptom of fHLH is hyperferritinemia, or an excess of ferritin in the bloodstream (Zhang, 2021). Ferritin is a protein in which ferric iron is stored (Kernan et al., 2017). The role of iron homeostasis in fHLH is currently unclear. My hypothesis is that iron homeostasis in fHLH will result from varying expression of ferritin encoding genes [FTL FTH], and subsequent protein concentrations.
Danio Rerio will be used as model organism
To test this relationship, Danio Rerio will be used as a model organism. Zebrafish is an excellent model due to its sensitivity to oxidative stress from ferric iron, as previous studies have also been completed with zebrafish to study the effects of ferric ions (Bohaud et al., 2021). Zebrafish is a popular choice for modeling immunology systems due to clear larval state. It also has a similar macrophage activity to humans (Ayaat et al., 2020). All of these reasons make zebrafish an ideal candidate for reverse genetics approaches.
PRF1 Interactions
PRF1 has similar protein networks in both Homo Sapiens and Danio Rerio, with both being heavily involved in T- cell proliferation pathways (green) as well as the innate immune response (blue) (Szklarczyk, 2023).
AIM 1
Identifying Protein Domain Essential for Pore Formation in Danio Rerio
Slide 1: Aim one will involve the identification of protein domains essential for pore formation in Danio Rerio. As previously determined using InterPro, the MACPF_2 domain is related to pore formation (Paysan-Lafosse et al. 2022).
Slide 2: Multiple sequence alignment of this region will be performed between the Danio Rerio and Homo Sapiens to identify possible regions to induce mutation to cause fHLH like symptoms in the Zebrafish. One identified mutation in Homo Sapiens that causes fHLH is the Glycine to Alanine mutation at the 445th location (Zhang 2011). The sequence alignment will be used to confirm that this region of interest has no gaps in Danio Rerio.
Slide 3: CRISPR/Cas9 will then be used to induce mutation in region of choice. Blood serum will be collected and analyzed in Zebrafish to confirm an excess of white blood cells is identified. We hypothesize that a mutation in this region would cause fHLH symptoms since the pore formation domain is crucial for cytotoxic activity.
Slide 2: Multiple sequence alignment of this region will be performed between the Danio Rerio and Homo Sapiens to identify possible regions to induce mutation to cause fHLH like symptoms in the Zebrafish. One identified mutation in Homo Sapiens that causes fHLH is the Glycine to Alanine mutation at the 445th location (Zhang 2011). The sequence alignment will be used to confirm that this region of interest has no gaps in Danio Rerio.
Slide 3: CRISPR/Cas9 will then be used to induce mutation in region of choice. Blood serum will be collected and analyzed in Zebrafish to confirm an excess of white blood cells is identified. We hypothesize that a mutation in this region would cause fHLH symptoms since the pore formation domain is crucial for cytotoxic activity.
Aim 2
Identifying Gene Expression in PRF1 and Iron Homeostasis Genes
Slide 1: Zebrafish spleen tissue of the induced knockouts as well as the control fish will be isolated using microscopy and a capillary pipette. The DNA of each cell will be amplified by PCR and sequenced using Illumina sequencing. Reverse transcription will be performed to result in an RNA library (Hwang et al., 2018).
Slide 2: Cluster data of PRF1 and iron homeostasis-associated genes will reveal any regulatory relationships between PRF1 mutation and ferritin (Hwang et al., 2018).
Slide 3: Confirmation of hyperferritinemia symptoms in knockout zebrafish will be completed with fluorescence assay targeting ferric iron. I hypothesize that Ferritin encoding genes will be highly associated with PRF1 perforin (Ayaat et al., 2020).
Slide 2: Cluster data of PRF1 and iron homeostasis-associated genes will reveal any regulatory relationships between PRF1 mutation and ferritin (Hwang et al., 2018).
Slide 3: Confirmation of hyperferritinemia symptoms in knockout zebrafish will be completed with fluorescence assay targeting ferric iron. I hypothesize that Ferritin encoding genes will be highly associated with PRF1 perforin (Ayaat et al., 2020).
Aim 3
Analysis of proteome via metabolic labeling of control/KO tissues
Slide 1: To approach this aim, tissue isolated from knockout zebrafish will be grown in medium with heavy arginine. Wild-type Dario Rerio tissue will be grown in a light arginine medium. After digestion with trypsin, mass spectroscopy will be completed on both experimental groups (Ong et al., 2007).
Slide 2: Volcano plots will be generated for analysis of fold increase/decrease protein expression (Choi et al., 2022). Labeling tissue experimental groups with arginine isotopes will reveal a fold increase or decrease in the system, which would reveal any fHLH associations with ferritin.
Slide 3:The fluorescence assay targeting ferric iron will once again be used as confirmation. I hypothesis increase in protein fold increase for ferritin will be observed in PRF1 perforin knockout tissues.
Slide 2: Volcano plots will be generated for analysis of fold increase/decrease protein expression (Choi et al., 2022). Labeling tissue experimental groups with arginine isotopes will reveal a fold increase or decrease in the system, which would reveal any fHLH associations with ferritin.
Slide 3:The fluorescence assay targeting ferric iron will once again be used as confirmation. I hypothesis increase in protein fold increase for ferritin will be observed in PRF1 perforin knockout tissues.
Future Directions
In conclusion, fHLH is an autoimmune condition where a PRF1 mutation can cause damage to the immune response. The relationship between this condition and high iron homeostasis is unknown. I explored Danio Rerio as a model organism for PRF1 mutation and how it can be used to elucidate this relationship. I proposed multiple genomics/proteomics approaches to exploring the relationship between these pathways. Future research should be targeted towards relieving symptoms of fHLH such as hyperferritinemia. This could be accomplished using a small molecule assay to discover any drugs or small molecules that may decrease said symptoms.
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