Extracellular mitochondria drive CD8 T cell dysfunction in trauma by upregulating CD39.

RATIONALE: The increased mortality and morbidity seen in critically injured patients appears associated with systemic inflammatory response syndrome (SIRS) and immune dysfunction, which ultimately predisposes to infection. Mitochondria released by injury could generate danger molecules, for example, ATP, which in turn would be rapidly scavenged by ectonucleotidases, expressed on regulatory immune cells. OBJECTIVE: To determine the association between circulating mitochondria, purinergic signalling and immune dysfunction after trauma. METHODS: We tested the impact of hepatocyte-derived free mitochondria on blood-derived and lung-derived CD8 T cells in vitro and in experimental mouse models in vivo. In parallel, immune phenotypic analyses were conducted on blood-derived CD8 T cells obtained from trauma patients. RESULTS: Isolated intact mitochondria are functional and generate ATP ex vivo. Extracellular mitochondria perturb CD8+ T cells in co-culture, inducing select features of immune exhaustion in vitro. These effects are modulated by scavenging ATP, modelled by addition of apyrase in vitro. Injection of intact mitochondria into recipient mice markedly upregulates the ectonucleotidase CD39, and other immune checkpoint markers in circulating CD8+ T cells. We note that mice injected with mitochondria, prior to instilling bacteria into the lung, exhibit more severe lung injury, characterised by elevated neutrophil influx and by changes in CD8+ T cell cytotoxic capacity. Importantly, the development of SIRS in injured humans, is likewise associated with disordered purinergic signalling and CD8 T cell dysfunction. CONCLUSION: These studies in experimental models and in a cohort of trauma patients reveal important associations between extracellular mitochondria, aberrant purinergic signalling and immune dysfunction. These pathogenic factors with immune exhaustion are linked to SIRS and could be targeted therapeutically.

Mitochondria Drive Immune Responses in Critical Disease.

Mitochondria engage in multiple cellular and extracellular signaling pathways ranging from metabolic control, antiviral and antibacterial host defense to the modulation of inflammatory responses following cellular damage and stress. The remarkable contributions of these organelles to innate and adaptive immunity, shape cell phenotype and modulate their functions during infection, after trauma and in the setting of inflammatory disease. We review the latest knowledge of mitochondrial biology and then discuss how these organelles may impact immune cells to drive aberrant immune responses in critical disease.

Adenosine deaminase 2 produced by infiltrative monocytes promotes liver fibrosis in nonalcoholic fatty liver disease.

Elevated circulating activity of adenosine deaminase 2 (ADA2) is associated with liver fibrosis in nonalcoholic fatty liver disease (NAFLD). In the liver of NAFLD patients, ADA2-positive portal macrophages are significantly associated with the degree of liver fibrosis. These liver macrophages are CD14- and CD16-positive and co-express chemokine receptors CCR2, CCR5, and CXCR3, indicating infiltrative monocyte origin. Human circulatory monocytes release ADA2 upon macrophage differentiation in vitro. When stimulated by recombinant human ADA2 (rhADA2), human monocyte-derived macrophages demonstrate upregulation of pro-inflammatory and pro-fibrotic genes, including PDGF-B, a key pro-fibrotic cytokine. This PDGF-B upregulation is reproduced by inosine, the enzymatic product of ADA2, but not adenosine, and is abolished by E359N, a loss-of-function mutation in ADA2. Finally, rhADA2 also stimulates PDGF-B production from Kupffer cells in primary human liver spheroids. Together, these data suggest that infiltrative monocytes promote fibrogenesis in NAFLD via ADA2-mediated autocrine/paracrine signaling culminating in enhanced PDGF-B production.

Trauma-induced heme release increases susceptibility to bacterial infection.

Infection is a common complication of major trauma that causes significantly increased morbidity and mortality. The mechanisms, however, linking tissue injury to increased susceptibility to infection remain poorly understood. To study this relationship, we present a potentially novel murine model in which a major liver crush injury is followed by bacterial inoculation into the lung. We find that such tissue trauma both impaired bacterial clearance and was associated with significant elevations in plasma heme levels. While neutrophil (PMN) recruitment to the lung in response to Staphylococcus aureus was unchanged after trauma, PMN cleared bacteria poorly. Moreover, PMN show > 50% less expression of TLR2, which is responsible, in part, for bacterial recognition. Administration of heme effectively substituted for trauma. Finally, day 1 trauma patients (n = 9) showed similar elevations in free heme compared with that seen after murine liver injury, and circulating PMN showed similar TLR2 reduction compared with volunteers (n = 6). These findings correlate to high infection rates.

Lack of antibodies against seasonal coronavirus OC43 nucleocapsid protein identifies patients at risk of critical COVID-19.

BACKGROUND: The vast majority of COVID-19 patients experience a mild disease. However, a minority suffers from critical disease with substantial morbidity and mortality. OBJECTIVES: To identify individuals at risk of critical COVID-19, the relevance of a seroreactivity against seasonal human coronaviruses was analyzed. METHODS: We conducted a multi-center non-interventional study comprising 296 patients with confirmed SARS-CoV-2 infections from four tertiary care referral centers in Germany and France. The ICU group comprised more males, whereas the outpatient group contained a higher percentage of females. For each patient, the serum or plasma sample obtained closest after symptom onset was examined by immunoblot regarding IgG antibodies against the nucleocapsid protein (NP) of HCoV 229E, NL63, OC43 and HKU1. RESULTS: Median age was 60 years (range 18-96). Patients with critical disease (n=106) had significantly lower levels of anti-HCoV OC43 nucleocapsid protein (NP)-specific antibodies compared to other COVID-19 inpatients (p=0.007). In multivariate analysis (adjusted for age, sex and BMI), OC43 negative inpatients had an increased risk of critical disease (adjusted odds ratio (AOR) 2.68 [95% CI 1.09 - 7.05]), higher than the risk by increased age or BMI, and lower than the risk by male sex. A risk stratification based on sex and OC43 serostatus was derived from this analysis. CONCLUSIONS: Our results suggest that prior infections with seasonal human coronaviruses can protect against a severe course of COVID-19. Therefore, anti-OC43 antibodies should be measured for COVID-19 inpatients and considered as part of the risk assessment for each patient. Hence, we expect individuals tested negative for anti-OC43 antibodies to particularly benefit from vaccination against SARS-CoV-2, especially with other risk factors prevailing.

Dysregulated Host Response in Severe Acute Respiratory Syndrome Coronavirus 2-Induced Critical Illness.

BACKGROUND: Impaired immune response has been reported to be the cause of the development of coronavirus disease 2019 (COVID-19)-related respiratory failure. Further studies are needed to understand the immunopathogenesis and to enable an improved stratification of patients who are at risk for critical illness. METHODS: Thirty-two severely ill patients hospitalized with COVID-19 were recruited in our center at the University Hospital Heidelberg. We performed a comprehensive analysis of immune phenotype, cytokine, and chemokine profiling and leukocyte transcripts in patients with severe COVID-19 and compared critically ill patients who required mechanical ventilation and high-flow oxygen therapy and noncritically ill patient who received low-flow oxygen therapy. RESULTS: Critically ill patients exhibited low levels of CD8 T cells and myeloid dendritic cells. We noted a pronounced CCR6+ TH17 phenotype in CD4 central memory cells and elevated circulating levels of interleukin-17 in the critical group. Gene expression of leukocytes derived from critically ill patients was characterized by an upregulation of proinflammatory cytokines and reduction of interferon (IFN)-responsive genes upon stimulation with Toll-like receptor 7/8 agonist. When correlating clinical improvement and immune kinetics, we found that CD8 T-cell subsets and myeloid dendritic cells significantly increased after disconnection from the ventilator. CONCLUSION: Critical illness was characterized by a TH17-mediated response and dysfunctional IFN-associated response, indicating an impaired capacity to mount antiviral responses during severe acute respiratory syndrome coronavirus 2 severe infection.

Global deletion of NTPDase3 protects against diet-induced obesity by increasing basal energy metabolism.

BACKGROUND: Ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3), also known as CD39L3, is the dominant ectonucleotidase expressed by beta cells in the islet of Langerhans and on nerves. NTPDase3 catalyzes the conversion of extracellular ATP and ADP to AMP and modulates purinergic signaling. Previous studies have shown that NTPDase3 decreases insulin release from beta-cells in vitro. This study aims to determine the impact of NTPDase3 in diet-induced obesity (DIO) and metabolism in vivo. METHODS: We developed global NTPDase3 deficient (Entpd3-/-) and islet beta-cell-specific NTPDase-3 deficient mice (Entpd3flox/flox,InsCre) using Ins1-Cre targeted gene editing to compare metabolic phenotypes with wildtype (WT) mice on a high-fat diet (HFD). RESULTS: Entpd3-/- mice exhibited similar growth rates compared to WT on chow diet. When fed HFD, Entpd3-/- mice demonstrated significant resistance to DIO. Entpd3-/- mice consumed more calories daily and exhibited less fecal calorie loss. Although Entpd3-/- mice had no increases in locomotor activity, the mice exhibited a significant increase in basal metabolic rate when on the HFD. This beneficial phenotype was associated with improved glucose tolerance, but not higher insulin secretion. In fact, Entpd3flox/flox,InsCre mice demonstrated similar metabolic phenotypes and insulin secretion compared to matched controls, suggesting that the expression of NTPDase3 in beta-cells was not the primary protective factor. Instead, we observed a higher expression of uncoupling protein 1 (UCP-1) in brown adipose tissue and an augmented browning in inguinal white adipose tissue with upregulation of UCP-1 and related genes involved in thermogenesis in Entpd3-/- mice. CONCLUSIONS: Global NTPDase3 deletion in mice is associated with resistance to DIO and obesity-associated glucose intolerance. This outcome is not driven by the expression of NTPDase3 in pancreatic beta-cells, but rather likely mediated through metabolic changes in adipocytes.

SARS-CoV-2 Seroprevalence and Clinical Features of COVID-19 in a German Liver Transplant Recipient Cohort: A Prospective Serosurvey Study.

BACKGROUND: In liver transplant (LT) recipients with severe coronavirus disease 2019 (COVID-19), fatal outcome has been reported in a substantial subset of patients. Whether LT recipients are at increased risk for severe COVID-19 compared with the general population is controversial. Here we report the results of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serosurvey in a large LT recipient cohort. METHODS: A total of 219 LT recipients were enrolled between May 5, 2020, and August 6, 2020, at the University Hospital Heidelberg. Serum blood samples were collected and tested for anti-SARS-CoV-2 IgG. SARS-CoV-2 RNA was detected in nasopharyngeal swabs using reverse transcription-polymerase chain reaction assays. RESULTS: Taking into account known risk factors of arterial hypertension, obesity, diabetes, or leukopenia, LT recipients a priori represented a high-risk cohort for severe COVID-19 with 101 of 219 (46.1%) presenting with more than 2 risk factors for severe COVID-19. Out of 219 LT recipients, 8 (3.7%) either had a positive test result for nasopharyngeal SARS-CoV-2 RNA or anti-SARS-CoV-2 serum IgG. Five of eight (62.5%) did not show any clinical signs of infection, three of eight (37.5%) had self-limited disease, and none required hospitalization for COVID-19. Two of eight (25%) had known exposure to infected health care staff as the probable source of infection. CONCLUSIONS: In summary, LT recipients showed a SARS-CoV-2 seroconversion rate similar to that of the general population with a substantial percentage of unrecognized infections.

Vitamin D Deficiency and Outcome of COVID-19 Patients.

Infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses an enormous challenge to health care systems throughout the world. Without causal treatment, identification of modifiable prognostic factors may help to improve outcomes. To explore possible associations of vitamin D (VitD) status with disease severity and survival, we studied 185 patients diagnosed with coronavirus disease 2019 (COVID-19) and treated at our center. VitD status at first presentation was assessed retrospectively using accredited laboratory methods. VitD deficiency was defined as serum total 25-hydroxyvitamin D level < 12 ng/mL (<30 nM). Primary endpoint was severe course of disease (i.e., need for invasive mechanical ventilation and/or death, IMV/D). Within a median observation period of 66 days (range 2-92), 23 patients required IMV. A total of 28 patients had IMV/D, including 16 deaths. Ninety-three (50%) patients required hospitalization (inpatient subgroup). A total of 41 (22%) patients were VitD deficient. When adjusted for age, gender, and comorbidities, VitD deficiency was associated with higher risk of IMV/D and death (HR 6.12, 95% CI 2.79-13.42, p < 0.001 and HR 14.73, 95% CI 4.16-52.19, p < 0.001, respectively). Similar correlations were observed in the inpatient subgroup. Our study demonstrates an association between VitD deficiency and severity/mortality of COVID-19, highlighting the need for interventional studies on VitD supplementation in SARS-CoV-2 infected individuals.

Daily high-dose aspirin does not lower APRI in the Aspirin-Myocardial Infarction Study.

Antiplatelet agents reduce liver fibrosis by inhibiting platelet activation and platelet-derived growth factor production. Previous cross-sectional epidemiological studies suggest that the use of aspirin is related to reduced liver fibrosis. The Aspirin-Myocardial Infarction Study (AMIS) aims to examine this relationship in a multicenter, randomized, double-blind and placebo-controlled trial. The existing clinical trial of aspirin was conducted to study the benefit of one gram aspirin daily among 4 524 individuals who had experienced at least one documented myocardial infarction. The aspartate aminotransferase (AST)-to-Platelet Ratio Index (APRI) was calculated at baseline and annually from the platelet count and AST levels. Participants in the AMIS trial had a mean baseline APRI of 0.34±0.36, and only 1% individuals had APRI scores higher than 1.0, a common cutoff for cirrhosis. The daily use of aspirin was associated with an increase, rather than a reduction of APRI, by 0.007 per year (95% CI 0.002-0.015, P=0.12). The use of aspirin did not significantly affect platelet counts. In a sensitivity analysis of individuals with probable significant fibrosis at baseline (APRI≥0.7), the aspirin group had a sustained reduction in APRI over time, although this change was not significant compared to that in the placebo group. In the AMIS trial, the daily use of high-dose aspirin did not significantly affect APRI, a surrogate index of liver fibrosis. This study highlights the need for de novo clinical trials to investigate the potential benefit of antiplatelet agents on liver fibrosis.

Circulating Phospholipid Patterns in NAFLD Patients Associated with a Combination of Metabolic Risk Factors.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is associated with inefficient macro- and micronutrient metabolism, and alteration of circulating phospholipid compositions defines the signature of NAFLD. This current study aimed to assess the pattern of serum phospholipids in the spectrum of NAFLD, and its related comorbidities and genetic modifications. METHODS: 97 patients with diagnosed NAFLD were recruited at a single center during 2013⁻2016. Based on histological and transient elastography assessment, 69 patients were divided into non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty liver (NAFL) subgroups. 28 patients served as healthy controls. Serum phospholipids were determined by liquid-chromatography mass spectrometry (LC-MS/MS). RESULTS: The total content of phosphatidylcholine (PC) and sphingomyelin in the serum was significantly increased in NAFL and NASH patients, compared to healthy controls. In addition, serum lysophospatidylethanolamine levels were significantly decreased in NAFL and NASH individuals. Circulating PC species, containing linoleic and α-linolenic acids, were markedly increased in NAFLD patients with hypertension, compared to NAFLD patients without hypertension. The pattern of phospholipids did not differ between NAFLD patients with diabetes and those without diabetes. However, NAFLD patients with hyperglycemia (blood glucose level (BGL) >100 mg/dL) exhibited significantly a higher amount of monounsaturated phosphatidylethanolamine than those with low blood glucose levels. In addition, NAFLD patients with proven GG-genotype of PNPLA3, who were at higher risk for the development of progressive disease with fibrosis, showed lower levels of circulating plasmalogens, especially 16:0, compared to those with CC- and CG-allele. CONCLUSIONS: Our extended lipidomic study presents a unique metabolic profile of circulating phospholipids associated with the presence of metabolic risk factors or the genetic background of NAFLD patients.