DOI: 10.5061/dryad.573n5tbkz
title: Sepsis restructures the mitochondrial calcium uniporter complex in the lymphoid tissues of mice and humans

description: 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.
description: # Sepsis restructures the mitochondrial calcium uniporter complex in the
 lymphoid tissues of mice and humans Dataset DOI:
 [10.5061/dryad.573n5tbkz](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: 1.
 plasma cytokine concentrations by ELISA 2. protein expression of
 components of the mitochondrial calcium uniporter (MCU) complex 3. RT-PCR
 of components of the MCU complex 4. protein expression of pyruvate
 dehydrogenase E1alpha 5. concentrations of NAD+, NADH, NADtotal, and ATP
 6. expression of cleaved caspase 3, total caspase 3, cleaved caspase 1,
 total caspase 1 7. immunofluorescence of rhod-2 am as a parameter of
 mitochondrial Ca2+ concentration 8. 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). 1. 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.