Anesthesia Research Centers

CD39 is an ectoenzyme in immune cells that regulates purinergic signaling by converting extracellular ATP into adenosine (ADO). Although first described on EBV-transformed B cells, CD39's role in humoral immunity remains unclear. Using murine infection models and human samples, we confirm and extend previous findings showing that high CD39 expression identifies antibody-secreting cells (ASC) across differentiation stages, including ASC derived from memory B cells, and in various tissues, regardless of the infection phase. CD39 was resistant to enzymatic digestion, facilitating ASC identification in processed tissues. We found that while CD39 was not essential for B-cell differentiation into ASC, it remained functionally active as an ectoenzyme. ASC as well as germinal center (GC) B cells expressed ADO receptors, making them responsive to ADO signaling. Consistently, systemic ADO administration impaired GC reactions without altering the ASC number in infected mice. However, in vitro, ADO reduces antibody production both in ASC and in B cells undergoing differentiation and also impairs the differentiation of activated B cells. Finally, B cell-specific CD39 deficiency increased GC B-cell frequencies in infected mice, likely due to reduced ADO levels. These findings highlight the relevance of the purinergic pathway in B-cell biology.

BACKGROUND: Calcific aortic valve stenosis is a global clinical burden, impacting around 2% of the population over 65 years of age. No pharmacotherapeutics exist, with surgical repair and transcatheter valve replacement being the only intervention. Females are underrepresented in studies of calcific aortic valve stenosis, leading to delay in timely intervention and increased mortality. Histopathology demonstrates female calcific aortic valve stenosis presents with decreased valvular calcification but increased fibrosis and severity of symptoms. We hypothesize that the underlying molecular mechanisms contributing to disease progression and fibrocalcific burden in aortic stenosis (AS) differ between male and female patients. Our goal for this study is to use previously acquired proteomic data sets of a clinically defined human AS cohort to examine sex disparities and underlying sex-specific disease signatures.

METHODS: Age-matched human AS tissue samples (n=14 males, n=4 females) were each segmented into nondiseased, fibrotic, and calcified disease stages and analyzed using LC-MS/ms proteomics and quantitative histopathology. AS plasma samples (n=32 males, n=20 females) were analyzed for circulating sex-specific biomarkers via LC-MS/ms.

RESULTS: Unbiased principal component analysis shows sex- and stage-specific proteome clustering. AS pathogenesis drove sex-specific disparities in the valvular proteome: 338/1503 total proteins were differentially enriched by sex across disease stages. Compared with sex-specific nondiseased controls, female fibrotic tissue resulted in 2.75-fold greater number of differentially enriched proteins than did male fibrotic tissue (female: 42, male: 16; P<0.05 threshold). In contrast, female calcific tissue identified 2.473-fold less differentially enriched proteins than male calcific tissue (female: 157, male 356; q<0.05 threshold). Functional Enrichment Analysis revealed specific proteins responsible for the exacerbated valvular fibrosis signature in females, implicated adenosine phosphate metabolism as a potential male-specific driver of AS, and further reinforced the shared contribution of aberrant lipid and cholesterol activity to AS progression in both sexes.

CONCLUSIONS: This proof-of-concept analysis allows for the identification of potential sex-specific protein drug targets implicated in AS pathobiology.

CD39 or NTPDase1 and other nucleoside triphosphate diphosphohydrolases (NTPDases), including NTPDase2, NTPDase3, and NTPDase8, regulate purinergic signaling through tuning the extracellular levels of purine nucleotides and nucleosides. Purinergic signaling regulates liver ischemia-reperfusion (I/R) injury, and CD39 is protective. However, the role of other NTPDases is unknown. In this study, we investigated the role of NTPDase2, NTPDase3, and NTPDase8. Global Entpd2-/-, Entpd3-/-, and Entpd8-/- and control wild type (WT) mice were subjected to liver I/R. In addition, WT and Entpd8-/- mice underwent global ischemia induced by hemorrhagic shock and resuscitation and injury evaluated. Bone marrow chimeric mice were generated to understand the role of NTPDase expression on hematopoietic cells in regulating liver injury. Although WT, Entpd2-/- and Entpd3-/- mice exhibited comparable levels of liver injury following local IR, Entpd8-/- mice had increased liver injury compared to WT mice. Studies with bone marrow chimeric mice indicated that NTPDase8 on parenchymal liver cells protected against hepatic injury. This was confirmed by single-nucleus RNAseq showing hepatocytes are the dominant cell type expressing NTPDase8 in the liver. Entpd8-/- mice after I/R injury were noted to have higher ATP concentrations in the liver and plasma. The P2 receptor antagonist suramin decreased liver injury in Entpd8-/- mice indicating P2 signaling contributes to liver injury in these mice. Finally, Entpd8-/- mice had increased liver injury compared to WT mice also after hemorrhagic shock and resuscitation. These findings highlight the differential roles of NTPDase family members in the liver, with parenchymal/hepatocyte expression of NTPDase8 emerging as a critical suppressor of the inflammatory and metabolic responses to hepatic I/R insult, even in the presence of vascular NTPDase1 expression.

Chagas disease, caused by Trypanosoma cruzi, is endemic to Latin America and is characterized by chronic inflammation of cardiac tissues due to parasite persistence. Hypoxia within infected tissues may trigger the stabilization of HIF-1 and be linked to ATP release. Extracellular ATP exhibits microbicidal effects but is scavenged by CD39 and CD73 ectonucleotidases, which ultimately generate adenosine (ADO), a potent immunosuppressor. Here, we comprehensively study the importance of HIF-1 stabilization and the CD39/CD73/ADO axis, on CD4+ T cells with the cytotoxic phenotype, in facilitating the persistence of T. cruzi. Myocardial infection induces prominent areas of hypoxia, which is concomitant with HIF-1α stabilization in T cells and linked to early expansion of CD39+CD73+CD4+ T cell infiltrating population. Functional assays further demonstrate that HIF-1 stabilization and CD73 activity are associated with impaired CD4+ T cell cytotoxic potential. RNA-Seq analysis reveals that HIF-1 and purinergic signaling pathways are overrepresented in cardiac tissues of patients with end-stage Chagas disease. The findings highlight a major effect of purinergic signaling on CD4+ T cells with potential cytotoxic capacity in the setting of T. cruzi infection and have translational implications for therapy.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a result of diffuse lung injury and dysregulated inflammation. Recent studies have demonstrated that B cells can perform anti-inflammatory and tissue-protective functions. We hypothesized that systemic B-cell administration could have therapeutic effects in hyperoxic acute lung injury.

METHODS: Acute lung injury was modeled in adult C57BL/6J male mice through continuous exposure to hyperoxia (FiO2 >90%). Mature B cells (CD45R+/CD19+) were purified from spleens of age- and sex-matched C57BL/6J mice. B cells (107) or saline were administered intravenously after 24 hours of hyperoxia. Hyperoxia exposure was continued for up to 96 hours. The effects of adoptive B-cell therapy were assessed using histologic, physiologic (pulse oximetry, echocardiography), and immunologic (flow cytometry) readouts.

RESULTS: Hyperoxia led to a 50% depletion of endogenous pulmonary B cells by day 3, from 30% to 15% CD45+ lung immune cells (95% confidence interval [CI], 6.16-24.45; P = .0017). B-cell administration ameliorated B-cell loss, improved lung injury scores (median score in saline-treated = 3.0 vs B-cell-treated = 2.67; P = .0101) and lung cellular infiltration (F [2,34] = 11.99; P = .0001). By day 3, B cells limited the duration of oxygen desaturations (difference 0.39 seconds; median length = 1.01 seconds in saline-treated vs 0.62 seconds in B-cell-treated; 95% CI, 0.02-0.73 seconds; P = .03) and their depth (median nadir = 82.0% in saline-treated vs 85.9% in B-cell-treated, 95% CI, -6.6% to -0.84%; P = .04). B-cell-treated mice showed a median 3.82% increase in left ventricular ejection fraction by day 3, compared to 12.35% in saline-treated mice (mean difference 7.32%; 95% CI, -5.0% to 19.6%; P = .23). Exogenous B cells represented less than 1.5% of pulmonary B cells on day 3. B-cell administration had homeostatic effects on relative abundance of pulmonary immune subsets affected by hyperoxia, including endogenous B cells, CD4+ T cells, natural killer (NK) cells, monocytes/macrophages, and neutrophils. Significant immunomodulatory effects of B-cell administration were observed in myeloid cells in the lungs and included reductions in the proportion of interleukin-17 (IL-17)-expressing Ly6Clo monocytes (F [2,14] = 19.02; P = .0001), alveolar macrophages (F [2,14] = 10.32; P = .0018), and neutrophils (F [2,14] = 6.621; P = .0095) as well as interferon-gamma (IFNγ)-expressing Ly6Clo monocytes (F [2,14] = 48.83; P = .0001).

CONCLUSIONS: Our data indicate that adoptive B-cell therapy ameliorates hyperoxic lung injury and may represent a novel treatment for ARDS.