Publications

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.

Microglia and the border-associated macrophages contribute to the modulation of cerebral blood flow, but the mechanisms have remained uncertain. Here, we show that microglia regulate the cerebral blood flow baseline and the responses to whisker stimulation or intra-cisternal magna injection of adenosine triphosphate, but not intra-cisternal magna injection of adenosine in mice model. Notably, microglia repopulation corrects these cerebral blood flow anomalies. The microglial-dependent regulation of cerebral blood flow requires the adenosine triphosphate-sensing P2RY12 receptor and ectonucleotidase CD39 that initiates the dephosphorylation of extracellular adenosine triphosphate into adenosine in both male and female mice. Pharmacological inhibition or CX3CR1-CreER-mediated deletion of CD39 mimics the cerebral blood flow anomalies in microglia-deficient mice and reduces the upsurges of extracellular adenosine following whisker stimulation. Together, these results suggest that the microglial CD39-initiated breakdown of extracellular adenosine triphosphate co-transmitter is an important step in neurovascular coupling and the regulation of cerebrovascular reactivity.

Stem cells reside in specialized microenvironments, termed niches, at several different locations in tissues1-3. The differential functions of heterogeneous stem cells and niches are important given the increasing clinical applications of stem-cell transplantation and immunotherapy. Whether hierarchical structures among stem cells at distinct niches exist and further control aspects of immune tolerance is unknown. Here we describe previously unknown new hierarchical arrangements in haematopoietic stem cells (HSCs) and bone marrow niches that dictate both regenerative potential and immune privilege. High-level nitric oxide-generating (NOhi) HSCs are refractory to immune attack and exhibit delayed albeit robust long-term reconstitution. Such highly immune-privileged, primitive NOhi HSCs co-localize with distinctive capillaries characterized by primary ciliated endothelium and high levels of the immune-checkpoint molecule CD200. These capillaries regulate the regenerative functions of NOhi HSCs through the ciliary protein IFT20 together with CD200, endothelial nitric oxide synthase and autophagy signals, which further mediate immunoprotection. Notably, previously described niche constituents, sinusoidal cells and type-H vessels2-10 co-localize with less immune-privileged and less potent NOlow HSCs. Together, we identify highly immune-privileged, late-rising primitive HSCs and characterize their immunoprotective niches comprising specialized vascular domains. Our results indicate that the niche orchestrates hierarchy in stem cells and immune tolerance, and highlight future immunotherapeutic targets.

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.

Extracellular diphosphate and triphosphate nucleotides are released from activated or injured cells to trigger vascular and immune P2 purinergic receptors, provoking inflammation and vascular thrombosis. These metabokines are scavenged by ectonucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1 or CD39). Further degradation of the monophosphate nucleoside end products occurs by surface ecto-5'-nucleotidase (NMPase) or CD73. These ectoenzymatic processes work in tandem to promote adenosinergic responses, which are immunosuppressive and antithrombotic. These homeostatic ectoenzymatic mechanisms are lost in the setting of oxidative stress, which exacerbates inflammatory processes. We have engineered bifunctional enzymes made up from ectodomains (ECDs) of CD39 and CD73 within a single polypeptide. Human alkaline phosphatase-ectodomain (ALP-ECD) and human acid phosphatase-ectodomain (HAP-ECD) fusion proteins were also generated, characterized, and compared with these CD39-ECD, CD73-ECD, and bifunctional fusion proteins. Through the application of colorimetrical functional assays and high-performance liquid chromatography kinetic assays, we demonstrate that the bifunctional ectoenzymes express high levels of CD39-like NTPDase activity and CD73-like NMPase activity. Chimeric CD39-CD73-ECD proteins were superior in converting triphosphate and diphosphate nucleotides into nucleosides when compared with ALP-ECD and HAP-ECD. We also note a pH sensitivity difference between the bifunctional fusion proteins and parental fusions, as well as ectoenzymatic property distinctions. Intriguingly, these innovative reagents decreased platelet activation to exogenous agonists in vitro. We propose that these chimeric fusion proteins could serve as therapeutic agents in inflammatory diseases, acting to scavenge proinflammatory ATP and also generate anti-inflammatory adenosine.