Sepsis restructures the mitochondrial calcium uniporter complex in the lymphoid tissues of mice and humans
Data files
Jul 02, 2025 version files 186.81 KB
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Figure_1A_(30_days).xlsx
10.31 KB
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Figure_1A_(7_days).xlsx
9.64 KB
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Figure_1B_(30_days__LPS_intratracheal).xlsx
10.51 KB
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Figure_2H_and_2I_(data_for_graphs).xlsx
11.40 KB
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Figure_2J_(data_for_graphs).xlsx
10.36 KB
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Figure_2K_(data_for_graphs).xlsx
9.60 KB
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Figure_3_(rhod-2).xlsx
10.94 KB
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Figure_4_data_for_graphs.xlsx
20.80 KB
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Figure_4J_data_for_graphs.xlsx
13.76 KB
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Figure_5_data_for_graphs.xlsx
18.26 KB
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Figure_6_data_for_graphs.xlsx
26.94 KB
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Figure_7_data_for_graphs.xlsx
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Figures_2B-F_(data_for_graphs).xlsx
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README.md
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Abstract
Survivors of sepsis suffer from an elevated risk of premature death that is not explained by a higher burden of chronic diseases prior to the infection. Nearly 1 out of 4 survivors have persistent elevations of inflammation biomarkers, such as interleukin (IL) 6. These observations suggest that sepsis imparts durable changes to organismal biology. Eukaryotic life depends upon ATP and calcium (Ca2+). During sepsis, mitochondrial dysfunction, a failure of Ca2+ homeostasis, and sustained elevations in cytosolic [Ca2+] occur. These insults may serve as sufficient pressure to select for cells uniquely able to adapt. In this study, we compare the spleen and bone marrow lymphoid tissues of murine and human survivors of intraabdominal sepsis and non-sepsis critical illness (i.e., sterile trauma). We observe that sepsis induces in lymphoid tissues a restructuring of the mitochondrial calcium uniporter (MCU) complex: the critical channel mediating the electrophoretic uptake of Ca2+ into the mitochondrion. We show these changes persist after clinical resolution of sepsis and lead to alterations in mitochondrial Ca2+ regulation, Ca2+ signaling, oxidative metabolism, and sensitivity to programmed cell death pathways. These biochemical changes manifest as fundamental alterations in phenotype: i.e., heightened systemic IL-6 concentration. Inhibiting lysosomal pathways partially restores the MCU complex stoichiometry, mitochondrial Ca2+ homeostasis, and lymphoid tissue phenotype to a sepsis naïve state.
Dataset DOI: 10.5061/dryad.573n5tbkz
Description of the data and file structure
We compared the lymphoid tissues (i.e., spleen, bone marrow) of mice that had survived to 7, 30, and 60 days days after either 1) cecal ligation puncture (CLP) followed by best clinical practices (i.e., control of the source of infection, resuscitation with balance crystalloids, 96 hours of antibiotics for the treatment of intraabdominal sepsis (CLP30d) or 2) sham laparotomy plus best clinical practices (Sham30d).
We performed the following analyses of tissue:
- plasma cytokine concentrations by ELISA
- protein expression of components of the mitochondrial calcium uniporter (MCU) complex
- RT-PCR of components of the MCU complex
- protein expression of pyruvate dehydrogenase E1alpha
- concentrations of NAD+, NADH, NADtotal, and ATP
- expression of cleaved caspase 3, total caspase 3, cleaved caspase 1, total caspase 1
- immunofluorescence of rhod-2 am as a parameter of mitochondrial Ca2+ concentration
- tissue expression of proteins mediating chaperone-mediated autophagy: LAMP2A, HSPA8, LC3-II
To test the hypothesis that MICU1 is constitutively reduced via heightened lysosomal degradation in sepsis survivors, we treated CLP30d mice with the lysosome inhibitors chloroquine and bafilomycin A1 (CQ+BafA1); the first dose was delivered after operative resection of the cecum (i.e., source control) and administration of best practices (i.e., volume resuscitation, antibiotics).
- we analyzed plasma and tissue for the same outcomes
Whether or not a similar restructuring of the MCU complex occurs in human sepsis survivors was studied by comparing MICU1:MCU expression in the peripheral blood mononuclear cells (PBMC) isolated from septic and trauma patients that had survived to day 30. We analyzed PBMC for expression of components of the MCU complex
We explored whether similar alterations occur with severe viral infection by measuring the expression of MICU1 and MCU longitudinally in the PBMCs of critically ill SARS-CoV2 ICU patients.
Files and variables
File: Figure_1A_(7_days).xlsx
Description: C57Bl/6 mice underwent cecal ligation and puncture (CLP) with best clinical practices or sham surgery with best clinical practices. At day 7, mice were euthanized, blood was collected, and plasma cytokines concentrations assayed by ELISA.
Variables
- TNFa, IL-6, and IL-10 plasma concentrations (pg/mL) - basal
File: Figure_1A_(30_days).xlsx
Description: C57Bl/6 mice underwent cecal ligation and puncture (CLP) with best clinical practices or sham surgery with best clinical practices. At day 30, mice were euthanized, blood was collected, and plasma cytokines concentrations assayed by ELISA.
Variables
- TNFa, IL-6, and IL-10 plasma concentrations (pg/mL) - basal
File: Figure_1B_(30_days__LPS_intratracheal).xlsx
Description: C57Bl/6 mice underwent cecal ligation and puncture (CLP) with best clinical practices or sham surgery with best clinical practices. At day 30, a nested, parallel cohort was administered intratracheally either LPS (3 mg/kg; i.t.) or equivolume 0.9% saline (NS). After 24 hours, mice were euthanized, blood was collected, and plasma cytokines concentrations assayed by ELISA.
Variables
- TNFa, IL-6, and IL-10 plasma concentrations (pg/mL) – endotoxemia.
File: Figures_2B-F_(data_for_graphs).xlsx
Description: CLP 30 days. C57Bl/6 mice underwent Sham or CLP and best clinical practices. At 30 days, mice were euthanized, spleen and bone marrow tissues were harvested, and total cell protein lysate isolated and analyzed by immunoblot for MCUR1, MICU1-3, EMRE, MCU, and TOM20.
Variables
- MICU1:TOM20, MCU:TOM20, MICU1:MCU, MCUR1:TOM20, MCUR1:MCU
File: Figure_2H_and_2I_(data_for_graphs).xlsx
Description: Endotoxemia 30 days. C57Bl/6 mice were injected with a single dose of LPS (3 mg/kg; i.p.). At 30 days, spleen and bone marrow tissues were harvested and analyzed for MCUR1, MICU1, MCU, and TOM20.
Variables
- MICU1:MCU, MICU1:TOM20
File: Figure_2J_(data_for_graphs).xlsx
Description: CLP 30 days - Mitochondrial protein. C57Bl/6 mice underwent Sham or CLP and best clinical practices. At 30 days, mice were euthanized, pleen and bone marrow tissues were harvested, and a mitochondria-enriched fraction was isolated and analyzed by immunoblot for MICU1, MCU, and TOM20.
Variables
- MICU1:MCU, MICU1:TOM20
File: Figure_2K_(data_for_graphs).xlsx
Description: *LP 30 days - RT-PCR. C57Bl/6 mice underwent Sham or CLP and best clinical practices. At 30 days, mice were euthanized, spleen and bone marrow tissues were harvested, and RT-PCR for MICU1 performed.
Variables
- MICU1 mRNA
File: Figure_3_(rhod-2).xlsx
Description: C57Bl/6 mice underwent Sham or CLP and best clinical practices. At 30 days, peritoneal macrophages were isolated from Sham and CLP mice, plated, and then loaded with Rhod-2 am for 30 minutes at 37°C. Fluorescence intensity as a marker of [Ca2+]m was imaged by spectrophotometry: ex/em 552nm/581nm.
RAW 264.7 macrophage cells were transfected with Non-target (NT), MICU1, MCUR1, or both MICU1 and MCUR1 siRNA. After 72 hours, cells were loaded with Rhod-2 am at 37°C. Fluorescence intensity was imaged by spectrophotometry: ex/em 552nm/581nm.
Variables
- Rhod-2 fluorescence intensity
File: Figure_4_data_for_graphs.xlsx
Description: C57Bl/6 mice underwent Sham30d or CLP30d and best practices and on day 30 were administered intratracheally either lipopolysaccharide (3 mg/kg; i.t.) or equivolume 0.9% normal saline (NS). Twenty-four hours later spleen and bone marrow tissues were harvested and analyzed.
Variables
- MICU1 and MCU expression. p-PDHE and PDHE expression. NADH (pmole/mg), NAD+ (pmole/mg), NAD+/NADH, NAD total (pmole/mg), and ATP (uM/mg) concentrations
File: Figure_4J_data_for_graphs.xlsx
Description: RAW 264.7 macrophage cells were transfected with Non-target (NT), MICU1, MCUR1, or both MICU1 and MCUR1 siRNA. After 72 hours cells were exposed to LPS (1ug/mL) or equivolume normal (0.9%) saline. Twenty-four hours later total cell lysate was harvested and analyzed.
Variables
- NADH, NAD+, NAD+/NADH, NAD (pmole/mg) total concentrations
File: Figure_5_data_for_graphs.xlsx
Description: C57Bl/6 mice underwent Sham or CLP and best practices. On day 30, mice were administered intratracheally lipopolysaccharide (4 mg/kg, i.t.) or equivolume 0.9% normal saline (NS). Twenty-four hours later spleen and bone marrow lymphoid tissues were harvested and analyzed by immunoblot for cleaved caspase 3 (cl-cas3), caspase 3 (cas 3), cleaved caspase 1 (cl-cas 1), caspase 1 (cas 1), and actin.
Variables
- cl-cas3, cas 3, cl-cas1, cas 1
File: Figure_6_data_for_graphs.xlsx
Description: C57Bl/6 mice underwent Sham or CLP and best practices. On day 30, mice were administered intratracheally LPS (4 mg/kg, i.t.) or equivolume 0.9% normal saline (NS). Twenty-four hours later spleen and bone marrow tissues were harvested and analyzed by immunoblot for LAMP2, LC3-I, LC3-II, and actin.
Variables
- LAMP2, LC3-I, LC3-II, and actin expression
- Spleen tissue was immunoprecipitated with anti-HSPA8 or isotype IgG control and analyzed by immunoblot for HSPA8, MICU1, LAMP2a, and MCU.
Description: HEK cells were incubated with CCCP, and at the times indicated, cell lysate was immunoprecipitated with anti-HSPA8 and analyzed for HSPA8, MICU1, and LAMP2a.
Variables
- LAMP2, LC3-I, LC3-II, and actin expression
Description: RAW 264.7 macrophage cells were transfected with Non-target (NT) or HSPA8 siRNA. After 72 hours, cells were stimulated with LPS (1ug/mL). After 24 hours, total cell lysate was harvested and analyzed by immunoblot for MICU1, MCU, and actin.
Variables
- MICU1, MCU, and actin expression
Description: C57Bl/6 mice underwent Sham30d or CLP30d. At days 2, 3, and 28 mice were administered intraperitoneally chloroquine and bafilomycin A1 or equivolume DMSO vehicle control. At day 30 spleen and bone marrow tissues were isolated and analyzed by immunoblot for MICU1, MCU, and TOM20. Plasma was isolated from blood and analyzed for cytokine concentrations.
Variables
- NAD+, NADH concentrations
- IL-6 and IL-10 plasma concentration (pg/mL)
File: Figure_7_data_for_graphs.xlsx
Description: Samples of blood were obtained from human subject participants at day 30 after ICU admission and a primary diagnosis of either trauma or sepsis. Peripheral blood mononuclear cells (PBMC) were isolated and analyzed by immunoblot for MICU1, MCU, and TOM20.
Variables
- MICU1, MCU, and TOM20 expression
Description: Samples of blood were obtained from participants at day 7, 28 and 84 after a first sample (Day 0) collected early after ICU admission. Peripheral blood mononuclear cells (PBMC) were isolated and analyzed by immunoblot for MICU1, MCU, and TOM20 and analyzed by flow cytometry for immune subtypes and for [Ca2+]m using Rhod-2 (ex/em 552nm/581nm)
Variables
- MICU1, MCU, and TOM20 expression
- rhod-2 relative fluorescence intensity
Human subjects data
The human subject study was approved by the Institutional Review Board (PRO#119120152). Written informed consent was obtained from all patients prior to inclusion. All ethical regulations relevant to human research participants were followed.