The Cardiac Inflammatory Response in Sterile and Viral Inflammation
Restricted (Penn State Only)
- Author:
- De Jeronimo Diaz, Cesar
- Graduate Program:
- Integrative and Biomedical Physiology (PHD)
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 27, 2023
- Committee Members:
- Vishal Singh, Outside Unit & Field Member
Connie Rogers, Major Field Member
Matam Kumar, Special Member
Donna Korzick, Chair & Dissertation Advisor
Kumble Prabhu, Major Field Member
Gregory Shearer, Program Head/Chair - Keywords:
- Left Ventricle
Hypertension
Flow Cytometry
NLRP3
SARS-CoV-2
Inflammation
Inflammasome
Salt Sensitivity
Macrophage - Abstract:
- The NACHT, LRR and PYD domain-containing protein 3 (NLRP3) inflammasome is a multimeric protein complex that initiates pyroptosis, an inflammatory form of cell death that produces a proinflammatory response via the processing and releasing IL-1ß and IL-18 via caspase-1 cleavage. The NLRP3 inflammasome is comprised of NLRP3, apoptosis-associated speck-like protein (ASC) and pro-caspase-1. In general, NLRP3 activation induces oligomerization resulting in ASC and pro-caspase-1 recruitment to the forming inflammasome. NLRP3 inflammasome assembly initiates pro-caspase-1 activation leading to pro-IL-1ß, pro-IL-18 and gasdermin D cleavage. Notably, the NLRP3 inflammasome is implicated in numerous cardiovascular diseases that include myocardial infarction (MI), myocarditis and hypertension. Various signaling motifs can activate NLRP3, which include oxidized mtDNA, K+ efflux, reactive oxygen species (ROS) and viral DNA. NLRP3 pharmacological inhibition is well-established to reduce infarct size in MI and attenuate left ventricle (LV) hypertrophy and cardiac fibrosis in pressure-overload injury. In addition, severe COVID-19 patients present elevated serum IL-1ß levels, which implicate the NLRP3 inflammasome in SARS-CoV-2 infection and pathophysiology. Interestingly, vitamin D supplementation is reported to attenuate inflammation in lung SARS-CoV-2 viral infection and is linked to improved outcomes in COVID-19 patients, which may be attributed to NLRP3 inhibition by the vitamin D receptor. On the contrary, NLRP3 contains numerous inflammasome independent functions that include transcriptional activity and promoting mitochondrial ROS production; however the mechanisms behind these functions are less well-characterized. We sought to investigate the NLRP3 inflammasome in numerous cardiovascular disease processes that include hypertension , SARS-CoV-2 viral infection, and MI. Study 1: We utilized NLRP3 KO and WT Dahl/SS rats to study hypertension disease progression. NLRP3 gene loss was induced in Dahl/SS rats by CRISPR mediated mutagenesis. Surprisingly, despite being fed low salt (0.12% NaCl) both WT and NLRP3 KO Dahl/SS rats developed hypertension, thus provided us an opportunity to investigate NLRP3 gene loss in salt-induced hypertension. Remarkably, we found that NLRP3 gene loss exacerbated LV hypertrophy compared to WT rats and did not attenuate cardiac fibrosis. In addition, we found that serum IL-18 levels were elevated in the NLRP3 KO vs WT group. Resident LV macrophages/monocytes were found to be polarized towards an anti-inflammatory M2 phenotype in NLRP3 KO rats. To our knowledge, this is the first time NLRP3 gene loss is demonstrated to worsen hypertension-induced LV hypertrophy and systemic inflammation. Study 2: Previous research investigating the effect of vitamin D supplementation in SARS-CoV-2 infected K18hACE2 mice indicates that vitamin D reduces lung inflammation by decreasing Ifnb mRNA expression. Moreover, current literature provides contradictory information on whether SARS-CoV-2 virus directly infects the K18hACE2 myocardium. We aimed to investigate whether the SARS-CoV-2 virus infects the LV and the effect of vitamin D supplementation on LV viral infection. K18hACE2 mice were intranasally infected with SARS-CoV-2 virus and sacrificed on day 6 and 14 post-infection. Mice were split into three vitamin D supplementation groups that included vitamin D deficient, vitamin D sufficient and vitamin D supplemented. We found that SARS-CoV-2 N-gene mRNA expression is not present in the LV of infected K18hACE2 mice. However, we observed that LV ACE2 protein expression was increased in SARS-CoV-2 infected mice compared to baseline. In addition, we observed no significant differences in LV CD68+ macrophages, LV H&E histology or cardiac fibrosis between infected and non-infected mice. Vitamin D supplementation was found to not provide anti-inflammatory effects to the LV in the parameters measured. Study 3: Our understanding of the temporal immune response to MI is largely based on permanent ligation rodent models, which may be due to exacerbated infarct size and inflammatory response. However, reperfusion of the ligated artery should be performed for clinical relevance. Evans Blue dye perfusion is required to visualize the infarcted and non-infarcted LV regions in ischemia-reperfusion studies, which may explain the bias towards the use of permanent ligation rodent models. Flow cytometry is extensively used to characterize immune cell dynamics due to its ability to detect various protein markers in single samples. However, Evans Blue perfusion presents a technical challenge since it produces an emission peak near 680nm when excited, which overlaps with numerous fluorophores. We sought to develop methodology to perform flow cytometry on single cell suspensions isolated from Evans Blue perfused LV from rats subjected to coronary artery ligation survival surgery. Evans Blue emission within 685nm and 735nm was observed in LV CD45+CD11b+ macrophages/monocytes isolated from Sprague Dawley and Dahl/SS rats. Interestingly, the Evans Blue emission intensity varied between both groups. We developed a flow cytometry panel for Dahl/SS rats that included CD45-APC-Cy7, CD11b-Pacific Blue, CD68-AF488 and CD206-PE to minimize Evans Blue emission interference. Using this panel we were able to identify and characterize LV CD45+CD11b+ macrophages/monocytes isolated from Evans Blue perfused hearts. In this study we demonstrate that flow cytometry on the Evans Blue perfused LV is feasible. Together, these studies provide novel insight onto an unexpected role for NLRP3 in LV hypertrophy disease progression, whether SARS-CoV-2 virus directly infects the LV and the effect of vitamin D supplementation on reducing cardiac inflammation, and methodology on performing flow cytometry on single cell suspensions isolated from Evans Blue perfused LV. These studies may be informative on identifying therapeutic targets for resistant hypertension, informing therapeutic strategies for SARS-CoV-2 viral infection, and providing technical knowledge for performing flow cytometry on Evans Blue perfused tissues.